Proteomics Core FacilityPublications
Identification of Regulatory Networks in HSCs and Their Immediate Progeny via Integrated Proteome, Transcriptome, and DNA Methylome Analysis.
Cabezas-Wallscheid, N., Klimmeck, D., Hansson, J., Lipka, D.B., Reyes, A., Wang, Q., Weichenhan, D., Lier, A., von Paleske, L., Renders, S., Wunsche, P., Zeisberger, P., Brocks, D., Gu, L., Herrmann, C., Haas, S., Essers, M.A., Brors, B., Eils, R., Huber, W., Milsom, M.D., Plass, C., Krijgsveld, J. & Trumpp, A.
Cell Stem Cell. 2014 Aug 20. pii: S1934-5909(14)00301-4. doi:10.1016/j.stem.2014.07.005.
In this study, we present integrated quantitative proteome, transcriptome, and methylome analyses of hematopoietic stem cells (HSCs) and four multipotent progenitor (MPP) populations. From the characterization of more than 6,000 proteins, 27,000 transcripts, and 15,000 differentially methylated regions (DMRs), we identified coordinated changes associated with early differentiation steps. DMRs show continuous gain or loss of methylation during differentiation, and the overall change in DNA methylation correlates inversely with gene expression at key loci. Our data reveal the differential expression landscape of 493 transcription factors and 682 lncRNAs and highlight specific expression clusters operating in HSCs. We also found an unexpectedly dynamic pattern of transcript isoform regulation, suggesting a critical regulatory role during HSC differentiation, and a cell cycle/DNA repair signature associated with multipotency in MPP2 cells. This study provides a comprehensive genome-wide resource for the functional exploration of molecular, cellular, and epigenetic regulation at the top of the hematopoietic hierarchy.
New advances in reproductive biomedicine.
Virant-Klun, I., Krijgsveld, J., Huntriss, J. & Rodgers, R.J.
Biomed Res Int. 2014;2014:529170. doi: 10.1155/2014/529170. Epub 2014 Jun 19. Europe PMC
Widespread Changes in the Posttranscriptional Landscape at the Drosophila Oocyte-to-Embryo Transition.
Kronja, I., Yuan, B., Eichhorn, S.W., Dzeyk, K., Krijgsveld, J., Bartel, D.P. & Orr-Weaver, T.L.
Cell Rep. 2014 May 28. pii: S2211-1247(14)00377-5. doi:10.1016/j.celrep.2014.05.002.
The oocyte-to-embryo transition marks the onset of development. The initial phase of this profound change from the differentiated oocyte to the totipotent embryo occurs in the absence of both transcription and mRNA degradation. Here we combine global polysome profiling, ribosome-footprint profiling, and quantitative mass spectrometry in a comprehensive approach to delineate the translational and proteomic changes that occur during this important transition in Drosophila. Our results show that PNG kinase is a critical regulator of the extensive changes in the translatome, acting uniquely at this developmental window. Analysis of the proteome in png mutants provided insights into the contributions of translation to changes in protein levels, revealing a compensatory dynamic between translation and protein turnover during proteome remodeling at the return to totipotency. The proteome changes additionally suggested regulators of meiosis and early embryogenesis, including the conserved H3K4 demethylase LID, which we demonstrated is required during this period despite transcriptional inactivity.
Proteomes of Animal Oocytes: What Can We Learn for Human Oocytes in the Fertilization Programme?
Virant-Klun, I. & Krijgsveld, J.
This is a review article.
Biomed Res Int. 2014;2014:856907. Epub 2014 Apr 3.
Oocytes are crucial cells for mammalian reproduction, yet the molecular principles underlying oocyte development are only partially understood. Therefore, contemporary proteomic approaches have been used increasingly to provide new insights into oocyte quality and maturation in various species such as mouse, pig, and cow. Especially, animal studies have helped in elucidating the molecular status of oocytes during in vitro maturation and other procedures of assisted reproduction. The aim of this review is to summarize the literature on mammalian oocyte proteome and secretome research in the light of natural and assisted reproduction and on lessons to be learned for human oocytes, which have so far remained inaccessible for proteome analysis.
Rapid temporal dynamics of transcription, protein synthesis and secretion during macrophage activation.
Eichelbaum, K. & Krijgsveld, J.
Mol Cell Proteomics. 2014 Jan 6.
Macrophages provide the first line of host defense by their capacity to react to an array of cytokines and bacterial components, requiring tight regulation of protein expression and secretion to invoke a properly tuned innate immune response. To capture the dynamics of this system, we introduce a novel method combining pulsed SILAC labeling with pulse-labeling using the methionine-analogue azidohomoalanine (AHA), allowing the enrichment of newly synthesized proteins via click-chemistry followed by their identification and quantification by mass spectrometry. We show that this permits the analysis of proteome changes at a rapid time scale evidenced by the detection of 4852 newly synthesized proteins after only a 20-minute SILAC pulse. We have applied this methodology to study proteome response during macrophage activation in a time-course manner. We have combined this with full proteome, transcriptome and secretome analyses producing an integrative analysis of the first 3 hours of lipopolysaccharides (LPS)-induced macrophage activation. We observed rapid induction of multiple processes well-known to TLR4 signaling, as well as anti-inflammatory proteins and proteins not associated with immune response before. By correlating transcriptional, translational and secretory events we derived novel mechanistic principles of processes specifically induced by LPS, including ectodomain shedding and proteolytic processing of transmembrane and extracellular proteins, and protein secretion independent of transcription. In conclusion, we demonstrate that the combination of pulsed AHA and pulsed SILAC labeling permits the detailed characterization of proteomic events at a rapid time scale. We anticipate that this approach will be very useful to probe immediate effects of cellular stimuli and to gain mechanistic insight of cellular perturbation in multiple biological systems. The data have been deposited to ProteomeXchange with identifier PXD000600.
Src kinase modulates the apoptotic p53 pathway by altering HIPK2 localization.
Polonio-Vallon, T., Kirkpatrick, J., Krijgsveld, J. & Hofmann, T.G.
Cell Cycle. 2014 Jan 1;13(1):115-25. doi: 10.4161/cc.26857. Epub 2013 Oct 25.
Non-receptor tyrosine kinase Src is a master regulator of cell proliferation. Hyperactive Src is a potent oncogene and a driver of cellular transformation and carcinogenesis. Homeodomain-interacting protein kinase 2 (HIPK2) is a tumor suppressor mediating growth suppression and apoptosis upon genotoxic stress through phosphorylation of p53 at Ser46. Here we show that Src phosphorylates HIPK2 and changes its subcellular localization. Using mass spectrometry we identified 9 Src-mediated Tyr-phosphorylation sites within HIPK2, 5 of them positioned in the kinase domain. By means of a phosphorylation-specific antibody we confirm that Src mediates phosphorylation of HIPK2 at Tyr354. We demonstrate that ectopic expression of Src increases the half-life of HIPK2 by interfering with Siah-1-mediated HIPK2 degradation. Moreover, we find that hyperactive Src binds HIPK2 and redistributes HIPK2 from the cell nucleus to the cytoplasm, where both kinases partially colocalize. Accordingly, we find that hyperactive Src decreases chemotherapeutic drug-induced p53 Ser46 phosphorylation and apoptosis activation. Together, our results suggest that Src kinase suppresses the apoptotic p53 pathway by phosphorylating HIPK2 and relocalizing the kinase to the cytoplasm.
Suppression of Mediator is regulated by Cdk8-dependent Grr1 turnover of the Med3 coactivator.
Gonzalez, D., Hamidi, N., Del Sol, R., Benschop, J.J., Nancy, T., Li, C., Francis, L., Tzouros, M., Krijgsveld, J., Holstege, F.C. & Conlan, R.S.
Proc Natl Acad Sci U S A. 2014 Feb 18;111(7):2500-5. doi:10.1073/pnas.1307525111. Epub 2014 Feb 3.
Mediator, an evolutionary conserved large multisubunit protein complex with a central role in regulating RNA polymerase II-transcribed genes, serves as a molecular switchboard at the interface between DNA binding transcription factors and the general transcription machinery. Mediator subunits include the Cdk8 module, which has both positive and negative effects on activator-dependent transcription through the activity of the cyclin-dependent kinase Cdk8, and the tail module, which is required for positive and negative regulation of transcription, correct preinitiation complex formation in basal and activated transcription, and Mediator recruitment. Currently, the molecular mechanisms governing Mediator function remain largely undefined. Here we demonstrate an autoregulatory mechanism used by Mediator to repress transcription through the activity of distinct components of different modules. We show that the function of the tail module component Med3, which is required for transcription activation, is suppressed by the kinase activity of the Cdk8 module. Med3 interacts with, and is phosphorylated by, Cdk8; site-specific phosphorylation triggers interaction with and degradation by the Grr1 ubiquitin ligase, thereby preventing transcription activation. This active repression mechanism involving Grr1-dependent ubiquitination of Med3 offers a rationale for the substoichiometric levels of the tail module that are found in purified Mediator and the corresponding increase in tail components seen in cdk8 mutants.
Combining Pulsed SILAC Labeling and Click-Chemistry for Quantitative Secretome Analysis.
Eichelbaum, K. & Krijgsveld, J.
Methods Mol Biol. 2014;1174:101-14. doi: 10.1007/978-1-4939-0944-5_7.
Secreted proteins, such as cytokines, chemokines, and hormones, exhibit central functions in intercellular communication, which is crucial to maintain homeostasis in every multicellular organism. A common approach to identify secreted proteins is by proteomic analysis of culture media after conditioning with a cell type of interest. This is preferably done in serum-free conditions to enable the detection of low-abundance secretory factors that would otherwise be masked by serum proteins. However, serum starvation introduces the risk of bringing cells in a stressed or perturbed state. A superior approach employs the enrichment of newly synthesized and secreted proteins from serum-containing growth medium. This is achieved by the combination of two metabolic labels: stable isotope-labeled amino acids for reliable quantification, and azidohomoalanine (AHA), an azide-bearing analogue of methionine, for the enrichment of newly synthesized and secreted proteins. This approach has been used to compare secretomes of multiple cell lines or to analyze proteins that are secreted upon a specific stimulation. Here we describe in detail the enrichment and quantification of newly synthesized and secreted proteins.
CHD4 is a RanGTP-dependent MAP that stabilizes microtubules and regulates bipolar spindle formation.
Yokoyama, H., Nakos, K., Santarella-Mellwig, R., Rybina, S., Krijgsveld, J., Koffa, M.D. & Mattaj, I.W.
Curr Biol. 2013 Dec 16;23(24):2443-51. doi: 10.1016/j.cub.2013.09.062. Epub 2013Nov 21.
BACKGROUND: Production of the GTP-bound form of the Ran GTPase (RanGTP) around chromosomes induces spindle assembly by activating nuclear localization signal (NLS)-containing proteins. Several NLS proteins have been identified as spindle assembly factors, but the complexity of the process led us to search for additional proteins with distinct roles in spindle assembly. RESULTS: We identify a chromatin-remodeling ATPase, CHD4, as a RanGTP-dependent microtubule (MT)-associated protein (MAP). MT binding occurs via the region containing an NLS and chromatin-binding domains. In Xenopus egg extracts and cultured cells, CHD4 largely dissociates from mitotic chromosomes and partially localizes to the spindle. Immunodepletion of CHD4 from egg extracts significantly reduces the quantity of MTs produced around chromatin and prevents spindle assembly. CHD4 RNAi in both HeLa and Drosophila S2 cells induces defects in spindle assembly and chromosome alignment in early mitosis, leading to chromosome missegregation. Further analysis in egg extracts and in HeLa cells reveals that CHD4 is a RanGTP-dependent MT stabilizer. Moreover, the CHD4-containing NuRD complex promotes organization of MTs into bipolar spindles in egg extracts. Importantly, this function of CHD4 is independent of chromatin remodeling. CONCLUSIONS: Our results uncover a new role for CHD4 as a MAP required for MT stabilization and involved in generating spindle bipolarity.
Proteomic analysis of cell fate decision.
Hansson, J. & Krijgsveld, J.
Curr Opin Genet Dev. 2013 Oct;23(5):540-7. doi: 10.1016/j.gde.2013.06.004. Epub2013 Aug 10.
