Increasing phosphoproteome coverage and identification of phosphorylation motifs through combination of different HPLC fractionation methods

Protein phosphorylation activates or deactivates many other proteins especially protein enzymes, and plays a significant role in a wide range of cellular processes. Recent advances in phosphopeptide enrichment procedures and mass spectrometry-based peptide sequencing techniques have enabled us to identify large number of protein phosphorylation sites. In this study, we combined three different HPLC techniques in fractionating enriched phosphopeptides before RPLC-MS/MS analysis, and found that although between 4000-5000 unique phosphopeptides could be identified following any of the HPLC fraction method, different HPLC method yielded a considerable amount of non-overlapping unique phosphopeptides. Combining data from all the HPLC methods, we were able to identify 9069 unique phosphopeptides and 3260 phosphoproteins covering 9463 unique phosphorylation sites, indicating that different HPLC methods are complementary to each other, and can be used together in order to increase the phosphoproteome coverage. A number of new phosphorylation sites and novel phosphorylation motifs were also discovered from our study.     

The phosphoproteome of a Chlamydomonas reinhardtii eyespot fraction includes key proteins of the light signaling pathway

Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. In a recent proteomic approach, we identified 202 proteins from a fraction enriched in eyespot apparatuses of Chlamydomonas reinhardtii. Among these proteins, five protein kinases and two protein phosphatases were present, indicating that reversible protein phosphorylation occurs in the eyespot. About 20 major phosphoprotein bands were detected in immunoblots of eyespot proteins with an anti-phosphothreonine antibody. Toward the profiling of the targets of protein kinases in the eyespot fraction, we analyzed its phosphoproteome. The solubilized proteins of the eyespot fraction were treated with the endopeptidases LysC and trypsin prior to enrichment of phosphopeptides with immobilized metal-ion affinity chromatography. Phosphopeptides were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS/MS as well as neutral-loss-triggered MS/MS/MS spectra. We were able to identify 68 different phosphopeptides along with 52 precise in vivo phosphorylation sites corresponding to 32 known proteins of the eyespot fraction. Among the identified phosphoproteins are enzymes of carotenoid and fatty acid metabolism, putative signaling components, such as a SOUL heme-binding protein, a Ca(2+)-binding protein, and an unusual protein kinase, but also several proteins with unknown function. Notably, two unique photoreceptors, channelrhodopsin-1 and channelrhodopsin-2, contain three and one phosphorylation sites, respectively. Phosphorylation of both photoreceptors occurs in the cytoplasmatic loop next to their seven transmembrane regions in a similar distance to that observed in vertebrate rhodopsins, implying functional importance for regulation of these directly light-gated ion channels relevant for the photoresponses of C. reinhardtii.     

Phosphoproteomic analysis of distinct tumor cell lines in response to nocodazole treatment

Here, we report for the first time a comparative phosphoproteomic analysis of distinct tumor cell lines in the presence or absence of the microtubule‐interfering agent nocodazole. In total, 1525 phosphorylation sites assigned to 726 phosphoproteins were identified using LC‐MS‐based technology following phosphopeptide enrichment. Analysis of the amino acid composition surrounding the identified in vivo phosphorylation sites revealed that they could be classified into two motif groups: pSer‐Pro and pSer‐Asp/Glu. Phosphoproteomic change resulting from nocodazole treatment varied among cell lines in terms of the numbers of total phosphopeptides identified, motif groups, and functional annotation groups; however, the cell lines were equally sensitive to nocodazole. The identified phosphoproteome subset contained major signaling proteins and proteins known to be involved in mitosis, but did not always exhibit the same changes in the tumor cells from nocodazole treatment. In spite of the complex changes observed in the phosphorylation of many of the proteins, possible common features induced by nocodazole were found, including phosphorylation of nucleophosmin (NPM) S254 and coatomer protein complex, subunit α (COPA) S173, suggesting that the events are not cell‐type specific but events generally occurring in mitosis or induced by a microtubule‐interfering agent. Further, temporal analysis of phosphoproteome change revealed that phosphorylation of NPM S254 and COPA S173 was observed from the early (6 h) and late (24 h) time point after nocodazole treatment, respectively, suggesting that NPM S254 may be involved in the induction of M‐phase arrest by nocodazole, whereas COPA S173 may be caused as a result of M‐phase arrest.     

