DOT1L regulates chromatin reorganization and gene expression during sperm differentiation.

Spermatozoa have a unique genome organization. Their chromatin is almost completely devoid of histones and is formed instead of protamines, which confer a high level of compaction and preserve paternal genome integrity until fertilization. Histone-to-protamine transition takes place in spermatids and is indispensable for the production of functional sperm. Here, we show that the H3K79-methyltransferase DOT1L controls spermatid chromatin remodeling and subsequent reorganization and compaction of the spermatozoon genome. Using a mouse model in which Dot1l is knocked-out (KO) in postnatal male germ cells, we found that Dot1l-KO sperm chromatin is less compact and has an abnormal content, characterized by the presence of transition proteins, immature protamine 2 forms and a higher level of histones. Proteomic and transcriptomic analyses performed on spermatids reveal that Dot1l-KO modifies the chromatin prior to histone removal and leads to the deregulation of genes involved in flagellum formation and apoptosis during spermatid differentiation. As a consequence of these chromatin and gene expression defects, Dot1l-KO spermatozoa have less compact heads and are less motile, which results in impaired fertility.

Chromatin phosphoproteomics unravels a function for AT-hook motif nuclear localized protein AHL13 in PAMP-triggered immunity.

In many eukaryotic systems during immune responses, mitogen-activated protein kinases (MAPKs) link cytoplasmic signaling to chromatin events by targeting transcription factors, chromatin remodeling complexes, and the RNA polymerase machinery. So far, knowledge on these events is scarce in plants and no attempts have been made to focus on phosphorylation events of chromatin-associated proteins. Here we carried out chromatin phosphoproteomics upon elicitor-induced activation of Arabidopsis The events in WT were compared with those in mpk3, mpk4, and mpk6 mutant plants to decipher specific MAPK targets. Our study highlights distinct signaling networks involving MPK3, MPK4, and MPK6 in chromatin organization and modification, as well as in RNA transcription and processing. Among the chromatin targets, we characterized the AT-hook motif containing nuclear localized (AHL) DNA-binding protein AHL13 as a substrate of immune MAPKs. AHL13 knockout mutant plants are compromised in pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species production, expression of defense genes, and PAMP-triggered immunity. Transcriptome analysis revealed that AHL13 regulates key factors of jasmonic acid biosynthesis and signaling and affects immunity toward Pseudomonas syringae and Botrytis cinerea pathogens. Mutational analysis of the phosphorylation sites of AHL13 demonstrated that phosphorylation regulates AHL13 protein stability and thereby its immune functions.

Analysis of the Arabidopsis coilin mutant reveals a positive role of AtCOILIN in plant immunity.

Biogenesis of ribonucleoproteins occurs in dynamic subnuclear compartments called Cajal bodies (CBs). COILIN is a critical scaffolding component essential for CB formation, composition, and activity. We recently showed that Arabidopsis (Arabidopsis thaliana) AtCOILIN is phosphorylated in response to bacterial elicitor treatment. Here, we further investigated the role of AtCOILIN in plant innate immunity. Atcoilin mutants are compromised in defense responses to bacterial pathogens. Besides confirming a role of AtCOILIN in alternative splicing (AS), Atcoilin showed differential expression of genes that are distinct from those of AS, including factors involved in RNA biogenesis, metabolism, plant immunity, and phytohormones. Atcoilin mutant plants have reduced levels of defense phytohormones. As expected, the mutant plants were more sensitive to the necrotrophic fungal pathogen Botrytis cinerea. Our findings reveal an important role for AtCOILIN in innate plant immunity.

Multi-omic analysis of gametogenesis reveals a novel signature at the promoters and distal enhancers of active genes.

Epigenetic regulation of gene expression is tightly controlled by the dynamic modification of histones by chemical groups, the diversity of which has largely expanded over the past decade with the discovery of lysine acylations, catalyzed from acyl-coenzymes A. We investigated the dynamics of lysine acetylation and crotonylation on histones H3 and H4 during mouse spermatogenesis. Lysine crotonylation appeared to be of significant abundance compared to acetylation, particularly on Lys27 of histone H3 (H3K27cr) that accumulates in sperm in a cleaved form of H3. We identified the genomic localization of H3K27cr and studied its effects on transcription compared to the classical active mark H3K27ac at promoters and distal enhancers. The presence of both marks was strongly associated with highest gene expression. Assessment of their co-localization with transcription regulators (SLY, SOX30) and chromatin-binding proteins (BRD4, BRDT, BORIS and CTCF) indicated systematic highest binding when both active marks were present and different selective binding when present alone at chromatin. H3K27cr and H3K27ac finally mark the building of some sperm super-enhancers. This integrated analysis of omics data provides an unprecedented level of understanding of gene expression regulation by H3K27cr in comparison to H3K27ac, and reveals both synergistic and specific actions of each histone modification.

