Phosphorylation site analysis of the anti-inflammatory and mRNA-destabilizing protein tristetraprolin

Tristetraprolin (TTP) is a member of the CCCH zinc finger proteins and is an anti-inflammatory protein. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. TTP binds to mRNA AU-rich elements with high affinity for UUAUUUAUU nucleotides and causes destabilization of those mRNA molecules. TTP is phosphorylated extensively in vivo and is a substrate for multiple protein kinases in vitro. A number of approaches have been used to identify its phosphorylation sites. This article highlights the recent progress and different approaches utilized for the identification of phosphorylation sites in mammalian TTP. Important but limited results are obtained using traditional methods, including in vivo labeling, site-directed mutagenesis, phosphopeptide mapping and protein sequencing. Mass spectrometry (MS), including MALDI/MS, MALDI/MS/MS, liquid chromatography/MS/MS, immobilized metal ion affinity chromatography (IMAC)/MALDI/MS/MS and multidimensional protein identification technology has led the way in identifying TTP phosphorylation sites. The combination of these approaches has identified multiple phosphorylation sites in mammalian TTP, some of which are predicted by motif scanning to be phosphorylated by several protein kinases. This information should provide the molecular basis for future investigation of TTP's regulatory functions in controlling proinflammatory cytokines.     

Phosphorylation site analysis of the anti-inflammatory and mRNA-destabilizing protein tristetraprolin

Tristetraprolin (TTP) is a member of the CCCH zinc finger proteins and is an anti-inflammatory protein. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. TTP binds to mRNA AU-rich elements with high affinity for UUAUUUAUU nucleotides and causes destabilization of those mRNA molecules. TTP is phosphorylated extensively in vivo and is a substrate for multiple protein kinases in vitro. A number of approaches have been used to identify its phosphorylation sites. This article highlights the recent progress and different approaches utilized for the identification of phosphorylation sites in mammalian TTP. Important but limited results are obtained using traditional methods, including in vivo labeling, site-directed mutagenesis, phosphopeptide mapping and protein sequencing. Mass spectrometry (MS), including MALDI/MS, MALDI/MS/MS, liquid chromatography/MS/MS, immobilized metal ion affinity chromatography (IMAC)/MALDI/MS/MS and multidimensional protein identification technology has led the way in identifying TTP phosphorylation sites. The combination of these approaches has identified multiple phosphorylation sites in mammalian TTP, some of which are predicted by motif scanning to be phosphorylated by several protein kinases. This information should provide the molecular basis for future investigation of TTP’s regulatory functions in controlling proinflammatory cytokines.     

生物质谱结合IMAC亲和提取和磷酸酶水解分析蛋白质磷酸化修饰

蛋白质磷酸化是生物体内非常重要的翻译后修饰方式 ,对磷酸化蛋白质的分析及磷酸化位点的确定有助于理解与其相关的生物功能。基质辅助激光解吸 /电离飞行时间质谱和电喷雾 四极杆 飞行时间质谱这两种生物质谱仪在蛋白质鉴定和翻译后修饰分析中发挥着重要作用。固相金属亲和色谱可选择性亲和提取肽混合物中的磷酸肽 ,结合磷酸酶水解实验和基质辅助激光解吸 /电离飞行时间质谱分析可确定肽混合物中的磷酸肽 ,最后用电喷雾 四极杆 飞行时间串联质谱分析磷酸肽的序列 ,结合数据库检索确定磷酸化位点。     

Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry

Global analyses of protein phosphorylation require specific enrichment methods because of the typically low abundance of phosphoproteins. To date, immobilized metal ion affinity chromatography (IMAC) for phosphopeptides has shown great promise for large-scale studies, but has a reputation for poor specificity. We investigated the potential of IMAC in combination with capillary liquid chromatography coupled to tandem mass spectrometry for the identification of plasma membrane phosphoproteins of Arabidopsis. Without chemical modification of peptides, over 75% pure phosphopeptides were isolated from plasma membrane digests and detected and sequenced by mass spectrometry. We present a scheme for two-dimensional peptide separation using strong anion exchange chromatography prior to IMAC that both decreases the complexity of IMAC-purified phosphopeptides and yields a far greater coverage of monophosphorylated peptides. Among the identified sequences, six originated from different isoforms of the plasma membrane H(+)-ATPase and defined two previously unknown phosphorylation sites at the regulatory C terminus. The potential for large-scale identification of phosphorylation sites on plasma membrane proteins will have wide-ranging implications for research in signal transduction, cell-cell communication, and membrane transport processes.     