Phosphoproteomic investigation of a solvent producing bacterium Clostridium acetobutylicum

In this study, we employed TiO? enrichment and high accuracy liquid chromatography-mass spectrometry-mass spectrometry to identify the phosphoproteome of Clostridium acetobutyicum ATCC824 in acidogenesis and solventogenesis. As many as 82 phosphopeptides in 61 proteins, with 107 phosphorylated sites on serine, threonine, or tyrosine, were identified with high confidence. We detected 52 phosphopeptides from 44 proteins in acidogenesis and 70 phosphopeptides from 51 proteins in solventogenesis, respectively. Bioinformatic analysis revealed most of the phosphoproteins located in cytoplasm and participated in carbon metabolism. Based on comparison between the two stages, we found 27 stage-specific phosphorylated proteins (10 in acidogenesis and 17 in solventogenesis), some of which were solvent production-related enzymes and metabolic regulators, showed significantly different phosphorylated status. Further analysis indicated that protein phosphorylation could be involved in the shift of stages or in solvent production pathway directly. Comparison against several other organisms revealed the evolutionary diversity among them on phosphorylation level in spite of their high homology on protein sequence level.     

Phosphoproteomic analysis of chromoplasts from sweet orange during fruit ripening

Like other types of plastids, chromoplasts have essential biosynthetic and metabolic activities which may be regulated via post‐translational modifications, such as phosphorylation, of their resident proteins. We here report a proteome‐wide mapping of in vivo phosphorylation sites in chromoplast‐enriched samples prepared from sweet orange [Citrus sinensis (L.) Osbeck] at different ripening stages by titanium dioxide‐based affinity chromatography for phosphoprotein enrichment with LC‐MS/MS. A total of 109 plastid‐localized phosphoprotein candidates were identified that correspond to 179 unique phosphorylation sites in 135 phosphopeptides. On the basis of Motif‐X analysis, two distinct types of phosphorylation sites, one as proline‐directed phosphorylation motif and the other as casein kinase II motif, can be generalized from these identified phosphopeptides. While most identified phosphoproteins show high homology to those already identified in plastids, approximately 22% of them are novel based on BLAST search using the public databases PhosPhAt and P³DB. A close comparative analysis showed that approximately 50% of the phosphoproteins identified in citrus chromoplasts find obvious counterparts in the chloroplast phosphoproteome, suggesting a rather high‐level of conservation in basic metabolic activities in these two types of plastids. Not surprisingly, the phosphoproteome of citrus chromoplasts is also characterized by the lack of phosphoproteins involved in photosynthesis and by the presence of more phosphoproteins implicated in stress/redox responses. This study presents the first comprehensive phosphoproteomic analysis of chromoplasts and may help to understand how phosphorylation regulates differentiation of citrus chromoplasts during fruit ripening.     

Immobilized metal affinity chromatography optimized for the analysis of extracellular phosphorylation

Phosphorylation is the most widely studied posttranslational modification. Its role within the cell has been the focus of numerous large‐scale studies. Recently there is growing evidence on the biological significance of extracellular phosphorylation. The analysis of these phosphopeptides is complicated by the abundance of glycosylation in the extracellular space, since glycopeptides are also enriched by the methods used for phosphopeptide isolation. Thus, we optimized IMAC for phosphorylation analysis of secreted proteins, specifically in human serum. Selectivity and efficiency of different enrichment conditions used in earlier large‐scale phosphoproteomic studies were evaluated. We found that minimizing hydrophilic interactions in the enrichment allowed selective phosphopeptide isolation. Using a two‐step IMAC enrichment protocol under these conditions led to the identification of ～100 phosphorylation sites from the tryptic digest of as little as 40 μL human serum.     