The Trihelix transcription factor GT2-like 1 (GTL1) promotes salicylic acid metabolism, and regulates bacterial-triggered immunity.

The Trihelix Transcription factor GT2-like 1 (GTL1) was previously shown to be a key regulator of ploidy-dependent trichome growth and drought tolerance. Here, we report that GTL1 plays an important role in coordinating plant immunity. We show that gtl1 mutants are compromised in the regulation of basal immunity, microbial pattern-triggered immunity (PTI) and effector-triggered RIN4-mediated immunity. Transcriptome analysis revealed that GTL1 positively regulates defense genes and inhibits factors that mediate growth and development. By performing hormonal measurements and chromatin-immunoprecipitation studies, we found GTL1 to coordinate genes involved in salicylic acid metabolism, transport and response. Interaction studies and comparative transcriptomics to known data sets revealed that GTL1 is part of the MPK4 pathway and regulates oppositely the expression of differentially expressed genes in mpk4 plants. We introduced the gtl1 mutation in the mpk4 mutant and thereby partially suppressed its dwarfism and the high resistance against a bacterial invader. Our data show that GTL1 is part of the MPK4 pathway and acts as a positive regulator of bacterial-triggered immunity and SA homeostasis.

Quantitative Phosphoproteomic Analysis Reveals Shared and Specific Targets of Arabidopsis Mitogen-Activated Protein Kinases (MAPKs) MPK3, MPK4, and MPK6.

In Arabidopsis, mitogen-activated protein kinases MPK3, MPK4, and MPK6 constitute essential relays for a variety of functions including cell division, development and innate immunity. Although some substrates of MPK3, MPK4 and MPK6 have been identified, the picture is still far from complete. To identify substrates of these MAPKs likely involved in cell division, growth and development we compared the phosphoproteomes of wild-type and mpk3, mpk4, and mpk6. To study the function of these MAPKs in innate immunity, we analyzed their phosphoproteomes following microbe-associated molecular pattern (MAMP) treatment. Partially overlapping substrates were retrieved for all three MAPKs, showing target specificity to one, two or all three MAPKs in different biological processes. More precisely, our results illustrate the fact that the entity to be defined as a specific or a shared substrate for MAPKs is not a phosphoprotein but a particular (S/T)P phosphorylation site in a given protein. One hundred fifty-two peptides were identified to be differentially phosphorylated in response to MAMP treatment and/or when compared between genotypes and 70 of them could be classified as putative MAPK targets. Biochemical analysis of a number of putative MAPK substrates by phosphorylation and interaction assays confirmed the global phosphoproteome approach. Our study also expands the set of MAPK substrates to involve other protein kinases, including calcium-dependent (CDPK) and sugar nonfermenting (SnRK) protein kinases.

Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins.

In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

Proteomic and phosphoproteomic analyses of chromatin-associated proteins from Arabidopsis thaliana.

The nucleus is the organelle where basically all DNA-related processes take place in eukaryotes, such as replication, transcription, and splicing as well as epigenetic regulation. The identification and description of the nuclear proteins is one of the requisites toward a comprehensive understanding of the biological functions accomplished in the nucleus. Many of the regulatory mechanisms of protein functions rely on their PTMs among which phosphorylation is probably one of the most important properties affecting enzymatic activity, interaction with other molecules, localization, or stability. So far, the nuclear and subnuclear proteome and phosphoproteome of the model plant Arabidopsis thaliana have been the subject of very few studies. In this work, we developed a purification protocol of Arabidopsis chromatin-associated proteins and performed proteomic and phosphoproteomic analyses identifying a total of 879 proteins of which 198 were phosphoproteins that were mainly involved in chromatin remodeling, transcriptional regulation, and RNA processing. From 230 precisely localized phosphorylation sites (phosphosites), 52 correspond to hitherto unidentified sites. This protocol and data thereby obtained should be a valuable resource for many domains of plant research.

Identification of novel PAMP-triggered phosphorylation and dephosphorylation events in Arabidopsis thaliana by quantitative phosphoproteomic analysis.