The field of proteomics is progressing at a rapid pace, developing from primarily a specialist technology to a valuable tool in biological research. Importantly, the establishment of mass spectrometry as a quantitative method, miniaturisation of liquid chromatography techniques, and improved sensitivity of mass-spectrometric instrumentation now enable near-complete monitoring of cellular proteome dynamics. An increasing number of studies are therefore now applying quantitative proteomics to study proteins and posttranslational modifications in stem cells, to reveal molecular mechanisms and pathways underlying pluripotency, differentiation and reprogramming.
The RNA-binding protein repertoire of embryonic stem cells.
Kwon, S.C., Yi, H., Eichelbaum, K., Fohr, S., Fischer, B., You, K.T., Castello, A., Krijgsveld, J., Hentze, M.W. & Kim, V.N.
Nat Struct Mol Biol. 2013 Sep;20(9):1122-30. doi: 10.1038/nsmb.2638. Epub 2013Aug 4.
RNA-binding proteins (RBPs) have essential roles in RNA-mediated gene regulation, and yet annotation of RBPs is limited mainly to those with known RNA-binding domains. To systematically identify the RBPs of embryonic stem cells (ESCs), we here employ interactome capture, which combines UV cross-linking of RBP to RNA in living cells, oligo(dT) capture and MS. From mouse ESCs (mESCs), we have defined 555 proteins constituting the mESC mRNA interactome, including 283 proteins not previously annotated as RBPs. Of these, 68 new RBP candidates are highly expressed in ESCs compared to differentiated cells, implicating a role in stem-cell physiology. Two well-known E3 ubiquitin ligases, Trim25 (also called Efp) and Trim71 (also called Lin41), are validated as RBPs, revealing a potential link between RNA biology and protein-modification pathways. Our study confirms and expands the atlas of RBPs, providing a useful resource for the study of the RNA-RBP network in stem cells.
Properties of isotope patterns and their utility for peptide identification in large-scale proteomic experiments.
Okawa, S., Fischer, B. & Krijgsveld, J.
Rapid Commun Mass Spectrom. 2013 May 15;27(9):1067-75. doi: 10.1002/rcm.6551.
RATIONALE: In proteomic experiments, isotope patterns are routinely generated for all detected peptides. While this pattern is determined by peptide composition, it has not been evaluated as a parameter that can help in the process of peptide identification. METHODS: First, we investigated how the relative isotope abundance (RIA) accuracy in proteomic data sets depends on the spectral intensity, resolution, and the number of mass spectrometry (MS) 1 scans, using an Orbitrap Velos mass spectrometer. Next, we explored the discriminatory power of isotope patterns in the context of proteome analyses of various complexities, either alone or in combination with a Mascot database search. Finally, we provide a theoretical framework for the required accuracies of both peptide mass and RIA for peptide identification. RESULTS: We demonstrate that the RIA error obtained in routine proteome analyses is 4-5%, and that this is only modestly influenced by spectral intensity, resolution, and the number of MS1 scans. While RIA alone has no discriminatory power, in a Mascot search isotope patterns can distinguish top scoring hits from runner-up hits in 70-95% of cases. Our theoretical approach shows that RIA accuracy needs to be ~0.2% in order to uniquely identify peptides in full proteomes. CONCLUSIONS: Our results demonstrate that isotope patterns can have discriminatory power when used in combination with a classical database search. Inclusion of this parameter in proteomic workflows may help to increase confidence in peptide identification, but in practical terms this will be limited to small proteomes.
Comprehensive histone phosphorylation analysis and identification of pf14-3-3 protein as a histone h3 phosphorylation reader in malaria parasites.
Dastidar, E.G., Dzeyk, K., Krijgsveld, J., Malmquist, N.A., Doerig, C., Scherf, A. & Lopez-Rubio, J.J.
PLoS One. 2013;8(1):e53179. doi: 10.1371/journal.pone.0053179. Epub 2013 Jan 7.
The important role of histone posttranslational modifications, particularly methylation and acetylation, in Plasmodium falciparum gene regulation has been established. However, the role of histone phosphorylation remains understudied. Here, we investigate histone phosphorylation utilizing liquid chromatography and tandem mass spectrometry to analyze histones extracted from asexual blood stages using two improved protocols to enhance preservation of PTMs. Enrichment for phosphopeptides lead to the detection of 14 histone phospho-modifications in P. falciparum. The majority of phosphorylation sites were observed at the N-terminal regions of various histones and were frequently observed adjacent to acetylated lysines. We also report the identification of one novel member of the P. falciparum histone phosphosite binding protein repertoire, Pf14-3-3I. Recombinant Pf14-3-3I protein bound to purified parasite histones. In silico structural analysis of Pf14-3-3 proteins revealed that residues responsible for binding to histone H3 S10ph and/or S28ph are conserved at the primary and the tertiary structure levels. Using a battery of H3 specific phosphopeptides, we demonstrate that Pf14-3-3I preferentially binds to H3S28ph over H3S10ph, independent of modification of neighbouring residues like H3S10phK14ac and H3S28phS32ph. Our data provide key insight into histone phosphorylation sites. The identification of a second member of the histone modification reading machinery suggests a widespread use of histone phosphorylation in the control of various nuclear processes in malaria parasites.
System-wide identification of RNA-binding proteins by interactome capture.
Castello, A., Horos, R., Strein, C., Fischer, B., Eichelbaum, K., Steinmetz, L.M., Krijgsveld, J. & Hentze, M.W.
Nat Protoc. 2013 Feb 14;8(3):491-500. doi: 10.1038/nprot.2013.020. Epub 2013 Feb14.
Owing to their preeminent biological functions, the repertoire of expressed RNA-binding proteins (RBPs) and their activity states are highly informative about cellular systems. We have developed a novel and unbiased technique, called interactome capture, for identifying the active RBPs of cultured cells. By making use of in vivo UV cross-linking of RBPs to polyadenylated RNAs, covalently bound proteins are captured with oligo(dT) magnetic beads. After stringent washes, the mRNA interactome is determined by quantitative mass spectrometry (MS). The protocol takes 3 working days for analysis of single proteins by western blotting, and about 2 weeks for the determination of complete cellular mRNA interactomes by MS. The most important advantage of interactome capture over other in vitro and in silico approaches is that only RBPs bound to RNA in a physiological environment are identified. When applied to HeLa cells, interactome capture revealed hundreds of novel RBPs. Interactome capture can also be broadly used to compare different biological states, including metabolic stress, cell cycle, differentiation, development or the response to drugs.
Origin of monocytes and macrophages in a committed progenitor.
Hettinger, J., Richards, D.M., Hansson, J., Barra, M.M., Joschko, A.C., Krijgsveld, J. & Feuerer, M.
Nat Immunol. 2013 Aug;14(8):821-30. doi: 10.1038/ni.2638. Epub 2013 Jun 30.
Monocytes, macrophages and dendritic cells (DCs) are developmentally related regulators of the immune system that share the monocyte-macrophage DC progenitor (MDP) as a common precursor. Unlike differentiation into DCs, the distal pathways for differentiation into monocytes and monocyte-derived macrophages are not fully elucidated. We have now demonstrated the existence of a clonogenic, monocyte- and macrophage-restricted progenitor cell derived from the MDP. This progenitor was a Ly6C(+) proliferating cell present in the bone marrow and spleen that generated the major monocyte subsets and macrophages, but not DCs or neutrophils. By in-depth quantitative proteomics, we characterized changes in the proteome during monocyte differentiation, which provided insight into the molecular principles of developing monocytes, such as their functional maturation. Thus, we found that monocytes and macrophages were renewed independently of DCs from a committed progenitor.
Developments in quantitative mass spectrometry for the analysis of proteome dynamics.
Hughes, C. & Krijgsveld, J.
Trends Biotechnol. 2012 Dec;30(12):668-76. doi: 10.1016/j.tibtech.2012.09.007.Epub 2012 Oct 27.
Two of the primary responses in a cell when perturbed are modulation of the dynamics of its constituent gene expression and protein abundance to restore steady-state homeostasis. To obtain a detailed model of the restoration of this balance, it is necessary to examine the kinetics of transcription and translation, thus going beyond establishing mere abundance levels of transcripts and proteins. In this review we discuss proteomic approaches that utilize genomic tagging and metabolic labeling to reveal turnover kinetics for cellular proteins in a high-throughput manner. Novel metabolic and multiplexed labeling techniques coupled to mass spectrometry, in combination with next-generation sequencing approaches, provide tools for studying the principles of cellular adaptation and dynamics in unprecedented detail.
Selective enrichment of newly synthesized proteins for quantitative secretome analysis.
Eichelbaum, K., Winter, M., Diaz, M.B., Herzig, S. & Krijgsveld, J.
Nat Biotechnol. 2012 Oct;30(10):984-90. doi: 10.1038/nbt.2356. Epub 2012 Sep 23.
Secreted proteins constitute a large and biologically important subset of proteins that are involved in cellular communication, adhesion and migration. Yet secretomes are understudied because of technical limitations in the detection of low-abundance proteins against a background of serum-containing media. Here we introduce a method that combines click chemistry and pulsed stable isotope labeling with amino acids in cell culture to selectively enrich and quantify secreted proteins. The combination of these two labeling approaches allows cells to be studied irrespective of the complexity of the background proteins. We provide an in-depth and differential secretome analysis of various cell lines and primary cells, quantifying secreted factors, including cytokines, chemokines and growth factors. In addition, we reveal that serum starvation has a marked effect on secretome composition. We also analyze the kinetics of protein secretion by macrophages in response to lipopolysaccharides.
Proteomic cornerstones of hematopoietic stem cell differentiation: distinct signatures of multipotent progenitors and myeloid committed cells.
Klimmeck, D., Hansson, J., Raffel, S., Vakhrushev, S.Y., Trumpp, A. & Krijgsveld, J.
Mol Cell Proteomics. 2012 Aug;11(8):286-302. doi: 10.1074/mcp.M111.016790. Epub2012 Mar 27.
Regenerative tissues such as the skin epidermis, the intestinal mucosa or the hematopoietic system are organized in a hierarchical manner with stem cells building the top of this hierarchy. Somatic stem cells harbor the highest self-renewal activity and generate a series of multipotent progenitors which differentiate into lineage committed progenitors and subsequently mature cells. In this report, we applied an in-depth quantitative proteomic approach to analyze and compare the full proteomes of ex vivo isolated and FACS-sorted populations highly enriched for either multipotent hematopoietic stem/progenitor cells (HSPCs, Lin(neg)Sca-1(+)c-Kit(+)) or myeloid committed precursors (Lin(neg)Sca-1(-)c-Kit(+)). By employing stable isotope dimethyl labeling and high-resolution mass spectrometry, more than 5000 proteins were quantified. From biological triplicate experiments subjected to rigorous statistical evaluation, 893 proteins were found differentially expressed between multipotent and myeloid committed cells. The differential protein content in these cell populations points to a distinct structural organization of the cytoskeleton including remodeling activity. In addition, we found a marked difference in the expression of metabolic enzymes, including a clear shift of specific protein isoforms of the glycolytic pathway. Proteins involved in translation showed a collective higher expression in myeloid progenitors, indicating an increased translational activity. Strikingly, the data uncover a unique signature related to immune defense mechanisms, centering on the RIG-I and type-1 interferon response systems, which are installed in multipotent progenitors but not evident in myeloid committed cells. This suggests that specific, and so far unrecognized, mechanisms protect these immature cells before they mature. In conclusion, this study indicates that the transition of hematopoietic stem/progenitors toward myeloid commitment is accompanied by a profound change in processing of cellular resources, adding novel insights into the molecular mechanisms at the interface between multipotency and lineage commitment.
Insights into RNA Biology from an Atlas of Mammalian mRNA-Binding Proteins.
Castello, A., Fischer, B., Eichelbaum, K., Horos, R., Beckmann, B.M., Strein, C., Davey, N.E., Humphreys, D.T., Preiss, T., Steinmetz, L.M., Krijgsveld, J. & Hentze, M.W.
Cell. 2012 Jun 8;149(6):1393-406. Epub 2012 May 31.