Mitochondrial phosphoproteome revealed by an improved IMAC method and MS/MS/MS

IMAC in combination with mass spectrometry is a promising approach for global analysis of protein phosphorylation. Nevertheless this approach suffers from two shortcomings: inadequate efficiency of IMAC and poor fragmentation of phosphopeptides in the mass spectrometer. Here we report optimization of the IMAC procedure using (32)P-labeled tryptic peptides and development of MS/MS/MS (MS3) for identifying phosphopeptide sequences and phosphorylation sites. The improved IMAC method allowed recovery of phosphorylated tryptic peptides up to approximately 77% with only minor retention of unphosphorylated peptides. MS3 led to efficient fragmentation of the peptide backbone in phosphopeptides for sequence assignment. Proteomics of mitochondrial phosphoproteins using the resulting IMAC protocol and MS3 revealed 84 phosphorylation sites in 62 proteins, most of which have not been reported before. These results revealed diverse phosphorylation pathways involved in the regulation of mitochondrial functions. Integration of the optimized batchwise IMAC protocol with MS3 offers a relatively simple and more efficient approach for proteomics of protein phosphorylation.     

Phosphoproteomic analysis of the mouse brain cytosol reveals a predominance of protein phosphorylation in regions of intrinsic sequence disorder

We analyzed the mouse forebrain cytosolic phosphoproteome using sequential (protein and peptide) IMAC purifications, enzymatic dephosphorylation, and targeted tandem mass spectrometry analysis strategies. In total, using complementary phosphoenrichment and LC-MS/MS strategies, 512 phosphorylation sites on 540 non-redundant phosphopeptides from 162 cytosolic phosphoproteins were characterized. Analysis of protein domains and amino acid sequence composition of this data set of cytosolic phosphoproteins revealed that it is significantly enriched in intrinsic sequence disorder, and this enrichment is associated with both cellular location and phosphorylation status. The majority of phosphorylation sites found by MS were located outside of structural protein domains (97%) but were mostly located in regions of intrinsic sequence disorder (86%). 368 phosphorylation sites were located in long regions of disorder (over 40 amino acids long), and 94% of proteins contained at least one such long region of disorder. In addition, we found that 58 phosphorylation sites in this data set occur in 14-3-3 binding consensus motifs, linear motifs that are associated with unstructured regions in proteins. These results demonstrate that in this data set protein phosphorylation is significantly depleted in protein domains and significantly enriched in disordered protein sequences and that enrichment of intrinsic sequence disorder may be a common feature of phosphoproteomes. This supports the hypothesis that disordered regions in proteins allow kinases, phosphatases, and phosphorylation-dependent binding proteins to gain access to target sequences to regulate local protein conformation and activity.     

Proteomic analysis of bovine conceptus fluids during early pregnancy

A proteomic analysis of bovine amniotic and allantoic fluids collected around Day 45 of gestation was performed using gel-based and LC-based MS workflows. A depletion/enrichment protocol using ultrafiltration under denaturing and reducing conditions produced an enriched fraction containing protein species predominantly between 5 and 50 kDa molecular weight. The analyses of conceptus fluid proteins were performed using two strategies; first, 2-DE coupled with MALDI-TOF-MS/MS and LC-ESI-MS/MS analysis of individual protein spots and second, a global protein snapshot of the enriched 5-50 kDa protein fraction by LC-ESI-MS/MS and LC-MALDI-TOF-MS/MS. Allocation of bovine specific protein identities was achieved by searching the Interactive Bovine In Silico SNP (IBISS) and NCBInr protein sequence databases resulting in the confident PMF identification and MS/MS confirmation of >200 2-DE generated allantoic fluids protein spots (74 individual protein species identified) and the MS/MS peptide identification of 105 LC-ESI-MS/MS generated protein identities. In total, the identity of 139 individual protein species from allantoic fluids was confirmed with peptide sequence probability MOWSE scores at the p<0.05 level or better. The comparison of bovine Day 45 amniotic and allantoic fluids protein profiles revealed differences between these two conceptus fluids in early pregnancy.     

