Large-scale Phosphoproteomic Analysis Research Articles

Arabidopsis hypocotyl growth in darkness requires the phosphorylation of a microtubule-associated protein.

Rapid hypocotyl elongation allows buried seedlings to emerge, where light triggers de-etiolation and inhibits hypocotyl growth mainly by photoreceptors. Phosphorylation/dephosphorylation events regulate many aspects of plant development. Only recently we have begun to uncover the earliest phospho-signaling responders to light. Here, we reported a large-scale phosphoproteomic analysis and identified 20 proteins that changed their phosphorylation pattern following a 20 min light pulse compared to darkness. Microtubule-associated proteins were highly overrepresented in this group. Among them, we studied CIP7 (COP1-INTERACTING-PROTEIN 7), which presented microtubule (MT) localization in contrast to the previous description. An isoform of CIP7 phosphorylated at Serine915 was detected in etiolated seedlings but was undetectable after a light pulse in the presence of photoreceptors, while CIP7 transcript expression decays with long light exposure. The short hypocotyl phenotype and rearrangement of MTs in etiolated cip7 mutants are complemented by CIP7-YFP and the phospho-mimetic CIP7S915D-YFP, but not the phospho-null CIP7S915A-YFP suggesting that the phosphorylated S915CIP7 isoform promotes hypocotyl elongation through MT reorganization in darkness. Our evidence on Serine915 of CIP7 unveils phospho-regulation of MT-based processes during skotomorphogenic hypocotyl growth.

Large-scale phosphoproteome analysis in wheat seedling leaves provides evidence for extensive phosphorylation of regulatory proteins during CWMV infection

BackgroundChinese wheat mosaic virus (CWMV) often causes severe damage to wheat (Triticum aestivum L.) growth and yield. It is well known that a successful infection in plants depends on a complex interaction between the host plant and the pathogen. Post-translational modification (PTM) of proteins is considered to be one of the main processes that decides the outcome of the plant-pathogen arms race during this interaction. Although numerous studies have investigated PTM in various organisms, there has been no large-scale phosphoproteomic analysis of virus-infected wheat plants. We therefore aimed to investigate the CWMV infection-induced phosphoproteomics changes in wheat by high-resolution liquid chromatography-tandem mass spectroscopy (LC–MS/MS) using affinity-enriched peptides followed by comprehensive bioinformatics analysis.ResultsThrough this study, a total of 4095 phosphorylation sites have been identified in 1968 proteins, and 11.6% of the phosphorylated proteins exhibited significant changes (PSPCs) in their phosphorylation levels upon CWMV infection. The result of Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that most of the PSPCs were associated with photosynthesis, plant-pathogen interactions, and MAPK signaling pathways. The protein–protein interaction (PPI) network analysis result showed that these PSPCs were mainly participated in the regulation of biosynthesis and metabolism, protein kinase activities, and transcription factors. Furthermore, the phosphorylation levels of TaChi1 and TaP5CS, two plant immunity-related enzymes, were significantly changed upon CWMV infection, resulting in a significant decrease in CWMV accumulation in the infected plants.ConclusionsOur results indicate that phosphorylation modification of protein plays a critical role in wheat resistance to CWMV infection. Upon CWMV infection, wheat plants will regulate the levels of extra- and intra-cellular signals and modifications of enzyme activities via protein phosphorylation. This novel information about the strategies used by wheat to resist CWMV infection will help researchers to breed new CWMV-resistant cultivars and to better understand the arms race between wheat and CWMV.

Phosphoproteomics analysis of the effect of target of rapamycin kinase inhibition on Cucumis sativus in response to Podosphaera xanthii

Target of rapamycin (TOR) kinase is a conserved sensor of cell growth in yeasts, plants, and mammals. Despite the extensive research on the TOR complex in various biological processes, large-scale phosphoproteomics analysis of TOR phosphorylation events upon environmental stress are scarce. Powdery mildew caused by Podosphaera xanthii poses a major threat to the quality and yield of cucumber (Cucumis sativus L.). Previous studies concluded that TOR participated in abiotic and biotic stress responses. Hence, studying the underlying mechanism of TOR-P. xanthii infection is particularly important. In this study, we performed a quantitative phosphoproteomics studies of Cucumis against P. xanthii attack under AZD-8055 (TOR inhibitor) pretreatment. A total of 3384 phosphopeptides were identified from the 1699 phosphoproteins. The Motif-X analysis showed high sensitivity and specificity of serine sites under AZD-8055-treatment or P. xanthii stress, and TOR exhibited a unique preference for proline at +1 position and glycine at −1 position to enhance the phosphorylation response to P. xanthii. The functional analysis suggested that the unique responses were attributed to proteins related to plant hormone signaling, mitogen-activated protein kinase cascade signaling, phosphatidylinositol signaling system, and circadian rhythm; and calcium signaling– and defense response–related proteins. Our results provided rich resources for understanding the molecular mechanism of how the TOR kinase controlled plant growth and stress adaptation.