RNA-binding proteins (RBPs) determine RNA fate from synthesis to decay. Employing two complementary protocols for covalent UV crosslinking of RBPs to RNA, we describe a systematic, unbiased, and comprehensive approach, termed "interactome capture," to define the mRNA interactome of proliferating human HeLa cells. We identify 860 proteins that qualify as RBPs by biochemical and statistical criteria, adding more than 300 RBPs to those previously known and shedding light on RBPs in disease, RNA-binding enzymes of intermediary metabolism, RNA-binding kinases, and RNA-binding architectures. Unexpectedly, we find that many proteins of the HeLa mRNA interactome are highly intrinsically disordered and enriched in short repetitive amino acid motifs. Interactome capture is broadly applicable to study mRNA interactome composition and dynamics in varied biological settings.
Proteomic analysis of stem cell differentiation and early development.
van Hoof, D., Krijgsveld, J. & Mummery, C.
Cold Spring Harb Perspect Biol. 2012 Mar 1;4(3). pii: a008177. doi:10.1101/cshperspect.a008177.
Genomics methodologies have advanced to the extent that it is now possible to interrogate the gene expression in a single cell but proteomics has traditionally lagged behind and required much greater cellular input and was not quantitative. Coupling protein with gene expression data is essential for understanding how cell behavior is regulated. Advances primarily in mass spectrometry have, however, greatly improved the sensitivity of proteomics methods over the last decade and the outcome of proteomic analyses can now also be quantified. Nevertheless, it is still difficult to obtain sufficient tissue from staged mammalian embryos to combine proteomic and genomic analyses. Recent developments in pluripotent stem cell biology have in part addressed this issue by providing surrogate scalable cell systems in which early developmental events can be modeled. Here we present an overview of current proteomics methodologies and the kind of information this can provide on the biology of human and mouse pluripotent stem cells.
Highly Coordinated Proteome Dynamics during Reprogramming of Somatic Cells to Pluripotency.
Hansson J., Rafiee M.R., Reiland S., Polo J.M., Gehring J., Okawa S., Huber W., Hochedlinger K., Krijgsveld J.
Cell Rep. 2012 Dec 27;2(6):1579-92. doi: 10.1016/j.celrep.2012.10.014.
Generation of induced pluripotent stem cells (iPSCs) is a process whose mechanistic underpinnings are only beginning to emerge. Here, we applied in-depth quantitative proteomics to monitor proteome changes during the course of reprogramming of fibroblasts to iPSCs. We uncover a two-step resetting of the proteome during the first and last 3 days of reprogramming, with multiple functionally related proteins changing in expression in a highly coordinated fashion. This comprised several biological processes, including changes in the stoichiometry of electron transport-chain complexes, repressed vesicle-mediated transport during the intermediate stage, and an EMT-like process in the late phase. In addition, we demonstrate that the nucleoporin Nup210 is essential for reprogramming by its permitting of rapid cellular proliferation and subsequent progression through MET. Along with the identification of proteins expressed in a stage-specific manner, this study provides a rich resource toward an enhanced mechanistic understanding of cellular reprogramming.
MSQuant: a platform for stable isotope-based quantitative proteomics.
Gouw, J.W. & Krijgsveld, J.
Methods Mol Biol. 2012;893:511-22. doi: 10.1007/978-1-61779-885-6_31.
Quantitative approaches in proteomics are emerging as a powerful tool to probe the dynamics of protein expression across biological conditions. Thereby, quantification helps to recognize proteins with potential biological relevance, which greatly aids in the design of follow-up experiments. Although multiple methods have been established that are based on stable-isotope labeling and label-free approaches, one of the remaining bottlenecks is the analysis and quantification of proteins in large datasets. MSQuant is a platform for protein quantification, capable of handling multiple labeling strategies and supporting several vendor data formats. Here, we report on the use and versatility of MSQuant.
Native thrombocidin-1 and unfolded thrombocidin-1 exert antimicrobial activity via distinct structural elements.
Kwakman, P.H., Krijgsveld, J., de Boer, L., Nguyen, L.T., Boszhard, L., Vreede, J., Dekker, H.L., Speijer, D., Drijfhout, J.W., te Velde, A.A., Crielaard, W., Vogel, H.J., Vandenbroucke-Grauls, C.M. & Zaat, S.A.
J Biol Chem. 2011 Dec 16;286(50):43506-14. Epub 2011 Oct 24.
Chemokines (chemotactic cytokines) can have direct antimicrobial activity, which is apparently related to the presence of a distinct positively charged patch on the surface. However, chemokines can retain antimicrobial activity upon linearization despite the loss of their positive patch, thus questioning the importance of this patch for activity. Thrombocidin-1 (TC-1) is a microbicidal protein isolated from human blood platelets. TC-1 only differs from the chemokine NAP-2/CXCL7 by a two-amino acid C-terminal deletion, but this truncation is crucial for antimicrobial activity. We assessed the structure-activity relationship for antimicrobial activity of TC-1. Reduction of the charge of the TC-1-positive patch by replacing lysine 17 with alanine reduced the activity against bacteria and almost abolished activity against the yeast Candida albicans. Conversely, augmentation of the positive patch by increasing charge density or size resulted in a 2-3-fold increased activity against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis but did not substantially affect activity against C. albicans. Reduction of TC-1 resulted in loss of the folded conformation, but this disruption of the positive patch did not affect antimicrobial activity. Using overlapping 15-mer synthetic peptides, we demonstrate peptides corresponding to the N-terminal part of TC-1 to have similar antimicrobial activity as intact TC-1. Although we demonstrate that the positive patch is essential for activity of folded TC-1, unfolded TC-1 retained antimicrobial activity despite the absence of a positive patch. This activity is probably exerted by a linear peptide stretch in the N-terminal part of the molecule. We conclude that intact TC-1 and unfolded TC-1 exert antimicrobial activity via distinct structural elements.
Progressive methylation of ageing histones by Dot1 functions as a timer.
De Vos, D., Frederiks, F., Terweij, M., van Welsem, T., Verzijlbergen, K.F., Iachina, E., de Graaf, E.L., Altelaar, A.F., Oudgenoeg, G., Heck, A.J., Krijgsveld, J., Bakker, B.M. & van Leeuwen, F.
EMBO Rep. 2011 Sep 1;12(9):956-62. doi: 10.1038/embor.2011.131.
Post-translational modifications of histone proteins have a crucial role in regulating gene expression. If efficiently re-established after chromosome duplication, histone modifications could help propagate gene expression patterns in dividing cells by epigenetic mechanisms. We used an integrated approach to investigate the dynamics of the conserved methylation of histone H3 Lys 79 (H3K79) by Dot1. Our results show that methylation of H3K79 progressively changes after histone deposition, which is incompatible with a rapid copy mechanism. Instead, methylation accumulates on ageing histones, providing the cell with a timer mechanism to directly couple cell-cycle length to changes in chromatin modification on the nucleosome core.
Tdrd1 acts as a molecular scaffold for Piwi proteins and piRNA targets in zebrafish.
Huang, H.Y., Houwing, S., Kaaij, L.J., Meppelink, A., Redl, S., Gauci, S., Vos, H., Draper, B.W., Moens, C.B., Burgering, B.M., Ladurner, P., Krijgsveld, J., Berezikov, E. & Ketting, R.F.
EMBO J. 2011 Jul 8;30(16):3298-308. doi: 10.1038/emboj.2011.228.
Piwi proteins function in an RNAi-like pathway that silences transposons. Piwi-associated RNAs, also known as piRNAs, act as a guide to identify Piwi targets. The tudor domain-containing protein Tdrd1 has been linked to this pathway but its function has thus far remained unclear. We show that zebrafish Tdrd1 is required for efficient Piwi-pathway activity and proper nuage formation. Furthermore, we find that Tdrd1 binds both zebrafish Piwi proteins, Ziwi and Zili, and reveals sequence specificity in the interaction between Tdrd1 tudor domains and symmetrically dimethylated arginines (sDMAs) in Zili. Finally, we show that Tdrd1 complexes contain piRNAs and RNA molecules that are longer than piRNAs. We name these longer transcripts Tdrd1-associated transcripts (TATs). TATs likely represent cleaved Piwi pathway targets and may serve as piRNA biogenesis intermediates. Altogether, our data suggest that Tdrd1 acts as a molecular scaffold for Piwi proteins, bound through specific tudor domain-sDMA interactions, piRNAs and piRNA targets.
The ecoresponsive genome of Daphnia pulex.
Colbourne, J.K., Pfrender, M.E., Gilbert, D., Thomas, W.K., Tucker, A., Oakley, T.H., Tokishita, S., Aerts, A., Arnold, G.J., Basu, M.K., Bauer, D.J., Caceres, C.E., Carmel, L., Casola, C., Choi, J.H., Detter, J.C., Dong, Q., Dusheyko, S., Eads, B.D., Frohlich, T., Geiler-Samerotte, K.A., Gerlach, D., Hatcher, P., Jogdeo, S., Krijgsveld, J., Kriventseva, E.V., Kultz, D., Laforsch, C., Lindquist, E., Lopez, J., Manak, J.R., Muller, J., Pangilinan, J., Patwardhan, R.P., Pitluck, S., Pritham, E.J., Rechtsteiner, A., Rho, M., Rogozin, I.B., Sakarya, O., Salamov, A., Schaack, S., Shapiro, H., Shiga, Y., Skalitzky, C., Smith, Z., Souvorov, A., Sung, W., Tang, Z., Tsuchiya, D., Tu, H., Vos, H., Wang, M., Wolf, Y.I., Yamagata, H., Yamada, T., Ye, Y., Shaw, J.R., Andrews, J., Crease, T.J., Tang, H., Lucas, S.M., Robertson, H.M., Bork, P., Koonin, E.V., Zdobnov, E.M., Grigoriev, I.V., Lynch, M. & Boore, J.L.
Science. 2011 Feb 4;331(6017):555-61. doi: 10.1126/science.1197761.
We describe the draft genome of the microcrustacean Daphnia pulex, which is only 200 megabases and contains at least 30,907 genes. The high gene count is a consequence of an elevated rate of gene duplication resulting in tandem gene clusters. More than a third of Daphnia's genes have no detectable homologs in any other available proteome, and the most amplified gene families are specific to the Daphnia lineage. The coexpansion of gene families interacting within metabolic pathways suggests that the maintenance of duplicated genes is not random, and the analysis of gene expression under different environmental conditions reveals that numerous paralogs acquire divergent expression patterns soon after duplication. Daphnia-specific genes, including many additional loci within sequenced regions that are otherwise devoid of annotations, are the most responsive genes to ecological challenges.
Defining pluripotent stem cells through quantitative proteomic analysis.
Reiland, S., Salekdeh, G.H. & Krijgsveld, J.
Expert Rev Proteomics. 2011 Feb;8(1):29-42. doi: 10.1586/epr.10.100.
Embryonic stem cells (ESCs) are at the center stage of intense research, inspired by their potential to give rise to all cell types of the adult individual. This property makes ESCs suitable candidates for generating specialized cells to replace damaged tissue lost after injury or disease. However, such clinical applications require a detailed insight of the molecular mechanisms underlying the self-renewal, expansion and differentiation of stem cells. This has gained further relevance since the introduction of induced pluripotent stem cells (iPSCs), which are functionally very similar to ESCs. The key property that iPSCs can be derived from somatic cells lifts some of the major ethical issues related to the need for embryos to generate ESCs. Yet, this has only increased the need to define the similarity of iPSCs and ESCs at the molecular level, both before and after they are induced to differentiate. In this article, we describe the proteomic approaches that have been used to characterize ESCs with regard to self-renewal and differentiation, with an emphasis on signaling cascades and histone modifications. We take this as a lead to discuss how quantitative proteomics can be deployed to study reprogramming and iPSC identity. In addition, we discuss how emerging proteomic technologies can become a useful tool to monitor the (de)differentiation status of ESCs and iPSCs.
Metabolic labeling of model organisms using heavy nitrogen (15N).
Gouw J.W., Tops B.B. & Krijgsveld J.
Methods Mol Biol. 2011;753:29-42. doi: 10.1007/978-1-61779-148-2_2.