Signaling cascades rely strongly on protein kinase-mediated substrate phosphorylation. Currently a major challenge in signal transduction research is to obtain high confidence substrate phosphorylation sites and assign them to specific kinases. In response to bacterial flagellin, a pathogen-associated molecular pattern (PAMP), we searched for rapidly phosphorylated proteins in Arabidopsis thaliana by combining multistage activation (MSA) and electron transfer dissociation (ETD) fragmentation modes, which generate complementary spectra and identify phosphopeptide sites with increased reliability. Of a total of 825 phosphopeptides, we identified 58 to be differentially phosphorylated. These peptides harbor kinase motifs of mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs), as well as yet unknown protein kinases. Importantly, 12 of the phosphopeptides show reduced phosphorylation upon flagellin treatment. Since protein abundance levels did not change, these results indicate that flagellin induces not only various protein kinases but also protein phosphatases, even though a scenario of inhibited kinase activity may also be possible.

Brassinosteroid-regulated GSK3/Shaggy-like kinases phosphorylate mitogen-activated protein (MAP) kinase kinases, which control stomata development in Arabidopsis thaliana.

Brassinosteroids (BRs) are steroid hormones that coordinate fundamental developmental programs in plants. In this study we show that in addition to the well established roles of BRs in regulating cell elongation and cell division events, BRs also govern cell fate decisions during stomata development in Arabidopsis thaliana. In wild-type A. thaliana, stomatal distribution follows the one-cell spacing rule; that is, adjacent stomata are spaced by at least one intervening pavement cell. This rule is interrupted in BR-deficient and BR signaling-deficient A. thaliana mutants, resulting in clustered stomata. We demonstrate that BIN2 and its homologues, GSK3/Shaggy-like kinases involved in BR signaling, can phosphorylate the MAPK kinases MKK4 and MKK5, which are members of the MAPK module YODA-MKK4/5-MPK3/6 that controls stomata development and patterning. BIN2 phosphorylates a GSK3/Shaggy-like kinase recognition motif in MKK4, which reduces MKK4 activity against its substrate MPK6 in vitro. In vivo we show that MKK4 and MKK5 act downstream of BR signaling because their overexpression rescued stomata patterning defects in BR-deficient plants. A model is proposed in which GSK3-mediated phosphorylation of MKK4 and MKK5 enables for a dynamic integration of endogenous or environmental cues signaled by BRs into cell fate decisions governed by the YODA-MKK4/5-MPK3/6 module.

Automated phosphopeptide identification using multiple MS/MS fragmentation modes.

Phosphopeptide identification is still a challenging task because fragmentation spectra obtained by mass spectrometry do not necessarily contain sufficient fragment ions to establish with certainty the underlying amino acid sequence and the precise phosphosite. To improve upon this, it has been suggested to acquire pairs of spectra from every phosphorylated precursor ion using different fragmentation modes, for example CID, ETD, and/or HCD. The development of automated tools for the interpretation of these paired spectra has however, until now, lagged behind. Using phosphopeptide samples analyzed by an LTQ-Orbitrap instrument, we here assess an approach in which, on each selected precursor, a pair of CID spectra, with or without multistage activation (MSA or MS2, respectively), are acquired in the linear ion trap. We applied this approach on phosphopeptide samples of variable proteomic complexity obtained from Arabidopsis thaliana . We present a straightforward computational approach to reconcile sequence and phosphosite identifications provided by the database search engine Mascot on the spectrum pairs, using two simple filtering rules, at the amino acid sequence and phosphosite localization levels. If multiple sequences and/or phosphosites are likely, they are reported in the consensus sequence. Using our program FragMixer, we could assess that on samples of moderate complexity, it was worth combining the two fragmentation schemes on every precursor ion to help efficiently identify amino acid sequences and precisely localize phosphosites. FragMixer can be flexibly configured, independently of the Mascot search parameters, and can be applied to various spectrum pairs, such as MSA/ETD and ETD/HCD, to automatically compare and combine the information provided by these more differing fragmentation modes. The software is openly accessible and can be downloaded from our Web site at http://proteomics.fr/FragMixer.

Interactome of the amyloid precursor protein APP in brain reveals a protein network involved in synaptic vesicle turnover and a close association with Synaptotagmin-1.