Vinculin phosphorylation impairs vascular endothelial junctions promoting atherosclerosis.

Atherosclerosis preferentially develops in arterial branches and curvatures where vascular endothelium is exposed to disturbed flow. In this study, the effects of disturbed flow on the regulation of vascular endothelial phosphoproteins and their contribution to therapeutic application in atherogenesis were elucidated. Porcine models, large-scale phosphoproteomics, transgenic mice, and clinical specimens were used to discover novel site-specific phosphorylation alterations induced by disturbed flow in endothelial cells (ECs). A large-scale phosphoproteomics analysis of native endothelium from disturbed (athero-susceptible) vs. pulsatile flow (athero-resistant) regions of porcine aortas led to the identification of a novel atherosclerosis-related phosphoprotein vinculin (VCL) with disturbed flow-induced phosphorylation at serine 721 (VCLS721p). The induction of VCLS721p was mediated by G-protein-coupled receptor kinase 2 (GRK2)S29p and resulted in an inactive form of VCL with a closed conformation, leading to the VE-cadherin/catenin complex disruption to enhance endothelial permeability and atherogenesis. The generation of novel apolipoprotein E-deficient (ApoE-/-) mice overexpressing S721-non-phosphorylatable VCL mutant in ECs confirmed the critical role of VCLS721p in promoting atherosclerosis. The administration of a GRK2 inhibitor to ApoE-/- mice suppressed plaque formation by inhibiting endothelial VCLS721p. Studies on clinical specimens from patients with coronary artery disease (CAD) revealed that endothelial VCLS721p is a critical clinicopathological biomarker for atherosclerosis progression and that serum VCLS721p level is a promising biomarker for CAD diagnosis. The findings of this study indicate that endothelial VCLS721p is a valuable hemodynamic-based target for clinical assessment and treatment of vascular disorders resulting from atherosclerosis.

LIMK2 promotes the metastatic progression of triple-negative breast cancer by activating SRPK1

Triple-negative breast cancer (TNBC) is a highly metastatic breast cancer subtype and due to the lack of hormone receptors and HER2 expression, TNBC has limited therapeutic options with chemotherapy being the primary choice for systemic therapy. LIM Domain Kinase 2 (LIMK2) is a serine/threonine kinase that plays an important role in the regulation of actin filament dynamics. Here, we show that LIM domain kinase 2 (LIMK2) is overexpressed in TNBC, and short-hairpin RNA (shRNA)-mediated LIMK2 knockdown or its pharmacological inhibition blocks metastatic attributes of TNBC cells. To determine the mechanism by which LIMK2 promotes TNBC metastatic progression, we performed stable isotope labeling by amino acids in cell culture (SILAC) based unbiased large-scale phosphoproteomics analysis. This analysis identified 258 proteins whose phosphorylation was significantly reduced due to LIMK2 inhibition. Among these proteins, we identified SRSF protein kinase 1 (SRPK1), which encodes for a serine/arginine protein kinase specific for the SR (serine/arginine-rich domain) family of splicing factors. We show that LIMK2 inhibition blocked SRPK1 phosphorylation and consequentially its activity. Furthermore, similar to LIMK2, genetic inhibition of SRPK1 by shRNAs or its pharmacological inhibition blocked the metastatic attributes of TNBC cells. Moreover, the pharmacological inhibition of LIMK2 blocked metastatic progression in mice without affecting primary tumor growth. In sum, these results identified LIMK2 as a facilitator of distal TNBC metastasis and a potential target for preventing TNBC metastatic progression.