Phosphoproteome analysis of the pathogenic bacterium Helicobacter pylori reveals over-representation of tyrosine phosphorylation and multiply phosphorylated proteins

Increasing evidence shows that protein phosphorylation on serine (Ser), threonine (Thr) and tyrosine (Tyr) residues is a major regulatory post-translational modification in the bacteria. To reveal the phosphorylation state in the Gram-negative pathogenic bacterium Helicobacter pylori, we carried out a global and site-specific phosphoproteomic analysis based on TiO(2) -phosphopeptide enrichment and high-accuracy LC-MS/MS determination. Eighty-two phosphopeptides from 67 proteins were identified with 126 phosphorylation sites, among which 79 class I sites were determined to have a distribution of 42.8:38.7:18.5% for the Ser/Thr/Tyr phosphorylation, respectively. The H. pylori phosphoproteome is characterized by comparably big size, high ratio of Tyr phosphorylation, high abundance of multiple phosphorylation sites in individual phosphopeptides and over-representation of membrane proteins. An interaction network covering 28 phosphoproteins was constructed with a total of 163 proteins centering on the major H. pylori virulence factor VacA, indicating that protein phosphorylation in H. pylori may be delicately controlled to regulate many aspects of the metabolic pathways and bacterial virulence.     

Qualitative and quantitative analyses of protein phosphorylation in naive and stimulated mouse synaptosomal preparations

Activity-dependent protein phosphorylation is a highly dynamic yet tightly regulated process essential for cellular signaling. Although recognized as critical for neuronal functions, the extent and stoichiometry of phosphorylation in brain cells remain undetermined. In this study, we resolved activity-dependent changes in phosphorylation stoichiometry at specific sites in distinct subcellular compartments of brain cells. Following highly sensitive phosphopeptide enrichment using immobilized metal affinity chromatography and mass spectrometry, we isolated and identified 974 unique phosphorylation sites on 499 proteins, many of which are novel. To further explore the significance of specific phosphorylation sites, we used isobaric peptide labels and determined the absolute quantity of both phosphorylated and non-phosphorylated peptides of candidate phosphoproteins and estimated phosphorylation stoichiometry. The analyses of phosphorylation dynamics using differentially stimulated synaptic terminal preparations revealed activity-dependent changes in phosphorylation stoichiometry of target proteins. Using this method, we were able to differentiate between distinct isoforms of Ca2+/calmodulin-dependent protein kinase (CaMKII) and identify a novel activity-regulated phosphorylation site on the glutamate receptor subunit GluR1. Together these data illustrate that mass spectrometry-based methods can be used to determine activity-dependent changes in phosphorylation stoichiometry on candidate phosphopeptides following large scale phosphoproteome analysis of brain tissue.     

Comprehensive identification of phosphorylation sites in postsynaptic density preparations

In the mammalian central nervous system, the structure known as the postsynaptic density (PSD) is a dense complex of proteins whose function is to detect and respond to neurotransmitter released from presynaptic axon terminals. Regulation of protein phosphorylation in this molecular machinery is critical to the activity of its components, which include neurotransmitter receptors, kinases/phosphatases, scaffolding molecules, and proteins regulating cytoskeletal structure. To characterize the phosphorylation state of proteins in PSD samples, we combined strong cation exchange (SCX) chromatography with IMAC. Initially, tryptic peptides were separated by cation exchange and analyzed by reverse phase chromatography coupled to tandem mass spectrometry, which led to the identification of phosphopeptides in most SCX fractions. Because each of these individual fractions was too complex to characterize completely in single LC-MS/MS runs, we enriched for phosphopeptides by performing IMAC on each SCX fraction, yielding at least a 3-fold increase in identified phosphopeptides relative to either approach alone (SCX or IMAC). This enabled us to identify at least one site of phosphorylation on 23% (287 of 1,264) of all proteins found to be present in the postsynaptic density preparation. In total, we identified 998 unique phosphorylated peptides, mapping to 723 unique sites of phosphorylation. At least one exact site of phosphorylation was determined on 62% (621 of 998) of all phosphopeptides, and approximately 80% of identified phosphorylation sites are novel.     