Phosphoproteomic analysis of synaptosomes from human cerebral cortex

Protein phosphorylation is a crucial post-translational modification mechanism in the regulation of synaptic organization and function. Here, we analyzed synaptosome fractions from human cerebral cortex obtained during therapeutic surgery. To minimize changes in the phosphorylation state of proteins, the tissue was homogenized within two minutes of excision. Synaptosomal proteins were digested with trypsin and phosphopeptides were isolated by immobilized metal affinity chromatography and analyzed by liquid chromatography and tandem mass spectrometry. The method allowed the detection of residues on synaptic proteins that were presumably phosphorylated in the intact cell, including synapsin 1, syntaxin 1, and SNIP, PSD-93, NCAM, GABA-B receptor, chaperone molecules, and protein kinases. Some of the residues identified are the same or homologous to sites that had been previously described to be phosphorylated in mammals whereas others appear to be novel sites which, to our knowledge, have not been reported previously. The study shows that new phosphoproteomic strategies can be used to analyze subcellular fractions from small amounts of tissue for the identification of phosphorylated residues for research and potentially for diagnostic purposes.     

Phosphoproteomics as a tool to unravel plant regulatory mechanisms

Reversible phosphorylation of proteins plays a key role in many regulatory processes that lie at the basis of life. With plants, much research has focused on protein kinases that are involved in the adaptation to different stress conditions, such as pathogen attack and cold. However, the substrates of these kinases are mostly unknown. With the recent advances in phosphoproteomic techniques, the large-scale identification of kinase substrates, including their phosphorylation sites, is finally possible. Studies in mainly non-plant systems have demonstrated the high potential of this method by uncovering numerous novel phosphorylation events. In this minireview, we focus on recent developments in the field of phosphoproteomics that are based on phosphopeptide isolation from complex mixtures by immobilized metal-affinity chromatography coupled to sequence identification by mass spectrometry. Combination of these methods with labelling techniques now allows quantitative analysis of phosphorylation between different samples. We discuss the potential of this technology to uncover entire phosphoproteomes and signalling pathways in plants in the future.     

Global phosphoproteome of HT-29 human colon adenocarcinoma cells

Phosphorylation events in cellular signaling cascades triggered by a variety of cellular stimuli modulate protein function, leading to diverse cellular outcomes including cell division, growth, death, and differentiation. Abnormal regulation of protein phosphorylation due to mutation or overexpression of signaling proteins often results in various disease states. We provide here a list of protein phosphorylation sites identified from HT-29 human colon adenocarcinoma cell line by immobilized metal affinity chromatography (IMAC) combined with liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis. In this study, proteins extracted from HT-29 whole cell lysates were digested with trypsin and carboxylate groups on the resulting peptides were converted to methyl esters. Derivatized phosphorylated peptides were enriched using Fe(3+)-chelated metal affinity resin. Phosphopeptides retained by IMAC were separated by high performance liquid chromatography (HPLC) and analyzed by electrospray ionization-quadrupole-time-of-flight (ESI-Q-TOF) mass spectrometry. We identified 238 phosphorylation sites, 213 of which could be conclusively localized to a single residue, from 116 proteins by searching MS/MS spectra against the human protein database using MASCOT. Peptide identification and phosphorylation site assignment were confirmed by manual inspection of the MS/MS spectra. Many of the phosphorylation sites identified in our results have not been described previously in the scientific literature. We attempted to ascribe functionality to the sites identified in this work by searching for potential kinase motifs with Scansite (http://scansite.mit.edu) and obtaining information on kinase substrate selectivity from Pattern Explorer (http://scansite.mit.edu/pe). The list of protein phosphorylation sites identified in the present experiment provides broad information on phosphorylated proteins under normal (asynchronous) cell culture conditions. Sites identified in this study may be utilized as surrogate bio-markers to assess the activity of selected kinases and signaling pathways from different cell states and exogenous stimuli.     