The Phosphoproteomic Response of Okra (Abelmoschus esculentus L.) Seedlings to Salt Stress

Soil salinization is a major environmental stresses that seriously threatens land use efficiency and crop yields worldwide. Although the overall response of plants to NaCl has been well studied, the contribution of protein phosphorylation to the detoxification and tolerance of NaCl in okra (Abelmoschus esculentus L.) seedlings is unclear. The molecular bases of okra seedlings’ responses to 300 mM NaCl stress are discussed in this study. Using a combination of affinity enrichment, tandem mass tag (TMT) labeling and high-performance liquid chromatography–tandem mass spectrometry analysis, a large-scale phosphoproteome analysis was performed in okra. A total of 4341 phosphorylation sites were identified on 2550 proteins, of which 3453 sites of 2268 proteins provided quantitative information. We found that 91 sites were upregulated and 307 sites were downregulated in the NaCl/control comparison group. Subsequently, we performed a systematic bioinformatics analysis including gene ontology annotation, domain annotation, subcellular localization, and Kyoto Encyclopedia of Genes and Genomes pathway annotation. The latter revealed that the differentially expressed proteins were most strongly associated with ‘photosynthesis antenna proteins’ and ‘RNA degradation’. These differentially expressed proteins probably play important roles in salt stress responses in okra. The results should help to increase our understanding of the molecular mechanisms of plant post-translational modifications in response to salt stress.

Exploring the nine realms of TOR.

The target of rapamycin (TOR) kinase regulates crucial cellular processes. Characterization of its signalling pathway in plants has lagged behind animals and yeast. A large-scale phosphoproteome and interactome analysis in Arabidopsis has now uncovered plant-specific and conserved TOR targets and interactors.

Systems-wide Analysis of Serine ADP-Ribosylation Reveals Widespread Occurrence and Site-Specific Overlap with Phosphorylation

ADP-ribosylation (ADPr) is a reversible posttranslational modification involved in a range of cellular processes. Here, we report system-wide identification of serine ADPr in human cells upon oxidative stress. High-resolution mass spectrometry and unrestricted data processing confirm that serine residues are the major target of ADPr in HeLa cells. Proteome-wide analysis identifies 3,090 serine ADPr sites, with 97% of acceptor sites modulating more than 2-fold upon oxidative stress, while treatment with the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib abrogates this induction. Serine ADPr predominantly targets nuclear proteins, while structural-predictive analyses reveal that serine ADPr preferentially targets disordered protein regions. The identified ADP-ribosylated serines significantly overlap with known phosphorylated serines, and large-scale phosphoproteomics analysis provides evidence for site-specific crosstalk between serine ADPr and phosphorylation. Collectively, we demonstrate that serine ADPr is a widespread modification and a major nuclear signaling response to oxidative stress, with a regulatory scope comparable to other extensive posttranslational modifications.

Abstract 208: Proteomic measurements of protein abundance and phosphorylation identify novel kinase-substrate relationships in ovarian cancer

Abstract As part of the Clinical Proteomic Tumor Analysis Consortium (CPTAC), we have recently published the first large-scale proteomic and phosphoproteomic analysis of high-grade serous ovarian tumors. We observed that phosphorylation status was an excellent indicator of pathway activity and could discriminate between patient survival times. This dataset covers tumor samples from 69 patients with deep phosphoproteomic depth (>20,000 phosphopeptides confidently identified). Our continuing analysis of this dataset, reported here, has revealed that the correlation between kinase protein abundance and abundance of phosphorylated target peptides is very low, indicating that kinase abundance is not a good proxy for phosphorylation status overall. However, highly correlated kinase-substrate pairs are significantly more likely to be true relationships (from existing knowledge), demonstrating that this method could be used to predict novel kinase targets in some cases. Using this approach we predicted novel kinase-target relationships and constructed a kinase activity network of ovarian cancer. To better analyze cancer-relevant pathway activity we developed a novel approach that characterizes correlation, differential abundance, and statistical interactions between components to analyze multiple omics types in the context of signaling and functional pathways. We used this approach, called the Layered Enrichment Analysis of Pathways (LEAP), to identify active pathways in molecular subtypes of ovarian cancer, contrasting observations in patients stratified for short versus long overall survival. This analysis resulted in a pre-treatment protein-based signature that is significantly predictive of overall survival. Our results show that integration of multiple omics types has the ability to inform our understanding of novel kinase-substrate interactions, and potentially identify novel interactions associated with patient survival. Note: This abstract was not presented at the meeting. Citation Format: Jason E. McDermott, Tao Liu, Samuel Payne, Vladislav Petyuk, Hui Zhang, Zhen Zhang, Daniel Chan, Richard Smith, Karin Rodland, Clinical Proteomic Tumor Analysis Consortium. Proteomic measurements of protein abundance and phosphorylation identify novel kinase-substrate relationships in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 208. doi:10.1158/1538-7445.AM2017-208