Quantitative proteomics aims to identify and quantify proteins in cells or organisms that have been obtained from different biological origin (e.g., "healthy vs. diseased"), that have received different treatments, or that have different genetic backgrounds. Protein expression levels can be quantified by labeling proteins with stable isotopes, followed by mass spectrometric analysis. Stable isotopes can be introduced in vitro by reacting proteins or peptides with isotope-coded reagents (e.g., iTRAQ, reductive methylation). A preferred way, however, is the metabolic incorporation of heavy isotopes into cells or organisms by providing the label, in the form of amino acids (such as in SILAC) or salts, in the growth media. The advantage of in vivo labeling is that it does not suffer from side reactions or incomplete labeling that might occur in chemical derivatization. In addition, metabolic labeling occurs at the earliest possible moment in the sample preparation process, thereby minimizing the error in quantitation. Labeling with the heavy stable isotope of nitrogen (i.e., (15)N) provides an efficient way for accurate protein quantitation. Where the application of SILAC is mostly restricted to cell culture, (15)N labeling can be used for micro-organisms as well as a number of higher (multicellular) organisms. The most prominent examples of the latter are Caenorhabditis elegans and Drosophila (fruit fly), two important model organisms for a range of regulatory processes underlying developmental biology. Here we describe in detail the labeling with (15)N atoms, with a particular focus on fruit flies and C. elegans. We also describe methods for the identification and quantitation of (15)N-labeled proteins by mass spectrometry and bioinformatic analysis.
Identification of cell surface proteins for antibody-based selection of human embryonic stem cell-derived cardiomyocytes.
Van Hoof, D., Dormeyer, W., Braam, S.R., Passier, R., Monshouwer-Kloots, J., Ward-van Oostwaard, D., Heck, A.J., Krijgsveld, J. & Mummery, C.L.
J Proteome Res. 2010 Mar 5;9(3):1610-8.
The absence of identified cell surface proteins and corresponding antibodies to most differentiated derivatives of human embryonic stem cells (hESCs) has largely limited selection of specific cell types from mixed cell populations to genetic approaches. Here, we describe the use of mass spectrometry (MS)-based proteomics on cell membrane proteins isolated from hESCs that were differentiated into cardiomyocytes to identify candidate proteins for this particular lineage. Quantitative MS distinguished cardiomyocyte-specific plasma membrane proteins that were highly enriched or detected only in cardiomyocytes derived from hESCs and human fetal hearts compared with a heterogeneous pool of hESC-derived differentiated cells. For several candidates, cardiomyocyte-specific expression and cell surface localization were verified by conventional antibody-based methodologies. Using an antibody against elastin microfibril interfacer 2 (EMILIN2), we demonstrate that cardiomyocytes can be sorted from live cell populations. Besides showing that MS-based membrane proteomics is a powerful tool to identify candidate proteins that allow purification of specific cell lineages from heterogeneous populations, this approach generated a plasma membrane proteome profile suggesting signaling pathways that control cell behavior.
A P4-ATPase protein interaction network reveals a link between aminophospholipid transport and phosphoinositide metabolism.
Puts, C.F., Lenoir, G., Krijgsveld, J., Williamson, P. & Holthuis, J.C.
J Proteome Res. 2010 Feb 5;9(2):833-42.
High-throughput analysis of protein-protein interactions can provide unprecedented insight into how cellular processes are integrated at the molecular level. Yet membrane proteins are often overlooked in these studies owing to their hydrophobic nature and low abundance. Here we used a proteomics-based strategy with the specific intention of identifying membrane-associated protein complexes. One important aspect of our approach is the use of chemical cross-linking to capture transient and low-affinity protein interactions that occur in living cells prior to cell lysis. We applied this method to identify binding partners of the yeast Golgi P(4)-ATPase Drs2p, a member of a conserved family of putative aminophospholipid transporters. Drs2p was endogeneously tagged with both a polyhistidine and a biotinylation peptide, allowing tandem-affinity purification of Drs2p-containing protein complexes under highly stringent conditions. Mass-spectrometric analysis of isolated complexes yielded one known and nine novel Drs2p binding partners. Binding specificity was verified by an orthogonal in vivo membrane protein interaction assay, confirming the efficacy of our method. Strikingly, three of the novel Drs2p interactors are involved in phosphoinositide metabolism. One of these, the phosphatidylinositol-4-phosphatase Sac1p, also displays genetic interactions with Drs2p. Together, these findings suggest that aminophospholipid transport and phosphoinositide metabolism are interconnected at the Golgi.
Worms from venus and mars: proteomics profiling of sexual differences in Caenorhabditis elegans using in vivo 15N isotope labeling.
Tops, B.B., Gauci, S., Heck, A.J. & Krijgsveld, J.
J Proteome Res. 2010 Jan;9(1):341-51.
Hermaphrodites of the nematode Caenorhabditis elegans produce both sperm and oocytes in the same germline. To investigate the process underlying spermatogenesis and oogenesis separately, we used a quantitative proteomics approach applied to two mutant worm lines (fem-3(q20) and fem-1(hc17)) developing only male and female germlines, respectively. We used stable isotopic labeling of whole animals by feeding them either (14)N or (15)N labeled Escherichia coli. This way, we could confidently identify and quantify 1040 proteins in two independent experiments. Of these, approximately 400 proteins showed significant differential expression between female-like and male-like animals. As expected, proteins linked to oogenesis were found to be highly upregulated in the feminized worms, whereas proteins involved in spermatogenesis were found to be highly upregulated in the masculinized worms. This was complemented by many proteins strongly enriched in either mutant. Although the function of the majority of these proteins is unknown, their expression profile indicates that they have an as yet unrecognized role in the development and/or function of the female- and male germline in C. elegans. We show that members of several protein complexes as well as functionally similar proteins show comparable abundance ratios, indicating coregulation of protein expression. Additional analysis comparing our protein data to a previously published microarray data set shows that mRNA and protein expression are poorly correlating. We provide one of the first examples of a large-scale quantitative proteomics experiment in C. elegans and show the potential and feasibility of an approach enabling system-wide accurate quantitative proteomics experiments in this model organism.
Quantitative proteomics by metabolic labeling of model organisms.
Gouw, J.W., Krijgsveld, J. & Heck, A.J.
Mol Cell Proteomics. 2010 Jan;9(1):11-24. Epub 2009 Nov 19.
In the biological sciences, model organisms have been used for many decades and have enabled the gathering of a large proportion of our present day knowledge of basic biological processes and their derailments in disease. Although in many of these studies using model organisms, the focus has primarily been on genetics and genomics approaches, it is important that methods become available to extend this to the relevant protein level. Mass spectrometry-based proteomics is increasingly becoming the standard to comprehensively analyze proteomes. An important transition has been made recently by moving from charting static proteomes to monitoring their dynamics by simultaneously quantifying multiple proteins obtained from differently treated samples. Especially the labeling with stable isotopes has proved an effective means to accurately determine differential expression levels of proteins. Among these, metabolic incorporation of stable isotopes in vivo in whole organisms is one of the favored strategies. In this perspective, we will focus on methodologies to stable isotope label a variety of model organisms in vivo, ranging from relatively simple organisms such as bacteria and yeast to Caenorhabditis elegans, Drosophila, and Arabidopsis up to mammals such as rats and mice. We also summarize how this has opened up ways to investigate biological processes at the protein level in health and disease, revealing conservation and variation across the evolutionary tree of life.
On-the-fly targeted selection of labeled peptides in liquid chromatography/mass spectrometry based quantitative proteomics.
Barbu, I.M., Smith, D.F., van Breukelen, B., van der Burgt, Y.E., Duursma, M.C., Heck, A.J., Heeren, R.M. & Krijgsveld, J.
Rapid Commun Mass Spectrom. 2010 Jan;24(2):239-41. Europe PMC
Novel Hsp90 partners discovered using complementary proteomic approaches.
Tsaytler, P.A., Krijgsveld, J., Goerdayal, S.S., Rudiger, S. & Egmond, M.R.
Cell Stress Chaperones. 2009 Nov;14(6):629-38. doi: 10.1007/s12192-009-0115-z.Epub 2009 Apr 26.
Hsp90 is an essential eukaryotic molecular chaperone that stabilizes a large set of client proteins, many of which are involved in various cellular signaling pathways. The current list of Hsp90 interactors comprises about 200 proteins and this number is growing steadily. In this paper, we report on the application of three complementary proteomic approaches directed towards identification of novel proteins that interact with Hsp90. These methods are coimmunoprecipitation, pull down with biotinylated geldanamycin, and immobilization of Hsp90beta on sepharose. In all, this study led to the identification of 42 proteins, including 18 proteins that had not been previously characterized as Hsp90 interactors. These novel Hsp90 partners not only represent abundant protein species, but several proteins were identified at low levels, among which signaling kinase Cdk3 and putative transcription factor tripartite motif-containing protein 29. Identification of tetratricopeptide-repeat-containing mitochondrial import receptor protein Tom34 suggests the involvement of Hsp90 in the early steps of translocation of mitochondrial preproteins. Taken together, our data expand the knowledge of the Hsp90 interactome and provide a further step in our understanding of the Hsp90 chaperone system.
Cooperative binding of two acetylation marks on a histone tail by a single bromodomain.
Moriniere, J., Rousseaux, S., Steuerwald, U., Soler-Lopez, M., Curtet, S., Vitte, A.L., Govin, J., Gaucher, J., Sadoul, K., Hart, D.J., Krijgsveld, J., Khochbin, S., Muller, C.W. & Petosa, C.
Nature. 2009 Oct 1;461(7264):664-8.
A key step in many chromatin-related processes is the recognition of histone post-translational modifications by effector modules such as bromodomains and chromo-like domains of the Royal family. Whereas effector-mediated recognition of single post-translational modifications is well characterized, how the cell achieves combinatorial readout of histones bearing multiple modifications is poorly understood. One mechanism involves multivalent binding by linked effector modules. For example, the tandem bromodomains of human TATA-binding protein-associated factor-1 (TAF1) bind better to a diacetylated histone H4 tail than to monoacetylated tails, a cooperative effect attributed to each bromodomain engaging one acetyl-lysine mark. Here we report a distinct mechanism of combinatorial readout for the mouse TAF1 homologue Brdt, a testis-specific member of the BET protein family. Brdt associates with hyperacetylated histone H4 (ref. 7) and is implicated in the marked chromatin remodelling that follows histone hyperacetylation during spermiogenesis, the stage of spermatogenesis in which post-meiotic germ cells mature into fully differentiated sperm. Notably, we find that a single bromodomain (BD1) of Brdt is responsible for selectively recognizing histone H4 tails bearing two or more acetylation marks. The crystal structure of BD1 bound to a diacetylated H4 tail shows how two acetyl-lysine residues cooperate to interact with one binding pocket. Structure-based mutagenesis that reduces the selectivity of BD1 towards diacetylated tails destabilizes the association of Brdt with acetylated chromatin in vivo. Structural analysis suggests that other chromatin-associated proteins may be capable of a similar mode of ligand recognition, including yeast Bdf1, human TAF1 and human CBP/p300 (also known as CREBBP and EP300, respectively). Our findings describe a new mechanism for the combinatorial readout of histone modifications in which a single effector module engages two marks on a histone tail as a composite binding epitope.
Phosphorylation dynamics during early differentiation of human embryonic stem cells.
Van Hoof, D., Munoz, J., Braam, S.R., Pinkse, M.W., Linding, R., Heck, A.J., Mummery, C.L. & Krijgsveld, J.
Cell Stem Cell. 2009 Aug 7;5(2):214-26.
Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during differentiation induced by bone morphogenetic protein (BMP) and removal of hESC growth factors. Of 5222 proteins identified, 1399 were phosphorylated on 3067 residues. Approximately 50% of these phosphosites were regulated within 1 hr of differentiation induction, revealing a complex interplay of phosphorylation networks spanning different signaling pathways and kinase activities. Among the phosphorylated proteins was the pluripotency-associated protein SOX2, which was SUMOylated as a result of phosphorylation. Using the data to predict kinase-substrate relationships, we reconstructed the hESC kinome; CDK1/2 emerged as central in controlling self-renewal and lineage specification. The findings provide new insights into how hESCs exit the pluripotent state and present the hESC (phospho)proteome resource as a complement to existing pluripotency network databases.
In vivo stable isotope labeling of fruit flies reveals post-transcriptional regulation in the maternal-to-zygotic transition.
Gouw, J.W., Pinkse, M.W., Vos, H.R., Moshkin, Y., Verrijzer, C.P., Heck, A.J. & Krijgsveld, J.