Profiling the Trypanosoma cruzi phosphoproteome

Protein phosphorylation is a reversible post-translational modification essential for the regulation of several signal transduction pathways and biological processes in the living cell. Therefore, the identification of protein phosphorylation sites is crucial to understand cell signaling control at the molecular level. Based on mass spectrometry, recent studies have reported the large-scale mapping of phosphorylation sites in various eukaryotes and prokaryotes. However, little is known about the impact of phosphorylation in protozoan parasites. To in depth characterize the phosphoproteome of Trypanosoma cruzi, a parasite of the Kinetoplastida class, protein samples from cells at different phases of the metacyclogenesis--differentiation process of the parasites from non-infective epimastigotes to infective metacyclic trypomastigotes--were enriched for phosphopeptides using TiO(2) chromatography and analyzed on an LTQ-Orbitrap mass spectrometer. In total, 1,671 proteins were identified, including 753 phosphoproteins, containing a total of 2,572 phosphorylation sites. The distribution of phosphorylated residues was 2,162 (84.1%) on serine, 384 (14.9%) on threonine and 26 (1.0%) on tyrosine. Here, we also report several consensus phosphorylation sequence motifs and as some of these conserved groups have enriched biological functions, we can infer the regulation by protein kinases of this functions. To our knowledge, our phosphoproteome is the most comprehensive dataset identified until now for Kinetoplastida species. Here we also were able to extract biological information and infer groups of sites phosphorylated by the same protein kinase. To make our data accessible to the scientific community, we uploaded our study to the data repositories PHOSIDA, Proteome Commons and TriTrypDB enabling researchers to access information about the phosphorylation sites identified here.     

磷酸化蛋白质组研究中的富集策略

蛋白质的磷酸化与去磷酸化过程,调控着包括信号转换、基因表达、细胞周期等诸多细胞过程。因此,对蛋白质磷酸化修饰的分析是蛋白质组研究中的重要内容。但由于磷酸化蛋白的丰度较低,难以用质谱直接检测。为了解决这个问题,改善质谱对磷酸肽的信号响应,需要对磷酸化蛋白质或磷酸肽进行富集。目前主要的富集方法包括免疫沉淀、固相金属离子亲和色谱、金属氧化物/氢氧化物亲和色谱等。     

Identification of phosphoproteins and their phosphorylation sites in the WEHI-231 B lymphoma cell line

A major goal of the Alliance for Cellular Signaling is to elaborate the components of signal transduction networks in model cell systems, including murine B lymphocytes. Due to the importance of protein phosphorylation in many aspects of cell signaling, the initial efforts have focused on the identification of phosphorylated proteins. In order to identify serine- and threonine-phosphorylated proteins on a proteome-wide basis, WEHI-231 cells were treated with calyculin A, a serine/threonine phosphatase inhibitor, to induce high levels of protein phosphorylation. Proteins were extracted from whole-cell lysates and digested with trypsin. Phosphorylated peptides were then enriched using immobilized metal affinity chromatography and identified by liquid chromatography-tandem mass spectrometry. A total of 107 proteins and 193 phosphorylation sites were identified using these methods. Forty-two of these proteins have been reported to be phosphorylated, but only some of them have been detected in B cells. Fifty-four of the identified proteins were not previously known to be phosphorylated. The remaining 11 phosphoproteins have previously only been characterized as novel cDNA or genomic sequences. Many of the identified proteins were phosphorylated at multiple sites. The proteins identified in this study significantly expand the repertoire of proteins known to be phosphorylated in B cells. The number of newly identified phosphoproteins indicates that B cell signaling pathways utilizing protein phosphorylation are likely to be more complex than previously appreciated.     

Identification of in vivo phosphorylation sites of MLK3 by mass spectrometry and phosphopeptide mapping

MLK3 is a serine/threonine protein kinase that functions as an upstream activator of the JNK pathway. Previous work has suggested that MLK3 is a multiphosphorylated protein. In this study, mass spectrometry coupled with comparative phosphopeptide mapping was used to directly characterize MLK3 in vivo phosphorylation sites. Various types of mass spectrometry were used to analyze MLK3 tryptic peptides separated by C18 reverse-phase HPLC, leading to the identification of Ser(524), Ser(654), Ser(705), Ser(740), Ser(758), Ser(770), Ser(793), and a site found on peptide Ser(11)-Arg(37) within a Gly-rich region as MLK3 phosphorylation sites. Additionally, porous graphitic carbon chromatography successfully retained and resolved phosphopeptides that had eluted along with nonvolatile salts and buffers in the flowthrough fractions from the C18 column. Following resolution by PGC chromatography, MALDI-MS in conjunction with alkaline phosphatase treatment identified Ser(555), Ser(556), Ser(724), and Ser(727) as sites of phosphorylation on MLK3. A proline residue immediately follows 7 of the 11 unambiguously identified phosphorylation sites, suggesting that MLK3 may be a target of proline-directed kinases. Finally, two-dimensional phosphopeptide mapping confirmed that phosphorylation of Ser(555) and Ser(556) of MLK3 is induced by the activated small GTPase Cdc42.     