Salt-induced phosphoproteomic changes in the hypothalamic paraventricular nucleus in rats with chronic renal failure

Hypothalamic paraventricular nucleus (PVN) is a cardiovascular regulating center within the brain, which plays a critical role in high salt-induced progression of chronic renal failure (CRF). However, the phosphoproteomic changes in the PVN caused by CRF remain unclear. This study aimed to perform large-scale phosphoproteomic analysis of PVN induced by CRF and high salt intake. In this study, eight weeks post 5/6 nephrectomy (CRF model) or sham operation, Sprague–Dawley rats were fed a high-salt (4%) or normal-salt (0.4%) diet for 3weeks. TiO2 enrichment, iTRAQ labeling, and liquid chromatography tandem mass spectrometry were applied for phosphoproteomic profiling of PVN. A total of 3723 unique phosphopeptides corresponding to 1530 phosphoproteins were identified. Compared with sham group, 133 upregulated and 141 downregulated phosphopeptides were identified in CRF group during normal-salt feeding. However, with a high-salt diet, 160 phosphopeptides were upregulated and 142 downregulated in the CRF group. Gene Ontology analysis revealed that these phosphoproteins were involved in binding, catalytic, transporter, and other molecular functions. Search Tool for the Retrieval of Interacting Genes protein–protein analysis showed direct or indirect functional links among 25 differentially expressed phosphoproteins in CRF rats compared with sham group. However, 24 differentially phosphorylated proteins induced by high salt intake were functionally linked in CRF animals. The altered phosphorylation levels of dynamin 1, TPPP and Erk1/2 were validated. Phosphoproteomic changes of PVN triggered by CRF and high salt-load have been investigated. It will provide new insight into pathogenetic mechanisms of development of chronic kidney disease and salt sensitivity.

Super-SILAC mix coupled with SIM/AIMS assays for targeted verification of phosphopeptides discovered in a large-scale phosphoproteome analysis of hepatocellular carcinoma

Plentiful studies have established a close association between aberrant phosphorylation and hepatocellular carcinoma (HCC). Here, we applied a quantitative phosphoproteomics platform combining dimethylation labeling and online 3D strong cation exchange chromatography (SCX)-titanium oxide (TiO2)/RP-LTQ-Orbitrap to compare phosphoproteomes between three pairs of HCC tissues and non-tumor counterparts. This analysis yielded 7868 quantifiable phosphopeptides and numerous up- or down-regulated candidates. Increased phosphorylation of LMNA and NIPA was confirmed using specific antibodies. To expand our verification capability, we evaluated the use of LTQ-Orbitrap run in SIM/Accurate inclusion mass screening (AIMS) mode with a super-SILAC mixture as an internal standard to quantify a subset of phosphopeptide candidates in HCC tissue samples. In sample I used for discovery experiment, we successfully quantified 32 (in SIM mode) and 30 (in AIMS mode) phosphopeptides with median coefficients of variation (CVs) of 7.5% and 8.3%, respectively. When the assay was applied to other three pairs of HCC specimens for verification experiment, 40 target phosphopeptides were quantified reliably (~7.5% CV), and more than half of them were differentially expressed between tumor and adjacent non-tumor tissues. Collectively, these results indicate the feasibility of using super-SILAC mix-SIM/AIMS assays for targeted verification of phosphopeptides discovered by large-scale phosphoproteome analyses of HCC specimens. SignificanceIn this study, we developed a strategy for conducting both discovery and targeted verification of deregulated phosphoproteins in HCC tissue specimens on LTQ-Orbitrap. This strategy allowed us to generate a quantitative HCC tissue phosphoproteome dataset containing significantly deregulated phosphoproteins that represents a valuable resource for the identification of potential HCC biomarkers and/or therapeutic targets. Furthermore, our proof-of-concept experiments demonstrated the feasibility of applying LTQ-Orbitrap, operated in SIM/AIMS mode, to multiplex and targeted verification of phosphopeptides in individual tissue specimens using a super-SILAC mix as an internal phosphopeptide standard. This method could be readily applied to verify dozens of phosphopeptide candidates in a larger HCC sample set.