Mol Cell Proteomics. 2009 Jul;8(7):1566-78. doi: 10.1074/mcp.M900114-MCP200. Epub2009 Mar 24.
An important hallmark in embryonic development is characterized by the maternal-to-zygotic transition (MZT) where zygotic transcription is activated by a maternally controlled environment. Post-transcriptional and translational regulation is critical for this transition and has been investigated in considerable detail at the gene level. We used a proteomics approach using metabolic labeling of Drosophila to quantitatively assess changes in protein expression levels before and after the MZT. By combining stable isotope labeling of fruit flies in vivo with high accuracy quantitative mass spectrometry we could quantify 2,232 proteins of which about half changed in abundance during this process. We show that approximately 500 proteins increased in abundance, providing direct evidence of the identity of proteins as a product of embryonic translation. The group of down-regulated proteins is dominated by maternal factors involved in translational control of maternal and zygotic transcripts. Surprisingly a direct comparison of transcript and protein levels showed that the mRNA levels of down-regulated proteins remained relatively constant, indicating a translational control mechanism specifically targeting these proteins. In addition, we found evidence for post-translational processing of cysteine proteinase-1 (Cathepsin L), which became activated during the MZT as evidenced by the loss of its N-terminal propeptide. Poly(A)-binding protein was shown to be processed at its C-terminal tail, thereby losing one of its protein-interacting domains. Altogether this quantitative proteomics study provides a dynamic profile of known and novel proteins of maternal as well as embryonic origin. This provides insight into the production, stability, and modification of individual proteins, whereas discrepancies between transcriptional profiles and protein dynamics indicate novel control mechanisms in genome activation during early fly development.
Orthogonal separation techniques for the characterization of the yeast nuclear proteome.
Gauci, S., Veenhoff, L.M., Heck, A.J. & Krijgsveld, J.
J Proteome Res. 2009 Jul;8(7):3451-63.
The presence of the nucleus is the distinguishing feature of eukaryotic cells, separating the genome from the cytoplasm. Key cellular events, including transcription, DNA replication, RNA-processing and ribosome biogenesis all take place in the nucleus. All of these processes can be regulated through controlled and bidirectional translocation of proteins across the nuclear envelope, making the nucleus a highly dynamic organelle. In this study, we present four orthogonal multidimensional separation techniques for the comprehensive characterization of the yeast nuclear proteome. By combining methods on the peptide level (SCX chromatography, isoelectric focusing) and protein level (SDS-PAGE, phosphocellulose chromatography) coupled with mass spectrometry, we identified 1889 proteins from highly purified nuclei, of which 1032 were previously annotated as nuclear proteins. In particular, the most successful setup was the use of phosphocellulose P11 chromatography in combination with SDS-PAGE and reversed phase chromatography. Phosphocellulose P11 chromatography has been classically used for the purification of functional protein complexes involved in transcription regulation. Here, by its coupling with LC-MS, this method resulted in approximately 1.5 times more protein identifications than the other three combined, thereby contributing significantly to the coverage of nuclear proteins. In addition, the use of this technique resulted in the enrichment of DNA binding proteins and proved to be a valuable tool for the simultaneous analysis of multiple protein complexes. The enrichment for specific nuclear complexes has resulted in high protein sequence coverage, which will be particularly useful for the detailed characterization of subunits.
An AC-5 cathepsin B-like protease purified from Haemonchus contortus excretory secretory products shows protective antigen potential for lambs.
De Vries, E., Bakker, N., Krijgsveld, J., Knox, D.P., Heck, A.J. & Yatsuda, A.P.
Vet Res. 2009 Jul-Aug;40(4):41. Epub 2009 Apr 30.
The immunogenic properties of cysteine proteases obtained from excretory/secretory products (ES) of Haemonchus contortus were investigated with a fraction purified with a recombinant H. contortus cystatin affinity column. The enrichment of H. contortus ES for cysteine protease was confirmed with substrate SDS-PAGE gels since the cystatin-binding fraction activity was three times higher than total ES, despite representing only 3% of total ES. This activity was inhibited by a specific cysteine protease inhibitor (E64) and by recombinant cystatin. The one-dimensional profile of the cystatin-binding fraction displayed a single band with a molecular mass of 43 kDa. Mass spectrometry showed this to be AC-5, a cathepsin B-like cysteine protease which had not been identified in ES products of H. contortus before. The cystatin binding fraction was tested as an immunogen in lambs which were vaccinated three times (week 0, 2.5 and 5), challenged with 10 000 L3 H. contortus (week 6) before necropsy and compared to unvaccinated challenge controls and another group given total ES (n = 10 per group). The group vaccinated with cystatin-binding proteins showed 36% and 32% mean worm burden and eggs per gram of faeces (EPG) reductions, respectively, compared to the controls but total ES was almost without effect. After challenge the cystatin-binding proteins induced significantly higher local and systemic ES specific IgA and IgG responses.
Exosomal secretion of cytoplasmic prostate cancer xenograft-derived proteins.
Jansen, F.H., Krijgsveld, J., van Rijswijk, A., van den Bemd, G.J., van den Berg, M.S., van Weerden, W.M., Willemsen, R., Dekker, L.J., Luider, T.M. & Jenster, G.
Mol Cell Proteomics. 2009 Jun;8(6):1192-205. doi: 10.1074/mcp.M800443-MCP200.Epub 2009 Feb 9.
Novel markers for prostate cancer (PCa) are needed because current established markers such as prostate-specific antigen lack diagnostic specificity and prognostic value. Proteomics analysis of serum from mice grafted with human PCa xenografts resulted in the identification of 44 tumor-derived proteins. Besides secreted proteins we identified several cytoplasmic proteins, among which were most subunits of the proteasome. Native gel electrophoresis and sandwich ELISA showed that these subunits are present as proteasome complexes in the serum from xenograft-bearing mice. We hypothesized that the presence of proteasome subunits and other cytoplasmic proteins in serum of xenografted mice could be explained by the secretion of small vesicles by cancer cells, so-called exosomes. Therefore, mass spectrometry and Western blotting analyses of the protein content of exosomes isolated from PCa cell lines was performed. This resulted in the identification of mainly cytoplasmic proteins of which several had previously been identified in the serum of xenografted mice, including proteasome subunits. The isolated exosomes also contained RNA, including the gene fusion TMPRSS2-ERG product. These observations suggest that although their function is not clearly defined cancer-derived exosomes offer possibilities for the identification of novel biomarkers for PCa.
Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach.
Gauci, S., Helbig, A.O., Slijper, M., Krijgsveld, J., Heck, A.J. & Mohammed, S.
Anal Chem. 2009 Jun 1;81(11):4493-501.
The analysis of proteome-wide phosphorylation events is still a major analytical challenge because of the enormous complexity of protein phosphorylation networks. In this work, we evaluate the complementarity of Lys-N, Lys-C, and trypsin with regard to their ability to contribute to the global analysis of the phosphoproteome. A refined version of low-pH strong cation exchange was used to efficiently separate N-terminally acetylated, phosphorylated, and nonmodified peptides. A total of 5036 nonredundant phosphopeptides could be identified with a false discovery rate of <1% from 1 mg of protein using a combination of the three enzymes. Our data revealed that the overlap between the phosphopeptide data sets generated with different proteases was marginal, whereas the overlap between two similarly generated tryptic data sets was found to be at least 4 times higher. In this way, the parallel use of Lys-N and trypsin enabled a 72% increase in the number of detected phosphopeptides as compared to trypsin alone, whereas a trypsin replicate experiment only led to a 25% increase. Thus, when focusing solely on the trypsin and Lys-N data, we identified 4671 nonredundant phosphopeptides. Further analysis of the detected sites showed that the Lys-N and trypsin data sets were enriched in significantly different phosphorylation motifs, further evidencing that multiprotease approaches are very valuable in phosphoproteome analyses.
Human Proteome Organisation's 7th World Congress, 2008.
Expert Rev Proteomics. 2008 Dec;5(6):775-7.
The annual world congress of Human Proteome Organisation (HUPO) is one of the premier meetings in proteomics. Rotating between Europe, North America and Asia/Oceana, this year's host city was Amsterdam, The Netherlands. Proteomics still being a rapidly evolving field, HUPO meetings provide a platform for technical advancements in protein purification and separation techniques, innovations in mass spectrometry and applications in bioinformatics and computational biology. A special focus of this year's meeting was on proteome biology, indicating that the state of technology has progressed to a level permitting interrogation of biological systems in a meaningful way.
A versatile peptide pI calculator for phosphorylated and N-terminal acetylated peptides experimentally tested using peptide isoelectric focusing.
Gauci, S., van Breukelen, B., Lemeer, S.M., Krijgsveld, J. & Heck, A.J.
Proteomics. 2008 Dec;8(23-24):4898-906.
We experimentally demonstrate the use of an in-house developed pI calculator which takes into account peptide PTM such as phosphorylation and N-terminal acetylation. The pI calculator was utilized for a large set of peptides derived from a complex zebrafish lysate fractionated using peptide IEF, whereby a good correlation between the calculated (theoretical) pI and the experimental pI could be established. This pI calculator permits the implementation of optimal pK values depending on the experimental conditions and a reliable calculation of peptide pI which can be utilized as a filtering technique in validating peptide identifications. Our data reveal that the shift due to a phosphorylation or N-terminal acetylation is highly dependent on the presence of acidic or basic residues in the peptide. Furthermore, using this pI calculator, we revealed previously unknown position-specific pKs of asparagine and carbamidomethylated cysteine depending on their location in the peptide. Collectively, this peptide pI calculator is a welcome addition to the versatility and robustness of IEF for the separation and confident identification of (post-translationally modified) peptides.
Feeder-free monolayer cultures of human embryonic stem cells express an epithelial plasma membrane protein profile.
Van Hoof, D., Braam, S.R., Dormeyer, W., Ward-van Oostwaard, D., Heck, A.J., Krijgsveld, J. & Mummery, C.L.
Stem Cells. 2008 Nov;26(11):2777-81. Epub 2008 Aug 14.
Human embryonic stem cells (hESCs) are often cocultured on mitotically inactive fibroblast feeder cells to maintain their undifferentiated state. Under these growth conditions, hESCs form multilayered colonies of morphologically heterogeneous cells surrounded by flattened mesenchymal cells. In contrast, hESCs grown in feeder cell-conditioned medium on Matrigel instead tend to grow as monolayers with uniform morphology. Using mass spectrometry and immunofluorescence microscopy, we showed that hESCs under these conditions primarily express proteins belonging to epithelium-related cell-cell adhesion complexes, including adherens junctions, tight junctions, desmosomes, and gap junctions. This indicates that monolayers of hESCs cultured under feeder-free conditions retain a homogeneous epithelial phenotype similar to that of the upper central cell layer of colonies maintained on feeder cells. Notably, feeder-free hESCs also coexpressed vimentin, which is usually associated with mesenchyme, suggesting that these cells may have undergone epithelium-to-mesenchyme transitions, indicating differentiation. However, if grown on a "soft" substrate (Hydrogel), intracellular vimentin levels were substantially reduced. Moreover, when hESCs were transferred back to feeder cells, expression of vimentin was again absent from the epithelial cell population. These results imply that on tissue culture substrates, vimentin expression is most likely a stress-induced response, unrelated to differentiation. Disclosure of potential conflicts of interest is found at the end of this article.
Optimizing identification and quantitation of 15N-labeled proteins in comparative proteomics.
Gouw, J.W., Tops, B.B., Mortensen, P., Heck, A.J. & Krijgsveld, J.
Anal Chem. 2008 Oct 15;80(20):7796-803. Epub 2008 Sep 23.
Comparative proteomics has emerged as a powerful approach to determine differences in protein abundance between biological samples. The introduction of stable-isotopes as internal standards especially paved the road for quantitative proteomics for comprehensive approaches to accurately determine protein dynamics. Metabolic labeling with (15)N isotopes is applied to an increasing number of organisms, including Drosophila, C. elegans, and rats. However, (15)N-enrichment is often suboptimal (<98%), which may hamper identification and quantitation of proteins. Here, we systematically investigated two independent (15)N-labeled data sets to explore the influence of heavy nitrogen enrichment on the number of identifications as well as on the error in protein quantitation. We show that specifically larger (15)N-labeled peptides are under-represented when compared to their (14)N counterparts and propose a correction method, which significantly increases the number of identifications. In addition, we developed a method that corrects for inaccurate peptide ratios introduced by incomplete (15)N enrichment. This results in improved accuracy and precision of protein quantitation. Altogether, this study provides insight into the process of protein identification and quantitation, and the methods described here can be used to improve both qualitative and quantitative data obtained by labeling with heavy nitrogen with enrichment less than 100%.