Knowledge of the protein networks interacting with the amyloid precursor protein (APP) in vivo can shed light on the physiological function of APP. To date, most proteins interacting with the APP intracellular domain (AICD) have been identified by Yeast Two Hybrid screens which only detect direct interaction partners. We used a proteomics-based approach by biochemically isolating tagged APP from the brains of transgenic mice and subjecting the affinity-purified complex to mass spectrometric (MS) analysis. Using two different quantitative MS approaches, we compared the protein composition of affinity-purified samples isolated from wild-type mice versus transgenic mice expressing tagged APP. This enabled us to assess truly enriched proteins in the transgenic sample and yielded an overlapping set of proteins containing the major proteins involved in synaptic vesicle endo- and exocytosis. Confocal microscopy analyses of cotransfected primary neurons showed colocalization of APP with synaptic vesicle proteins in vesicular structures throughout the neurites. We analyzed the interaction of APP with these proteins using pulldown experiments from transgenic mice or cotransfected cells followed by Western blotting. Synaptotagmin-1 (Stg1), a resident synaptic vesicle protein, was found to directly bind to APP. We fused Citrine and Cerulean to APP and the candidate proteins and measured fluorescence resonance energy transfer (FRET) in differentiated SH-SY5Y cells. Differentially tagged APPs showed clear sensitized FRET emission, in line with the described dimerization of APP. Among the candidate APP-interacting proteins, again only Stg1 was in close proximity to APP. Our results strongly argue for a function of APP in synaptic vesicle turnover in vivo. Thus, in addition to the APP cleavage product Aß, which influences synaptic transmission at the postsynapse, APP interacts with the calcium sensor of synaptic vesicles and might thus play a role in the regulation of synaptic vesicle exocytosis.

Linking the proteins--elucidation of proteome-scale networks using mass spectrometry.

Proteomes are intricate. Typically, thousands of proteins interact through physical association and post-translational modifications (PTMs) to give rise to the emergent functions of cells. Understanding these functions requires one to study proteomes as "systems" rather than collections of individual protein molecules. The abstraction of the interacting proteome to "protein networks" has recently gained much attention, as networks are effective representations, that lose specific molecular details, but provide the ability to see the proteome as a whole. Mostly two aspects of the proteome have been represented by network models: proteome-wide physical protein-protein-binding interactions organized into Protein Interaction Networks (PINs), and proteome-wide PTM relations organized into Protein Signaling Networks (PSNs). Mass spectrometry (MS) techniques have been shown to be essential to reveal both of these aspects on a proteome-wide scale. Techniques such as affinity purification followed by MS have been used to elucidate protein-protein interactions, and MS-based quantitative phosphoproteomics is critical to understand the structure and dynamics of signaling through the proteome. We here review the current state-of-the-art MS-based analytical pipelines for the purpose to characterize proteome-scale networks.

Analysis of human C1q by combined bottom-up and top-down mass spectrometry: detailed mapping of post-translational modifications and insights into the C1r/C1s binding sites.

C1q is a subunit of the C1 complex, a key player in innate immunity that triggers activation of the classical complement pathway. Featuring a unique structural organization and comprising a collagen-like domain with a high level of post-translational modifications, C1q represents a challenging protein assembly for structural biology. We report for the first time a comprehensive proteomics study of C1q combining bottom-up and top-down analyses. C1q was submitted to proteolytic digestion by a combination of collagenase and trypsin for bottom-up analyses. In addition to classical LC-MS/MS analyses, which provided reliable identification of hydroxylated proline and lysine residues, sugar loss-triggered MS(3) scans were acquired on an LTQ-Orbitrap (Linear Quadrupole Ion Trap-Orbitrap) instrument to strengthen the localization of glucosyl-galactosyl disaccharide moieties on hydroxylysine residues. Top-down analyses performed on the same instrument allowed high accuracy and high resolution mass measurements of the intact full-length C1q polypeptide chains and the iterative fragmentation of the proteins in the MS(n) mode. This study illustrates the usefulness of combining the two complementary analytical approaches to obtain a detailed characterization of the post-translational modification pattern of the collagen-like domain of C1q and highlights the structural heterogeneity of individual molecules. Most importantly, three lysine residues of the collagen-like domain, namely Lys(59) (A chain), Lys(61) (B chain), and Lys(58) (C chain), were unambiguously shown to be completely unmodified. These lysine residues are located about halfway along the collagen-like fibers. They are thus fully available and in an appropriate position to interact with the C1r and C1s protease partners of C1q and are therefore likely to play an essential role in C1 assembly.

CK2ß Is a Gatekeeper of Focal Adhesions Regulating Cell Spreading.