Comprehensive Analysis of the Membrane Phosphoproteome Regulated by Oligogalacturonides in Arabidopsis thaliana.

Early changes in the Arabidopsis thaliana membrane phosphoproteome in response to oligogalacturonides (OGs), a class of plant damage-associated molecular patterns (DAMPs), were analyzed by two complementary proteomic approaches. Differentially phosphorylated sites were determined through phosphopeptide enrichment followed by LC-MS/MS using label-free quantification; differentially phosphorylated proteins were identified by 2D-DIGE combined with phospho-specific fluorescent staining (phospho-DIGE). This large-scale phosphoproteome analysis of early OG-signaling enabled us to determine 100 regulated phosphosites using LC-MS/MS and 46 differential spots corresponding to 34 pdhosphoproteins using phospho-DIGE. Functional classification showed that the OG-responsive phosphoproteins include kinases, phosphatases and receptor-like kinases, heat shock proteins (HSPs), reactive oxygen species (ROS) scavenging enzymes, proteins related to cellular trafficking, transport, defense and signaling as well as novel candidates for a role in immunity, for which elicitor-induced phosphorylation changes have not been shown before. A comparison with previously identified elicitor-regulated phosphosites shows only a very limited overlap, uncovering the immune-related regulation of 70 phosphorylation sites and revealing novel potential players in the regulation of elicitor-dependent immunity.

Quantitative Phosphoproteomic Study Reveals that Protein Kinase A Regulates Neural Stem Cell Differentiation Through Phosphorylation of Catenin Beta-1 and Glycogen Synthase Kinase 3β.

Protein phosphorylation is central to the understanding of multiple cellular signaling pathways responsible for regulating the self-renewal and differentiation of neural stem cells (NSCs). Here we performed a large-scale phosphoproteomic analysis of rat fetal NSCs using strong cation exchange chromatography prefractionation and citric acid-assisted two-step enrichment with TiO2 strategy followed by nanoLC-MS/MS analysis. Totally we identified 32,546 phosphosites on 5,091 phosphoproteins, among which 23,945 were class I phosphosites, and quantified 16,000 sites during NSC differentiation. More than 65% of class I phosphosites were novel when compared with PhosphoSitePlus database. Quantification results showed that the early and late stage of NSC differentiation differ greatly. We mapped 69 changed phosphosites on 20 proteins involved in Wnt signaling pathway, including S552 on catenin beta-1 (Ctnnb1) and S9 on glycogen synthase kinase 3β (Gsk3β). Western blotting and real-time PCR results proved that Wnt signaling pathway plays critical roles in NSC fate determination. Furthermore, inhibition and activation of PKA dramatically affected the phosphorylation state of Ctnnb1 and Gsk3β, which regulates the differentiation of NSCs. Our data provides a valuable resource for studying the self-renewal and differentiation of NSCs. Stem Cells 2016;34:2090-2101.

Quantitative Analysis of Tissue Samples by Combining iTRAQ Isobaric Labeling with Selected/Multiple Reaction Monitoring (SRM/MRM).

Mass spectrometry-based phosphoproteomics is an indispensible technique used in the discovery and quantification of phosphorylation events on proteins in biological samples. The application of this technique to tissue samples is especially useful for the discovery of biomarkers as well as biological studies. We herein describe the application of a large-scale phosphoproteome analysis and SRM/MRM-based quantitation to develop a strategy for the systematic discovery and validation of biomarkers using tissue samples.

Targeted Phosphoproteome Analysis Using Selected/Multiple Reaction Monitoring (SRM/MRM).

Mass spectrometry-based phosphoproteomics has been rapidly spread based on the advancement of mass spectrometry and development of efficient enrichment techniques for phosphorylated proteins or peptides. Non-targeted approach has been employed in most of the studies for phosphoproteome analysis. However, targeted approach using selected/multiple reaction monitoring (SRM/MRM) is an indispensible technique used for the quantitation of known targets especially when we have many samples to quantitate phosphorylation events on proteins in biological or clinical samples. We herein describe the application of a large-scale phosphoproteome analysis and SRM-based quantitation for the systematic discovery and validation of biomarkers.