A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.
Dormeyer, W., van Hoof, D., Mummery, C.L., Krijgsveld, J. & Heck, A.J.
Proteomics. 2008 Oct;8(19):4036-53.
The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological mechanisms that regulate proliferation and differentiation. The comparison of their membrane proteomes will help unravel the biological principles of pluripotency, and the identification of biomarker proteins in their plasma membranes is considered a crucial step to fully exploit pluripotent cells for therapeutic purposes. For these tasks, membrane proteomics is the method of choice, but as indicated by the scarce identification of membrane and plasma membrane proteins in global proteomic surveys it is not an easy task. In this minireview, we first describe the general challenges of membrane proteomics. We then review current sample preparation steps and discuss protocols that we found particularly beneficial for the identification of large numbers of (plasma) membrane proteins in human tumour- and embryo-derived stem cells. Our optimized assembled protocol led to the identification of a large number of membrane proteins. However, as the composition of cells and membranes is highly variable we still recommend adapting the sample preparation protocol for each individual system.
Proteomics and human embryonic stem cells.
Van Hoof, D., Heck, A.J., Krijgsveld, J. & Mummery, C.L.
Stem Cell Res. 2008 Sep;1(3):169-82. Epub 2008 May 29.
The derivation of human embryonic stem cells (hESCs) brought cell therapy-based regenerative medicine significantly closer to clinical application. However, expansion of undifferentiated cells and their directed differentiation in vitro have proven difficult to control. This is mainly because of a lack of knowledge of the intracellular signaling events that direct these complex processes. Additionally, extracellular factors, either secreted by feeder cells that support self-renewal and maintain pluripotency or present in serum supplementing proprietary culture media, that influence hESC behavior are largely unknown. Xeno-free media that effectively support long-term hESC self-renewal and differentiation to specific types of specialized cells are only slowly becoming available. Microarray-based transcriptome analyses have produced valuable gene expression profiles of hESCs and indicated changes in transcription that occur during differentiation. However, proteins are the actual effectors of these events and changes in their levels do not always match changes in their corresponding mRNA. Furthermore, information on posttranslational modifications that influence the activity of pivotal proteins is still largely missing. Over the years, mass spectrometry has experienced major breakthroughs in high-throughput identification of proteins and posttranslational modifications in cells under different conditions. Mass spectrometry-based proteomic techniques are being applied with increasing frequency to analyze hESCs, as well as media conditioned by feeder cells, and have generated proteome profiles that not only support, but also complement, existing microarray data. In this review, the various proteomic studies on hESCs and feeder cells are discussed. In a meta-analysis, comparison of published data sets distinguished 32 intracellular proteins and 16 plasma membrane proteins that are present in multiple hESC lines but not in differentiated cells, which were therefore likely to include proteins important for hESCs. In addition, 13 and 24 proteins, respectively, were commonly found in different feeder cell lines of mouse and human origin, some of which may be extracellular signaling molecules that play a key role in the undifferentiated propagation of hESCs. These findings underscore the power of mass spectrometry-based techniques to identify novel proteins associated with hESCs by studying these cells in an unbiased, discovery-oriented manner on a proteome-wide scale.
Immediate protein targets of photodynamic treatment in carcinoma cells.
Tsaytler, P.A., C O'Flaherty, M., Sakharov, D.V., Krijgsveld, J. & Egmond, M.R.
J Proteome Res. 2008 Sep;7(9):3868-78. Epub 2008 Jul 25.
Oxidative stress induced in tumor cells undergoing photodynamic treatment (PDT) leads to extensive modification of many proteins in these cells. Protein oxidation mainly gives rise to formation of carbonyls and oxidized thiols. The immediate targets of PDT-induced protein oxidation in A431 tumor cells have been identified using a proteomic approach involving selective biotinylation, affinity purification and mass spectrometric identification of modified proteins. In all, 314 proteins were shown to undergo PDT-mediated oxidative modifications. While abundant structural proteins and chaperones represented a significant fraction of the carbonylated proteins, labeling of proteins containing oxidized thiols allowed identification of many proteins at low abundance and those involved in signaling and redox homeostasis. On the basis of the identification of these proteins, several likely mechanisms of PDT-induced triggering of apoptosis were put forward. This may not only lead to a further understanding of the complex network of cellular responses to oxidative stress, but it may also help in detailed targeting of photodynamic treatment applied to cancer.
Plasma membrane proteomics of human embryonic stem cells and human embryonal carcinoma cells.
Dormeyer, W., van Hoof, D., Braam, S.R., Heck, A.J., Mummery, C.L. & Krijgsveld, J.
J Proteome Res. 2008 Jul;7(7):2936-51. Epub 2008 May 20.
Human embryonic stem cells (hESCs) are of immense interest in regenerative medicine as they can self-renew indefinitely and can give rise to any adult cell type. Human embryonal carcinoma cells (hECCs) are the malignant counterparts of hESCs found in testis tumors. hESCs that have acquired chromosomal abnormalities in culture are essentially indistinguishable from hECC. Direct comparison of karyotypically normal hESCs with hECCs could lead to understanding differences between their mechanisms of growth control and contribute to implementing safe therapeutic use of stem cells without the development of germ cell cancer. While several comparisons of hECCs and hESCs have been reported, their cell surface proteomes are largely unknown, partly because plasma membrane proteomics is still a major challenge. Here, we present a strategy for the identification of plasma membrane proteins that has been optimized for application to the relatively small numbers of stem cells normally available, and that does not require tedious cell fractionation. The method led to the identification of 237 and 219 specific plasma membrane proteins in the hESC line HUES-7 and the hECC line NT2/D1, respectively. In addition to known stemness-associated cell surface markers like ALP, CD9, and CTNNB, a large number of receptors, transporters, signal transducers, and cell-cell adhesion proteins were identified. Our study revealed that several Hedgehog and Wnt pathway members are differentially expressed in hESCs and hECCs including NPC1, FZD2, FZD6, FZD7, LRP6, and SEMA4D, which play a pivotal role in stem cell self-renewal and cancer growth. Various proteins encoded on chromosome 12p, duplicated in testicular cancer, were uniquely identified in hECCs. These included GAPDH, LDHB, YARS2, CLSTN3, CSDA, LRP6, NDUFA9, and NOL1, which are known to be upregulated in testicular cancer. Distinct HLA molecules were revealed on the surface of hESCs and hECCs, despite their low abundance. Results were compared with genomic and proteomic data sets reported previously for mouse ESCs, hECCs, and germ cell tumors. Our data provides a surface signature for HUES-7 and NT2/D1 cells and distinguishes normal hESCs from hECCs, helping explain their 'benign' versus 'malignant' nature.
Nonprocessive methylation by Dot1 leads to functional redundancy of histone H3K79 methylation states.
Frederiks, F., Tzouros, M., Oudgenoeg, G., van Welsem, T., Fornerod, M., Krijgsveld, J. & van Leeuwen, F.
Nat Struct Mol Biol. 2008 Jun;15(6):550-7. Epub 2008 May 30.
Whereas mono-, di- and trimethylation states of lysines on histones typically have specific functions, no specific functions have been attributed so far to the different methylation states of histone H3 Lysine 79 (H3K79) generated by Dot1. Here we show that Dot1, in contrast to other known histone methyltransferases, introduces multiple methyl groups via a nonprocessive mechanism. The kinetic mechanism implies that the H3K79 methylation states cannot be generated independently, suggesting functional redundancy. Indeed, gene silencing in yeast, which is dependent on Dot1, relied on global H3K79 methylation levels and not on one specific methylation state. Furthermore, our findings suggest that histone H2B ubiquitination affects H3K79 trimethylation by enhancing synthesis of all H3K79 methylation states. Our results suggest that multiple methylation of H3K79 leads to a binary code, which is expected to limit the possibilities for regulation by putative demethylases or binding proteins.
The time is right: proteome biology of stem cells.
Whetton, A.D., Williamson, A.J., Krijgsveld, J., Lee, B.H., Lemischka, I., Oh, S., Pera, M., Mummery, C. & Heck, A.J.
Cell Stem Cell. 2008 Mar 6;2(3):215-7.
In stem cell biology, there is a growing need for advanced technologies that may help to unravel the molecular mechanisms of self-renewal and differentiation. Proteomics, the comprehensive analysis of proteins, is such an emerging technique. To facilitate interactions between specialists in proteomics and stem cell biology,a new initiative has been undertaken, supported by the Human Proteome Organization (HUPO) and the International Society for Stem Cell Research (ISSCR). Here we present the Proteome Biology of Stem Cells Initiative (PBSCI) and report on its goals and future activities.
Proteome biology of stem cells: a new joint HUPO and ISSCR initiative.
Krijgsveld, J., Whetton, A.D., Lee, B., Lemischka, I., Oh, S., Pera, M., Mummery, C. & Heck, A.J.
Mol Cell Proteomics. 2008 Jan;7(1):204-5. Europe PMC
Targeted analysis of protein termini.
Dormeyer, W., Mohammed, S., Breukelen, B., Krijgsveld, J. & Heck, A.J.
J Proteome Res. 2007 Dec;6(12):4634-45. Epub 2007 Oct 10.
We describe a targeted analysis of protein isoforms by selective enrichment and identification of in vivo acetylated protein N-termini and protein C-termini. Our method allows the characterization of these protein termini regardless of their annotation in protein databases and requires no chemical derivatization. Using an iterative database search strategy that takes account of the enrichment protocol, 263 IPI annotated and 87 unpredicted acetylated N-termini were identified in the crude membrane fraction of human embryonic carcinoma cells. The N-acetylated peptides conform to the reported criteria for in vivo modification. In addition, 168 IPI annotated and 193 unpredicted C-termini were identified. Additionally, and for the first time, we also report on in vivo N-terminal propionylation. The significant number of unknown protein N- and C-termini suggests a high degree of novel transcription independent of annotated gene boundaries and/or specific protein processing. Biological relevance of several of these unpredicted protein termini could be curated from the literature, adding further weight to the argument to go beyond routine database search strategies. Our method will improve the correct annotation of genes and proteins in databases.
Proteome biology of stem cells.
Heck, A.J., Mummery, C., Whetton, A., Oh, S., Lee, B., Pera, M., Lemischka, I. & Krijgsveld, J.
Stem Cell Res. 2007 Oct;1(1):7-8. Epub 2007 Aug 24.
The notion that integration of cutting-edge technologies in stem cell research would be enhanced by proteomic analyses has emanated from rapid advances in proteome technology. These advances have increased the probability that basic properties of stem cells will be elucidated more effectively, leading to acceleration toward novel stem cell therapies. We have therefore sought to establish a world-wide alliance of proteomics and stem cell researchers, which has resulted in the foundation of an initiative supported by the Human Proteome Organisation (HUPO) and the International Society for Stem Cell Research (ISSCR) called the Proteome Biology of Stem Cells Initiative. Here we report on the rationale and goals of this initiative.
An experimental correction for arginine-to-proline conversion artifacts in SILAC-based quantitative proteomics.
Van Hoof, D., Pinkse, M.W., Oostwaard, D.W., Mummery, C.L., Heck, A.J. & Krijgsveld, J.
Nat Methods. 2007 Sep;4(9):677-8. Europe PMC
A high-quality catalog of the Drosophila melanogaster proteome.
Brunner, E., Ahrens, C.H., Mohanty, S., Baetschmann, H., Loevenich, S., Potthast, F., Deutsch, E.W., Panse, C., de Lichtenberg, U., Rinner, O., Lee, H., Pedrioli, P.G., Malmstrom, J., Koehler, K., Schrimpf, S., Krijgsveld, J., Kregenow, F., Heck, A.J., Hafen, E., Schlapbach, R. & Aebersold, R.
Nat Biotechnol. 2007 May;25(5):576-83. Epub 2007 Apr 22.