Reference-facilitated phosphoproteomics: fast and reliable phosphopeptide validation by microLC-ESI-Q-TOF MS/MS

Recent advances in instrument control and enrichment procedures have enabled us to quantify large numbers of phosphoproteins and record site-specific phosphorylation events. An intriguing problem that has arisen with these advances is to accurately validate where phosphorylation events occur, if possible, in an automated manner. The problem is difficult because MS/MS spectra of phosphopeptides are generally more complicated than those of unmodified peptides. For large scale studies, the problem is even more evident because phosphorylation sites are based on single peptide identifications in contrast to protein identifications where at least two peptides from the same protein are required for identification. To address this problem we have developed an integrated strategy that increases the reliability and ease for phosphopeptide validation. We have developed an off-line titanium dioxide (TiO(2)) selective phosphopeptide enrichment procedure for crude cell lysates. Following enrichment, half of the phosphopeptide fractionated sample is enzymatically dephosphorylated, after which both samples are subjected to LC-MS/MS. From the resulting MS/MS analyses, the dephosphorylated peptide is used as a reference spectrum against the original phosphopeptide spectrum, in effect generating two peptide spectra for the same amino acid sequence, thereby enhancing the probability of a correct identification. The integrated procedure is summarized as follows: 1) enrichment for phosphopeptides by TiO(2) chromatography, 2) dephosphorylation of half the sample, 3) LC-MS/MS-based analysis of phosphopeptides and corresponding dephosphorylated peptides, 4) comparison of peptide elution profiles before and after dephosphorylation to confirm phosphorylation, and 5) comparison of MS/MS spectra before and after dephosphorylation to validate the phosphopeptide and its phosphorylation site. This phosphopeptide identification represents a major improvement as compared with identifications based only on single MS/MS spectra and probability-based database searches. We investigated an applicability of this method to crude cell lysates and demonstrate its application on the large scale analysis of phosphorylation sites in differentiating mouse myoblast cells.     

Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism

Most regulatory pathways are governed by the reversible phosphorylation of proteins. Recent developments in mass spectrometry-based technology allow the large-scale analysis of protein phosphorylation. Here, we show the application of immobilized metal affinity chromatography to purify phosphopeptides from Arabidopsis extracts. Phosphopeptide sequences were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS/MS). A total of 79 unique phosphorylation sites were determined in 22 phosphoproteins with a putative role in RNA metabolism, including splicing of mRNAs. Among these phosphoproteins, 12 Ser/Arg-rich (SR) splicing factors were identified. A conserved phosphorylation site was found in most of the phosphoproteins, including the SR proteins, suggesting that these proteins are targeted by the same or a highly related protein kinase. To test this hypothesis, Arabidopsis SR protein-specific kinase 4 (SRPK4) that was initially identified as an interactor of SR proteins was tested for its ability to phosphorylate the SR protein RSp31. In vitro kinase assays showed that all in vivo phosphorylation sites of RSp31 were targeted by SRPK4. These data suggest that the plant mRNA splicing machinery is a major target of phosphorylation and that a considerable number of proteins involved in RNA metabolism may be targeted by SRPKs.     