Quantitative phosphoproteome analysis of lysophosphatidic acid induced chemotaxis applying dual-step (18)O labeling coupled with immobilized metal-ion affinity chromatography

Reversible protein phosphorylation is a central cellular regulatory mechanism in modulating protein activity and propagating signals within cellular pathways and networks. Development of more effective methods for the simultaneous identification of phosphorylation sites and quantification of temporal changes in protein phosphorylation could provide important insights into molecular signaling mechanisms in various cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its application for comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) gradient stimulation. The approach combines trypsin-catalyzed (16)O/ (18)O labeling plus (16)O/ (18)O-methanol esterification for quantitation, a macro-immobilized metal-ion affinity chromatography trap for phosphopeptide enrichment, and LC-MS/MS analysis. LC separation and MS/MS are followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer. A variety of phosphorylated proteins were identified and quantified including receptors, kinases, proteins associated with small GTPases, and cytoskeleton proteins. A number of hypothetical proteins were also identified as differentially expressed followed by LPA stimulation, and we have shown evidence of pseudopodia subcellular localization of one of these candidate proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with LPA gradient sensing and cell chemotaxis.     

Large-scale phosphotyrosine proteomic profiling of rat renal collecting duct epithelium reveals predominance of proteins involved in cell polarity determination

Although extensive phosphoproteomic information is available for renal epithelial cells, previous emphasis has been on phosphorylation of serines and threonines with little focus on tyrosine phosphorylation. Here we have carried out large-scale identification of phosphotyrosine sites in pervanadate-treated native inner medullary collecting ducts of rat, with a view towards identification of physiological processes in epithelial cells that are potentially regulated by tyrosine phosphorylation. The method combined antibody-based affinity purification of tyrosine phosphorylated peptides coupled with immobilized metal ion chromatography to enrich tyrosine phosphopeptides, which were identified by LC-MS/MS. A total of 418 unique tyrosine phosphorylation sites in 273 proteins were identified. A large fraction of these sites have not been previously reported on standard phosphoproteomic databases. All results are accessible via an online database: http://helixweb.nih.gov/ESBL/Database/iPY/. Analysis of surrounding sequences revealed four overrepresented motifs: [D/E]xxY*, Y*xxP, DY*, and Y*E, where the asterisk symbol indicates the site of phosphorylation. These motifs plus contextual information, integrated using the NetworKIN tool, suggest that the protein tyrosine kinases involved include members of the insulin- and ephrin-receptor kinase families. Analysis of the gene ontology (GO) terms and KEGG pathways whose protein elements are overrepresented in our data set point to structures involved in epithelial cell-cell and cell-matrix interactions ("adherens junction," "tight junction," and "focal adhesion") and to components of the actin cytoskeleton as major sites of tyrosine phosphorylation in these cells. In general, these findings mesh well with evidence that tyrosine phosphorylation plays a key role in epithelial polarity determination.     

In vivo phosphorylation of human erythrocyte spectrin occurs in a sequential manner

Spectrin is the major component of the erythrocyte membrane skeleton and exists as a 526 kDa alphabeta heterodimer. The 246 kDa beta-chain of human spectrin is phosphorylated near the C-terminus, but the exact phosphorylation sites are unknown and the role of this phosphorylation is not fully characterized. In this study, we produced a monoclonal antibody, Sp316, capable of recognizing the C-terminal region of beta-spectrin regardless of its phosphorylation state and used it to purify the phosphorylated region after 2-nitro-5-thiocyanobenzoic acid cleavage of spectrin. Two-dimensional gels, mass spectrometry, and reversed-phase high-performance liquid chromatography were used to characterize these phosphorylation states. Only about 1.5% of spectrin isolated from fresh blood is unphosphorylated, about 9% has more than four phosphates per molecule, and the majority of the protein has one to four phosphates per molecule. A total of six phosphorylation sites were identified by tandem mass spectrometry. Quantitative analysis of the phosphorylation states by reversed-phase high-performance liquid chromatography revealed that phosphorylation of beta-spectrin occurs in a sequential manner where each specific site is completely phosphorylated before the next site is modified. The first phosphorylation event occurs on Ser-2114, followed by Ser-2125, Ser-2123, Ser-2128, Ser-2117, and Thr-2110. The identification of the specific phosphorylated beta-spectrin residues and the ordered sequence of phosphorylation events in vivo should provide an invaluable basis for further studies of the role of these posttranslational modifications in spectrin function in situ.     