Understanding how proteins and their complex interaction networks convert the genomic information into a dynamic living organism is a fundamental challenge in biological sciences. As an important step towards understanding the systems biology of a complex eukaryote, we cataloged 63% of the predicted Drosophila melanogaster proteome by detecting 9,124 proteins from 498,000 redundant and 72,281 distinct peptide identifications. This unprecedented high proteome coverage for a complex eukaryote was achieved by combining sample diversity, multidimensional biochemical fractionation and analysis-driven experimentation feedback loops, whereby data collection is guided by statistical analysis of prior data. We show that high-quality proteomics data provide crucial information to amend genome annotation and to confirm many predicted gene models. We also present experimentally identified proteotypic peptides matching approximately 50% of D. melanogaster gene models. This library of proteotypic peptides should enable fast, targeted and quantitative proteomic studies to elucidate the systems biology of this model organism.
Novel binding partners of Ldb1 are required for haematopoietic development.
Meier, N., Krpic, S., Rodriguez, P., Strouboulis, J., Monti, M., Krijgsveld, J., Gering, M., Patient, R., Hostert, A. & Grosveld, F.
Development. 2006 Dec;133(24):4913-23. Epub 2006 Nov 15.
Ldb1, a ubiquitously expressed LIM domain binding protein, is essential in a number of tissues during development. It interacts with Gata1, Tal1, E2A and Lmo2 to form a transcription factor complex regulating late erythroid genes. We identify a number of novel Ldb1 interacting proteins in erythroleukaemic cells, in particular the repressor protein Eto-2 (and its family member Mtgr1), the cyclin-dependent kinase Cdk9, and the bridging factor Lmo4. MO-mediated knockdowns in zebrafish show these factors to be essential for definitive haematopoiesis. In accordance with the zebrafish results these factors are coexpressed in prehaematopoietic cells of the early mouse embryo, although we originally identified the complex in late erythroid cells. Based on the change in subcellullar localisation of Eto-2 we postulate that it plays a central role in the transition from the migration and expansion phase of the prehaematopoietic cells to the establishment of definitive haematopoietic stem cells.
Mass spectrometric identification of human prostate cancer-derived proteins in serum of xenograft-bearing mice.
van den Bemd, G.J., Krijgsveld, J., Luider, T.M., van Rijswijk, A.L., Demmers, J.A. & Jenster, G.
Mol Cell Proteomics. 2006 Oct;5(10):1830-9. Epub 2006 May 19.
Lack of sensitivity and specificity of current tumor markers has intensified research efforts to find new biomarkers. The identification of potential tumor markers in human body fluids is hampered by large variability and complexity of both control and patient samples, laborious biochemical analyses, and the fact that the identified proteins are unlikely produced by the diseased cells but are due to secondary body defense mechanisms. In a new approach presented here, we eliminate these problems by performing proteomic analysis in a prostate cancer xenograft model in which human prostate cancer cells form a tumor in an immune-incompetent nude mouse. Using this concept, proteins present in mouse serum that can be identified as human will, by definition, originate from the human prostate cancer xenograft and might have potential diagnostic and prognostic value. Using one-dimensional gel electrophoresis, liquid chromatography, and mass spectrometry, we identified tumor-derived human nm23/nucleoside-diphosphate kinase (NME) in the serum of a nude mouse bearing the androgen-independent human prostate cancer xenograft PC339. NME is known to be involved in the metastatic potential of several tumor cells, including prostate cancer cells. Furthermore we identified six human enzymes involved in glycolysis (fructose-bisphosphate aldolase A, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, alpha enolase, and lactate dehydrogenases A and B) in the serum of the tumor-bearing mice. The presence of human NME and glyceraldehyde-3-phosphate dehydrogenase in the serum of PC339-bearing mice was confirmed by Western blotting. Although the putative usefulness of these proteins in predicting prognosis of prostate cancer remains to be determined, the present data illustrate that our approach is a promising tool for the focused discovery of new prostate cancer biomarkers.
Embryonic stem cell proteomics.
Van Hoof, D., Mummery, C.L., Heck, A.J. & Krijgsveld, J.
Expert Rev Proteomics. 2006 Aug;3(4):427-37.
Human embryonic stem cells potentially represent an unlimited source of cells and tissues for regenerative medicine. Understanding signaling events that drive proliferation and specialization of these cells into various differentiated derivatives is of utmost importance for controlling their behavior in vitro. Major progress has been made in unraveling these signaling events with large-scale studies at the transcriptional level, but analysis of protein expression, interaction and modification has been more limited, since it requires different strategies. Recent advances in mass spectrometry-based proteomics indicate that proteome characterization can contribute significantly to our understanding of embryonic stem cell biology. In this article, we review mass spectrometry-based studies of human and mouse embryonic stem cells and their differentiated progeny, as well as studies of conditioned media that have been reported to support self-renewal of the undifferentiated cells in the absence of the more commonly used feeder cells. In addition, we make concise comparisons with related transcriptome profiling reports.
A quest for human and mouse embryonic stem cell-specific proteins.
Van Hoof, D., Passier, R., Ward-Van Oostwaard, D., Pinkse, M.W., Heck, A.J., Mummery, C.L. & Krijgsveld, J.
Mol Cell Proteomics. 2006 Jul;5(7):1261-73. Epub 2006 Apr 6.
Embryonic stem cells (ESCs) are of immense interest as they can proliferate indefinitely in vitro and give rise to any adult cell type, serving as a potentially unlimited source for tissue replacement in regenerative medicine. Extensive analyses of numerous human and mouse ESC lines have shown generic similarities and differences at both the transcriptional and functional level. However, comprehensive proteome analyses are missing or are restricted to mouse ESCs. Here we have used an extensive proteomic approach to search for ESC-specific proteins by analyzing the differential protein expression profiles of human and mouse ESCs and their differentiated derivatives. The data sets comprise 1,775 non-redundant proteins identified in human ESCs, 1,532 in differentiated human ESCs, 1,871 in mouse ESCs, and 1,552 in differentiated mouse ESCs with a false positive rate of <0.2%. Comparison of the data sets distinguished 191 proteins exclusively identified in both human and mouse ESCs but not in their differentiated derivatives. Besides well known ESC benchmarks, this subset included many uncharacterized proteins, some of which may be novel ESC-specific markers. To complement the mass spectrometric approach, differential expression of a selection of these proteins was confirmed by Western blotting, immunofluorescence confocal microscopy, and fluorescence-activated cell sorting. Additionally two other independently isolated and cultured human ESC lines as well as their differentiated derivatives were monitored for differential expression of selected proteins. Some of these proteins were identified exclusively in ESCs of all three human lines and may thus serve as generic ESC markers. Our wide scale proteomic approach enabled us to screen thousands of proteins rapidly and select putative ESC-associated proteins for further analysis. Validation by three independent conventional protein analysis techniques shows that our methodology is robust, provides an excellent tool to characterize ESCs at the protein level, and may disclose novel ESC-specific benchmarks.
In-gel isoelectric focusing of peptides as a tool for improved protein identification.
Krijgsveld, J., Gauci, S., Dormeyer, W. & Heck, A.J.
J Proteome Res. 2006 Jul;5(7):1721-30.
In the analysis of proteins in complex samples, pre-fractionation is imperative to obtain the necessary depth in the number of reliable protein identifications by mass spectrometry. Here we explore isoelectric focusing of peptides (peptide IEF) as an effective fractionation step that at the same time provides the added possibility to eliminate spurious peptide identifications by filtering for pI. Peptide IEF in IPG strips is fast and sharply confines peptides to their pI. We have evaluated systematically the contribution of pI filtering and accurate mass measurements on the total number of protein identifications in a complex protein mixture (Drosophila nuclear extract). At the same time, by varying Mascot identification cutoff scores, we have monitored the false positive rate among these identifications by searching reverse protein databases. From mass spectrometric analyses at low mass accuracy using an LTQ ion trap, false positive rates can be minimized by filtering of peptides not focusing at their expected pI. Analyses using an LTQ-FT mass spectrometer delivers low false positive rates by itself due to the high mass accuracy. In a direct comparison of peptide IEF with SDS-PAGE as a pre-fractionation step, IEF delivered 25% and 43% more proteins when identified using FT-MS and LTQ-MS, respectively. Cumulatively, 2190 non redundant proteins were identified in the Drosophila nuclear extract at a false positive rate of 0.5%. Of these, 1751 proteins (80%) were identified after peptide IEF and FT-MS alone. Overall, we show that peptide IEF allows to increase the confidence level of protein identifications, and is more sensitive than SDS-PAGE.
Identification of secreted cysteine proteases from the parasitic nematode Haemonchus contortus detected by biotinylated inhibitors.
Yatsuda, A.P., Bakker, N., Krijgsveld, J., Knox, D.P., Heck, A.J. & de Vries, E.
Infect Immun. 2006 Mar;74(3):1989-93.
Seven cathepsin B-like cysteine proteases (CBLs) were identified from the immunoprotective excretory-secretory products of Haemonchus contortus. Two-dimensional (2-D) zymography and biotinylated inhibitors were employed to localize active CBLs in 2-D protein gels. Mass spectrometry provided the identification of AC-4, HMCP1, HMCP2, and GCP7 as well as three novel CBLs encoded by clustered expressed sequence tags.
GATA-1 forms distinct activating and repressive complexes in erythroid cells.
Rodriguez, P., Bonte, E., Krijgsveld, J., Kolodziej, K.E., Guyot, B., Heck, A.J., Vyas, P., de Boer, E., Grosveld, F. & Strouboulis, J.
EMBO J. 2005 Jul 6;24(13):2354-66. Epub 2005 May 26.
GATA-1 is essential for the generation of the erythroid, megakaryocytic, eosinophilic and mast cell lineages. It acts as an activator and repressor of different target genes, for example, in erythroid cells it represses cell proliferation and early hematopoietic genes while activating erythroid genes, yet it is not clear how both of these functions are mediated. Using a biotinylation tagging/proteomics approach in erythroid cells, we describe distinct GATA-1 interactions with the essential hematopoietic factor Gfi-1b, the repressive MeCP1 complex and the chromatin remodeling ACF/WCRF complex, in addition to the known GATA-1/FOG-1 and GATA-1/TAL-1 complexes. Importantly, we show that FOG-1 mediates GATA-1 interactions with the MeCP1 complex, thus providing an explanation for the overlapping functions of these two factors in erythropoiesis. We also show that subsets of GATA-1 gene targets are bound in vivo by distinct complexes, thus linking specific GATA-1 partners to distinct aspects of its functions. Based on these findings, we suggest a model for the different roles of GATA-1 in erythroid differentiation.
Mass spectrometric analysis of the Schistosoma mansoni tegumental sub-proteome.
van Balkom, B.W., van Gestel, R.A., Brouwers, J.F., Krijgsveld, J., Tielens, A.G., Heck, A.J. & van Hellemond, J.J.
J Proteome Res. 2005 May-Jun;4(3):958-66.
Schistosoma mansoni is a parasitic worm that lives in the blood vessels of its host. We mapped the S. mansoni tegumental outer-surface structure proteome by 1D SDS-PAGE and LC-MS/MS and an EST-database from the ongoing genome-sequencing project. We identified 740 proteins of which 43 were tegument-specific. Many of these proteins show no homology to any nonschistosomal protein, demonstrating that the schistosomal outer-surface comprises specific and unique proteins, likely to be critical for parasite survival.
GMP synthetase stimulates histone H2B deubiquitylation by the epigenetic silencer USP7.
van der Knaap, J.A., Kumar, B.R., Moshkin, Y.M., Langenberg, K., Krijgsveld, J., Heck, A.J., Karch, F. & Verrijzer, C.P.
Mol Cell. 2005 Mar 4;17(5):695-707.
The packaging of eukaryotic genomic DNA into chromatin is modulated through a range of posttranslational histone modifications. Among these, the role of histone ubiquitylation remains poorly understood. Here, we show that the essential Drosophila ubiquitin-specific protease 7 (USP7) contributes to epigenetic silencing of homeotic genes by Polycomb (Pc). We purified USP7 from embryo nuclear extracts as a stable heteromeric complex with guanosine 5'-monophosphate synthetase (GMPS). The USP7-GMPS complex catalyzed the selective deubiquitylation of histone H2B, but not H2A. Biochemical assays confirmed the tight association between USP7 and GMPS in Drosophila embryo extracts. Similar to USP7, mutations in GMPS acted as enhancers of Pc in vivo. USP7 binding to GMPS was required for histone H2B deubiquitylation and strongly augmented deubiquitylation of the human tumor suppressor p53. Thus, GMPS can regulate the activity of a ubiquitin protease. Collectively, these results implicate a biosynthetic enzyme in chromatin control via ubiquitin regulation.