Gel-based and gel-free identification of proteins and phosphopeptides during egg-to-larva transition in polychaete Neanthes arenaceodentata

The polychaete Neanthes arenaceodentata- is cosmopolitan in distribution-, has been used as a laboratory test animal. Life history of this species has several unique features; the female dies after spawning and the male incubates the fertilized eggs through the 21-segmented stage. The larvae leave the tube and commence feeding. Changes in protein abundance and phosphorylation were examined during early development of N. arenaceodentata. A gel-based approach and gel-free enrichment of phosphopeptides coupled with mass spectrometry were used to identify proteins and phosphopeptides in fertilized ova and larval stages. Patterns of proteins and phosphoproteins changed from fertilized ova to larval stages. Twelve proteins occurred in phosphorylated form and nine as stage specific proteins. Cytoskeletal proteins have exhibited differential phosphorylation from ova to larval stages; whereas, other proteins exhibited stage-specific phosphorylation patterns. Ten phosphopeptides were identified that showed phosphorylation sites on serine or threonine residues. Sixty percent of the identified proteins were related to structural reorganization and others with protein synthesis, stress response and attachment. The abundance and distribution of two cytoskeleton proteins were examined further by 2-DE Western blot analysis. This is the first report on changes in protein expression and phosphorylation sites at Thr/Ser in early development of N. arenaceodentata. The 2-DE proteome maps and identified phosphoproteins contributes toward understanding the state of fertilized ova and early larval stages and serves as a basis for further studies on proteomics changes under different developmental conditions in this and other polychaete species.     

Large-scale analysis of phosphorylated proteins in maize leaf

Phosphorylation is an ubiquitous regulatory mechanism governing the activity, subcellular localization, and intermolecular interactions of proteins. To identify a broad range of phosphoproteins from Zea mays, we enriched phosphopeptides from Zea mays leaves using titanium dioxide microcolumns and then extensively fractionated and identified the phosphopeptides by mass spectrometry. A total of 165 unique phosphorylation sites with a putative role in biological processes were identified in 125 phosphoproteins. Most of these proteins are involved in metabolism, including carbohydrate and protein metabolism. We identified novel phosphorylation sites on translation initiation factors, splicing factors, nucleolar RNA helicases, and chromatin-remodeling proteins such as histone deacetylases. Intriguingly, we also identified phosphorylation sites on several proteins associated with photosynthesis, and we speculate that these sites may be involved in carbohydrate metabolism or electron transport. Among these phosphoproteins, phosphoenolpyruvate carboxylase and NADH: nitrate reductase (NR) which catalyzes the rate-limiting and regulated step in the pathway of inorganic nitrogen assimilation were identified. A conserved phosphorylation site was found in the cytochrome b5 heme-binding domain of NADH: nitrate reductase, suggesting that NADH: nitrate reductase is phosphorylated by the same protein kinase or highly related kinases. These data demonstrate that the pathways that regulate diverse processes in plants are major targets of phosphorylation.     

The serine/threonine/tyrosine phosphoproteome of the model bacterium Bacillus subtilis

Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is well established as a key regulatory posttranslational modification in eukaryotes, but little is known about its extent and function in prokaryotes. Although protein kinases and phosphatases have been predicted and identified in a variety of bacterial species, classical biochemical approaches have so far revealed only a few substrate proteins and even fewer phosphorylation sites. Bacillus subtilis is a model Gram-positive bacterium in which two-dimensional electrophoresis-based studies suggest that the Ser/Thr/Tyr phosphorylation should be present on more than a hundred proteins. However, so far only 16 phosphorylation sites on eight of its proteins have been determined, mostly in in vitro studies. Here we performed a global, gel-free, and site-specific analysis of the B. subtilis phosphoproteome using high accuracy mass spectrometry in combination with biochemical enrichment of phosphopeptides from digested cell lysates. We identified 103 unique phosphopeptides from 78 B. subtilis proteins and determined 78 phosphorylation sites: 54 on serine, 16 on threonine, and eight on tyrosine. Detected phosphoproteins are involved in a wide variety of metabolic processes but are enriched in carbohydrate metabolism. We report phosphorylation sites on almost all glycolytic and tricarboxylic acid cycle enzymes, several kinases, and members of the phosphoenolpyruvate-dependent phosphotransferase system. This significantly enlarged number of bacterial proteins known to be phosphorylated on Ser/Thr/Tyr residues strongly supports the emerging view that protein phosphorylation is a general and fundamental regulatory process, not restricted only to eukaryotes, and opens the way for its detailed functional analysis in bacteria.