CK2 is a hetero-tetrameric serine/threonine protein kinase made up of two CK2α/α' catalytic subunits and two CK2ß regulatory subunits. The free CK2α subunit and the tetrameric holoenzyme have distinct substrate specificity profiles, suggesting that the spatiotemporal organization of the individual CK2 subunits observed in living cells is crucial in the control of the many cellular processes that are governed by this pleiotropic kinase. Indeed, previous studies reported that the unbalanced expression of CK2 subunits is sufficient to drive epithelial to mesenchymal transition (EMT), a process involved in cancer invasion and metastasis. Moreover, sub-stoichiometric expression of CK2ß compared to CK2α in a subset of breast cancer tumors was correlated with the induction of EMT markers and increased epithelial cell plasticity in breast carcinoma progression. Phenotypic changes of epithelial cells are often associated with the activation of phosphotyrosine signaling. Herein, using phosphotyrosine enrichment coupled with affinity capture and proteomic analysis, we show that decreased expression of CK2ß in MCF10A mammary epithelial cells triggers the phosphorylation of a number of proteins on tyrosine residues and promotes the striking activation of the FAK1-Src-PAX1 signaling pathway. Moreover, morphometric analyses also reveal that CK2ß loss increases the number and the spatial distribution of focal adhesion signaling complexes that coordinate the adhesive and migratory processes. Together, our findings allow positioning CK2ß as a gatekeeper for cell spreading by restraining focal adhesion formation and invasion of mammary epithelial cells.

Isolation and characterization of plant protein complexes by mass spectrometry.

The components that enable cells and organisms to fulfill a plethora of chemical and physical reactions, including their ability to metabolize, replicate, repair and communicate with their environment are mostly based on the functioning of highly complex cellular machines which are to a large extent composed of proteins. With the development of MS techniques compatible with the analysis of minute amounts of biological material, it has become more and more feasible to dissect the composition and modification of these protein machineries. Indeed, new purification methods of protein complexes followed by MS analysis together with the genomic sequencing of various organisms - and in particular of crop species - now provide unforeseen insight to understand biological processes at a molecular level. We here review the current state of the art of in vivo protein complex isolation and their MS-based analytical characterization, emphasizing on the tandem affinity purification approach.

Mapping surface accessibility of the C1r/C1s tetramer by chemical modification and mass spectrometry provides new insights into assembly of the human C1 complex.

C1, the complex that triggers the classic pathway of complement, is a 790-kDa assembly resulting from association of a recognition protein C1q with a Ca(2+)-dependent tetramer comprising two copies of the proteases C1r and C1s. Early structural investigations have shown that the extended C1s-C1r-C1r-C1s tetramer folds into a compact conformation in C1. Recent site-directed mutagenesis studies have identified the C1q-binding sites in C1r and C1s and led to a three-dimensional model of the C1 complex (Bally, I., Rossi, V., Lunardi, T., Thielens, N. M., Gaboriaud, C., and Arlaud, G. J. (2009) J. Biol. Chem. 284, 19340-19348). In this study, we have used a mass spectrometry-based strategy involving a label-free semi-quantitative analysis of protein samples to gain new structural insights into C1 assembly. Using a stable chemical modification, we have compared the accessibility of the lysine residues in the isolated tetramer and in C1. The labeling data account for 51 of the 73 lysine residues of C1r and C1s. They strongly support the hypothesis that both C1s CUB(1)-EGF-CUB(2) interaction domains, which are distant in the free tetramer, associate with each other in the C1 complex. This analysis also provides the first experimental evidence that, in the proenzyme form of C1, the C1s serine protease domain is partly positioned inside the C1q cone and yields precise information about its orientation in the complex. These results provide further structural insights into the architecture of the C1 complex, allowing significant improvement of our current C1 model.

Small Mass but Strong Information: Diagnostic Ions Provide Crucial Clues to Correctly Identify Histone Lysine Modifications.

(1) Background: The proteomic analysis of histones constitutes a delicate task due to the combination of two factors: slight variations in the amino acid sequences of variants and the multiplicity of post-translational modifications (PTMs), particularly those occurring on lysine residues. (2) Methods: To dissect the relationship between both aspects, we carefully evaluated PTM identification on lysine 27 from histone H3 (H3K27) and the artefactual chemical modifications that may lead to erroneous PTM determination. H3K27 is a particularly interesting example because it can bear a range of PTMs and it sits nearby residues 29 and 31 that vary between H3 sequence variants. We discuss how the retention times, neutral losses and immonium/diagnostic ions observed in the MS/MS spectra of peptides bearing modified lysines detectable in the low-mass region might help validate the identification of modified sequences. (3) Results: Diagnostic ions carry key information, thereby avoiding potential mis-identifications due to either isobaric PTM combinations or isobaric amino acid-PTM combinations. This also includes cases where chemical formylation or acetylation of peptide N-termini artefactually occurs during sample processing or simply in the timeframe of LC-MS/MS analysis. Finally, in the very subtle case of positional isomers possibly corresponding to a given mass of lysine modification, the immonium and diagnostic ions may allow the identification of the in vivo structure.