Munc13-4 is an effector of rab27a and controls secretion of lysosomes in hematopoietic cells.
Neeft, M., Wieffer, M., de Jong, A.S., Negroiu, G., Metz, C.H., van Loon, A., Griffith, J., Krijgsveld, J., Wulffraat, N., Koch, H., Heck, A.J., Brose, N., Kleijmeer, M. & van der Sluijs, P.
Mol Biol Cell. 2005 Feb;16(2):731-41. Epub 2004 Nov 17.
Griscelli syndrome type 2 (GS2) is a genetic disorder in which patients exhibit life-threatening defects of cytotoxic T lymphocytes (CTLs) whose lytic granules fail to dock on the plasma membrane and therefore do not release their contents. The disease is caused by the absence of functional rab27a, but how rab27a controls secretion of lytic granule contents remains elusive. Mutations in Munc13-4 cause familial hemophagocytic lymphohistiocytosis subtype 3 (FHL3), a disease phenotypically related to GS2. We show that Munc13-4 is a direct partner of rab27a. The two proteins are highly expressed in CTLs and mast cells where they colocalize on secretory lysosomes. The region comprising the Munc13 homology domains is essential for the localization of Munc13-4 to secretory lysosomes. The GS2 mutant rab27aW73G strongly reduced binding to Munc13-4, whereas the FHL3 mutant Munc13-4Delta608-611 failed to bind rab27a. Overexpression of Munc13-4 enhanced degranulation of secretory lysosomes in mast cells, showing that it has a positive regulatory role in secretory lysosome fusion. We suggest that the secretion defects seen in GS2 and FHL3 have a common origin, and we propose that the rab27a/Munc13-4 complex is an essential regulator of secretory granule fusion with the plasma membrane in hematopoietic cells. Mutations in either of the two genes prevent formation of this complex and abolish secretion.
Mass spectrometry-based quantitative proteomics.
Heck, A.J. & Krijgsveld, J.
Expert Rev Proteomics. 2004 Oct;1(3):317-26.
A major aim of present-day proteomics is to study changes in protein expression levels at a global level, ideally monitoring all proteins present in cells or tissue. Mass spectrometry is a well-respected technology in proteomics that is widely used for the identification of proteins. More recently, methodologies have been introduced showing that mass spectrometry can also be used for protein quantification. This article reviews various mass spectrometry-based technologies in quantitative proteomics, highlighting several interesting applications in areas ranging from cell biology to clinical applications.
Quantitative cancer proteomics: stable isotope labeling with amino acids in cell culture (SILAC) as a tool for prostate cancer research.
Everley, P.A., Krijgsveld, J., Zetter, B.R. & Gygi, S.P.
Mol Cell Proteomics. 2004 Jul;3(7):729-35. Epub 2004 Apr 21.
Microarrays have been the primary means for large-scale analyses of genes implicated in cancer progression. However, more recently a need has been recognized for investigating cancer development directly at the protein level. In this report, we have applied a comparative proteomic technique to the study of metastatic prostate cancer. This technology, termed stable isotope labeling with amino acids in cell culture (SILAC), has recently gained popularity for its ability to compare the expression levels of hundreds of proteins in a single experiment. SILAC makes use of (12)C- and (13)C-labeled amino acids added to the growth media of separately cultured cell lines, giving rise to cells containing either "light" or "heavy" proteins, respectively. Upon mixing lysates collected from these cells, proteins can be identified by tandem mass spectrometry. The incorporation of stable isotopes also allows for a quantitative comparison between the two samples. Using this method, we compared the expression levels for more than 440 proteins in the microsomal fractions of prostate cancer cells with varying metastatic potential. Of these, 60 were found elevated greater than 3-fold in the highly metastatic cells, whereas 22 were reduced by equivalent amounts. Western blotting provided further confirmation of the mass spectrometry-based quantification. Our results demonstrate the applicability of this novel approach toward the study of cancer progression using defined cell lines.
Differential targeting of two distinct SWI/SNF-related Drosophila chromatin-remodeling complexes.
Mohrmann, L., Langenberg, K., Krijgsveld, J., Kal, A.J., Heck, A.J. & Verrijzer, C.P.
Mol Cell Biol. 2004 Apr;24(8):3077-88.
The SWI/SNF family of ATP-dependent chromatin-remodeling factors plays a central role in eukaryotic transcriptional regulation. In yeast and human cells, two subclasses have been recognized: one comprises yeast SWI/SNF and human BAF, and the other includes yeast RSC and human PBAF. Therefore, it was puzzling that Drosophila appeared to contain only a single SWI/SNF-type remodeler, the Brahma (BRM) complex. Here, we report the identification of two novel BRM complex-associated proteins: Drosophila Polybromo and BAP170, a conserved protein not described previously. Biochemical analysis established that Drosophila contains two distinct BRM complexes: (i) the BAP complex, defined by the presence of OSA and the absence of Polybromo and BAP170, and (ii) the PBAP complex, containing Polybromo and BAP170 but lacking OSA. Determination of the genome-wide distributions of OSA and Polybromo on larval salivary gland polytene chromosomes revealed that BAP and PBAP display overlapping but distinct distribution patterns. Both complexes associate predominantly with regions of open, hyperacetylated chromatin but are largely excluded from Polycomb-bound repressive chromatin. We conclude that, like yeast and human cells, Drosophila cells express two distinct subclasses of the SWI/SNF family. Our results support a close reciprocity of chromatin regulation by ATP-dependent remodelers and histone-modifying enzymes.
Lung proteome alterations in a mouse model for nonallergic asthma.
Houtman, R., Krijgsveld, J., Kool, M., Romijn, E.P., Redegeld, F.A., Nijkamp, F.P., Heck, A.J. & Humphery-Smith, I.
Proteomics. 2003 Oct;3(10):2008-18.
A mouse model for nonatopic asthma was employed to study the alterations of the lung proteome to gain insight into the underlying molecular mechanisms of disease pathophysiology post-challenge. Lung samples from asthmatic and control mice were used to generate 24 high quality two-dimensional electrophoresis gels wherein 2115 proteins were examined for disease relevance. In total, 23 proteins were significantly up- or down-regulated following hapten-challenge of dinitro-fluorobenzene-hypersensitive mice. Twenty proteins were identified by mass spectrometry, of which 18 could be linked to asthma related symptoms, such as stress and inflammation, lung detoxification, plasma exudation and/or tissue remodeling. As such, proteomics was clearly vindicated as a means of studying this complex disease phenomenon. The proteins found in this study may not necessarily play a role in the immunological mechanisms and/or pathophysiology of asthma development. However, they may prove useful as surrogate biomarkers for quantitatively monitoring disease state progression or response to therapy. The mathematics of achieving statistical confidence from low numbers of gel replicates containing large numbers of independent variables stress the need for high numbers of replicates to better sample the population of proteins revealed by two-dimensional gel electrophoresis.
Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomics.
Krijgsveld, J., Ketting, R.F., Mahmoudi, T., Johansen, J., Artal-Sanz, M., Verrijzer, C.P., Plasterk, R.H. & Heck, A.J.
Nat Biotechnol. 2003 Aug;21(8):927-31. Epub 2003 Jul 13.
A crucial issue in comparative proteomics is the accurate quantification of differences in protein expression levels. To achieve this, several methods have been developed in which proteins are labeled with stable isotopes either in vivo via metabolic labeling or in vitro by protein derivatization. Although metabolic labeling is the only way to obtain labeling of all proteins, it has thus far only been applied to single- celled organisms and cells in culture. Here we describe quantitative 15N metabolic labeling of the multicellular organisms Caenorhabditis elegans, a nematode, and Drosophila melanogaster, the common fruit fly, achieved by feeding them on 15N-labeled Escherichia coli and yeast, respectively. The relative abundance of individual proteins obtained from different samples can then be determined by mass spectrometry (MS). The applicability of the method is exemplified by the comparison of protein expression levels in two C. elegans strains, one with and one without a germ line. The methodology described provides tools for accurate quantitative proteomic studies in these model organisms.
Recent liquid chromatographic-(tandem) mass spectrometric applications in proteomics.
Romijn, E.P., Krijgsveld, J. & Heck, A.J.
J Chromatogr A. 2003 Jun 6;1000(1-2):589-608.
Conventional proteomics makes use of two-dimensional gel electrophoresis followed by mass spectrometric analysis of typtic fragments derived from in-gel digestion of proteins. Although being a very strong technique capable of separating and visualizing hundreds of proteins, 2D-gel electrophoresis has some well-documented disadvantages as well. More recently, liquid chromatographic-(tandem) mass spectrometric techniques have been developed to overcome some of the shortcomings of 2D-gel electrophoresis. In this review we have described several recent applications of liquid chromatography-(tandem) mass spectrometry in the field of proteomics and especially in the field of membrane proteomics, quantitative proteomics and in the analysis of post-translational modifications.
Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice.
de Boer, E., Rodriguez, P., Bonte, E., Krijgsveld, J., Katsantoni, E., Heck, A., Grosveld, F. & Strouboulis, J.
Proc Natl Acad Sci U S A. 2003 Jun 24;100(13):7480-5. Epub 2003 Jun 11.
Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated templates. Here, we describe an approach for the single-step purification of transcription factor complex(es) based on specific in vivo biotinylation. We expressed the bacterial BirA biotin ligase in mammalian cells and demonstrated very efficient biotinylation of a hematopoietic transcription factor bearing a small (23-aa) artificial peptide tag. Biotinylation of the tagged transcription factor altered neither the factor's protein interactions or DNA binding properties in vivo nor its subnuclear distribution. Using this approach, we isolated the biotin-tagged transcription factor and at least one other known interacting protein from crude nuclear extracts by direct binding to streptavidin beads. Finally, this method works efficiently in transgenic mice, thus raising the prospect of using biotinylation tagging in protein complex purification directly from animal tissues. Therefore, BirA-mediated biotinylation of tagged proteins provides the basis for the single-step purification of proteins from mammalian cells.
Comprehensive analysis of the secreted proteins of the parasite Haemonchus contortus reveals extensive sequence variation and differential immune recognition.
Yatsuda, A.P., Krijgsveld, J., Cornelissen, A.W., Heck, A.J. & de Vries, E.
J Biol Chem. 2003 May 9;278(19):16941-51. Epub 2003 Feb 7.
Haemonchus contortus is a nematode that infects small ruminants. It releases a variety of molecules, designated excretory/secretory products (ESP), into the host. Although the composition of ESP is largely unknown, it is a source of potential vaccine components because ESP are able to induce up to 90% protection in sheep. We used proteomic tools to analyze ESP proteins and determined the recognition of these individual proteins by hyperimmune sera. Following two-dimensional electrophoresis of ESP, matrix-assisted laser desorption ionization time-of-flight and liquid chromatography-tandem mass spectrometry were used for protein identification. Few sequences of H. contortus have been determined. Therefore, the data base of expressed sequence tags (dbEST) and a data base consisting of contigs from Haemonchus ESTs were also consulted for identification. Approximately 200 individual spots were observed in the two-dimensional gel. Comprehensive proteomics analysis, combined with bioinformatic search tools, identified 107 proteins in 102 spots. The data include known as well as novel proteins such as serine, metallo- and aspartyl proteases, in addition to H. contortus ESP components like Hc24, Hc40, Hc15, and apical gut GA1 proteins. Novel proteins were identified from matches with H. contortus ESTs displaying high similarity with proteins like cyclophilins, nucleoside diphosphate kinase, OV39 antigen, and undescribed homologues of Caenorhabditis elegans. Of special note is the finding of microsomal peptidase H11, a vaccine candidate previously regarded as a "hidden antigen" because it was not found in ESP. Extensive sequence variation is present in the abundant Hc15 proteins. The Hc15 isoforms are differentially recognized by hyperimmune sera, pointing to a possible specific role of Hc15 in the infectious process and/or in immune evasion. This concept and the identification of multiple novel immune-recognized components in ESP should assist future vaccine development strategies.