Identification of phosphorylation sites in the polo-like kinases Plx1 and Plk1 by a novel strategy based on element and electrospray high resolution mass spectrometry

A novel strategy for the determination of protein phosphorylation sites is described and applied to the polo-like kinases Plx1 (Xenopus laevis) and Plk1 (Homo sapiens). The strategy comprises the sequential application of the following techniques: proteolytic digestion, capillary liquid chromatography (LC)-inductively coupled plasma mass spectrometry with phosphorus detection, capillary LC-electrospray mass spectrometry and electrospray tandem mass spectrometry. In this approach, phosphopeptides are generated, their elution time in capillary LC is determined, candidate phosphopeptides at the corresponding elution times are identified, and positive identification and sequencing of phosphopeptides is performed in the last step of the analysis. Using this technique, Ser25/26, Ser326, and Ser340 were identified as phosphorylation sites in recombinant Plx1, and Ser340 was identified as the major phosphorylation site in a kinase-dead mutant of Plx1 expressed in okadaic acid-treated Sf9 insect cells. A site corresponding to Ser326 in Plx1 was also shown to be phosphorylated in the human polo-like kinase Plk1 (Ser335). Element mass spectrometry with phosphorus detection provides a quantitative phosphorylation profile of all phosphorylation sites accessible by LC.     

Protein phosphorylation and expression profiling by Yin-yang multidimensional liquid chromatography (Yin-yang MDLC) mass spectrometry

A system which consisted of multidimensional liquid chromatography (Yin-yang MDLC) coupled with mass spectrometry was used for the identification of peptides and phosphopeptides. The multidimensional liquid chromatography combines the strong-cation exchange (SCX), strong-anion exchange (SAX), and reverse-phase methods for the separation. Protein digests were first loaded on an SCX column. The flow-through peptides from SCX were collected and further loaded on an SAX column. Both columns were eluted by offline pH steps, and the collected fractions were identified by reverse-phase liquid chromatography tandem mass spectrometry. Comprehensive peptide identification was achieved by the Yin-yang MDLC-MS/MS for a 1 mg mouse liver. In total, 14 105 unique peptides were identified with high confidence, including 13 256 unmodified peptides and 849 phosphopeptides with 809 phosphorylated sites. The SCX and SAX in the Yin-Yang system displayed complementary features of binding and separation for peptides. When coupled with reverse-phase liquid chromatography mass spectrometry, the SAX-based method can detect more extremely acidic (pI < 4.0) and phosphorylated peptides, while the SCX-based method detects more relatively basic peptides (pI > 4.0). In total, 134 groups of phosphorylated peptide isoforms were obtained, with common peptide sequences but different phosphorylated states. This unbiased profiling of protein expression and phosphorylation provides a powerful approach to probe protein dynamics, without using any prefractionation and chemical derivation.     

Proteomic analysis of in vivo phosphorylated synaptic proteins

In the nervous system, protein phosphorylation is an essential feature of synaptic function. Although protein phosphorylation is known to be important for many synaptic processes and in disease, little is known about global phosphorylation of synaptic proteins. Heterogeneity and low abundance make protein phosphorylation analysis difficult, particularly for mammalian tissue samples. Using a new approach, combining both protein and peptide immobilized metal affinity chromatography and mass spectrometry data acquisition strategies, we have produced the first large scale map of the mouse synapse phosphoproteome. We report over 650 phosphorylation events corresponding to 331 sites (289 have been unambiguously assigned), 92% of which are novel. These represent 79 proteins, half of which are novel phosphoproteins, and include several highly phosphorylated proteins such as MAP1B (33 sites) and Bassoon (30 sites). An additional 149 candidate phosphoproteins were identified by profiling the composition of the protein immobilized metal affinity chromatography enrichment. All major synaptic protein classes were observed, including components of important pre- and postsynaptic complexes as well as low abundance signaling proteins. Bioinformatic and in vitro phosphorylation assays of peptide arrays suggest that a small number of kinases phosphorylate many proteins and that each substrate is phosphorylated by many kinases. These data substantially increase existing knowledge of synapse protein phosphorylation and support a model where the synapse phosphoproteome is functionally organized into a highly interconnected signaling network.