Quantitative analysis of protein complex constituents and their phosphorylation states on a LTQ-Orbitrap instrument.

Cellular functions are largely carried out by noncovalent protein complexes that may exist within the cell as stable modules or as assemblies of dynamically changing composition, whose formation and decomposition are triggered in response to extracellular stimuli. The protein constituents of complexes often exhibit post-translational modifications such as phosphorylation that can impact their ability to interact with other proteins and thus to form multicomponent complexes. A complete characterization of a particular protein complex thus requires determining both, the identity of interacting proteins and their covalent modifications, in terms of attachment sites and stoichiometry. We have previously developed a protocol which identifies genuine constituents of partially purified protein complexes and concurrently determines their phosphorylation sites and levels in a single LC-MS/MS analysis performed on a MALDI-TOF/TOF instrument (Pflieger, D.; Junger, M. A.; Muller, M.; Rinner, O.; Lee, H.; Gehrig, P. M.; Gstaiger, M.; Aebersold, R. Mol. Cell. Proteomics 2008 , 7 , 326 - 346). The method combines fourplex iTRAQ labeling (isobaric tags for relative and absolute quantification) and phosphatase treatment of peptide samples derived from the tryptic digestion of isolated complexes. To test the performances of this method with nanoESI and different peptide fragmentation modes, possibly better suited for the identification of phosphorylated sequences than MALDI-TOF/TOF-MS, we have implemented it on the nanoESI-LTQ-Orbitrap instrument. The model protein beta-casein was used to optimize the conditions with respect to sensitivity and quantitative accuracy: a combination of CID fragmentation in the linear ion trap and Higher energy Collision Dissociation (HCD) appeared optimal to obtain reliable and robust identification and quantification data. The optimized conditions were then applied to identify and estimate the respective levels of phosphorylation sites on the purified, autoactivated tyrosine kinase domain of Fibroblast Growth Factor Receptor 3 (FGFR3-KD) and to analyze complexes formed around the insulin receptor substrate homologue CHICO immunopurified from Drosophila melanogaster cells that were either stimulated with insulin or left untreated. These new analyses allowed us to improve the assignment of the phosphorylation sites of some peptides previously detected by MALDI-TOF/TOF analysis and to identify additional phosphorylated sequences in CHICO and in the insulin receptor.

Quantitative proteomic analysis of protein complexes: concurrent identification of interactors and their state of phosphorylation.

Protein complexes have largely been studied by immunoaffinity purification and (mass spectrometric) analysis. Although this approach has been widely and successfully used it is limited because it has difficulties reliably discriminating true from false protein complex components, identifying post-translational modifications, and detecting quantitative changes in complex composition or state of modification of complex components. We have developed a protocol that enables us to determine, in a single LC-MALDI-TOF/TOF analysis, the true protein constituents of a complex, to detect changes in the complex composition, and to localize phosphorylation sites and estimate their respective stoichiometry. The method is based on the combination of fourplex iTRAQ (isobaric tags for relative and absolute quantification) isobaric labeling and protein phosphatase treatment of substrates. It was evaluated on model peptides and proteins and on the complex Ccl1-Kin28-Tfb3 isolated by tandem affinity purification from yeast cells. The two known phosphosites in Kin28 and Tfb3 could be reproducibly shown to be fully modified. The protocol was then applied to the analysis of samples immunopurified from Drosophila melanogaster cells expressing an epitope-tagged form of the insulin receptor substrate homologue Chico. These experiments allowed us to identify 14-3-3epsilon, 14-3-3zeta, and the insulin receptor as specific Chico interactors. In a further experiment, we compared the immunopurified materials obtained from tagged Chico-expressing cells that were either treated with insulin or left unstimulated. This analysis showed that hormone stimulation increases the association of 14-3-3 proteins with Chico and modulates several phosphorylation sites of the bait, some of which are located within predicted recognition motives of 14-3-3 proteins.