Abstract LB-C12: Functional proteomics elucidates signaling adaptation driven by combination therapy in BRAF mutant melanoma cell line models

Abstract Targeted kinase inhibition is a promising treatment modality for melanoma, which has significantly increased available clinical strategies and improved survival outcomes for patients, whose tumors harbor the BRAF V600E mutation. However, responses are transient and multiple mechanisms for drug resistance can contribute to therapeutic escape. Therefore, a novel multiplexed proteomics approach provides an optimal choice for comprehensively dissecting multiple mechanisms of resistance to BRAFi and combination therapy within one experiment. A large-scale quantitative expression proteomics and phosphoproteomics analysis (SysQuant) was carried out on BRAF V600E mutant melanoma cell lines treated with two different clinically relevant kinase inhibitor combinations: 1) BRAFi/MEKi and 2) BRAFi/PI3Ki. Changes in protein expression and phosphorylation in response to each treatment are determined by comparison to vehicle controls. Cell line models all harbor BRAF V600E mutations, but differ in PTEN status (A375 is WT, while WM793 is PTEN null). Briefly, cells were treated with either drug combination (as above) and harvested at 1hr, 6hrs, 24hrs and 48hrs post-treatment. Control cells were treated with DMSO. Samples were lysed, reduced, alkylated and digested with trypsin. Tryptic peptides from each sample were chemically labeled or “barcoded” with TMT-10plex reagents (4 BRAFi/MEKi time points, 4 BRAFi/PI3Ki time points, 1 DMSO control and a pooled reference sample) and combined for LC-MS/MS. After peptide fractionation with strong cation exchange chromatography and phosphopeptide enrichment, LC-MS/MS peptide sequencing and relative quantification was performed using an Orbitrap Fusion mass spectrometer (Thermo). Raw MS data was searched by Proteome Discoverer and analyzed by in-house R-scripts and Perseus statistical software package. Pathway analysis was done in GeneGO (Metacore). The SysQuant workflow identified >9,000 protein groups and >17,000 unique phosphosites per cell line across different treatments. Principal component analysis of the phosphoproteomics data revealed signaling differences across different treatment conditions and drug combinations are mainly driven by BRAFi and the time post-treatment. K-means clustering was also used to examine trends in the data; as an example, this technique could be used to track signaling changes that correlate with reduction and recovery in ERK signaling. The SysQuant approach provides a systems view of global signaling changes occurring in response to drug treatment. The ability to multiplex samples with TMT allows quantitative deduction of protein expression and phosphorylation patterns that are common or unique in different cell lines, time post-treatment and the effect of combination treatments. This proteomics approach ties protein expression and phosphorylation status in response to combination therapy and generates several hypotheses for further testing with the goal of developing novel combination therapy strategies. Citation Format: Ritin Sharma, Inna Fedorenko, Sasa Koncarevic, Vikram Mitra, Stefan Selzer, Gitte Boehm, Ian Pike, Keiran Smalley, John M. Koomen. Functional proteomics elucidates signaling adaptation driven by combination therapy in BRAF mutant melanoma cell line models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C12.

Hydrophilic Carboxyl Cotton Chelator for Titanium(IV) Immobilization and Its Application as Novel Fibrous Sorbent for Rapid Enrichment of Phosphopeptides.

Sample preparation methods with high selectivity, efficiency, and matrix resistance are essential for phosphoproteomic analysis. In this study, carboxyl cotton chelator-titanium(IV) (CCC-Ti4+) fibers, a novel CCC-based fibrous sorbent with excellent biocompatibility, were successfully synthesized on the basis of the coordination effect between double carboxyl groups on CCC and Ti4+. The synthesis of CCC-Ti4+ fibers was easy, and the incorporated titanium content was high. On the basis of immobilized metal ion affinity chromatography (IMAC), CCC-Ti4+ fibers were used for specific capture of phosphopeptides using a lab-in-syringe solid-phase extraction (SPE) from multiple biological samples, including standard protein digests, nonfat milk digests, human serum, and animal tissue. The proposed sorbent exhibited high selectivity (β-casein/BSA=1:1000) and good sensitivity (10 fmol) in phosphopeptides analysis. Meanwhile, the lab-in-syringe SPE greatly simplified the entire process of enrichment. Thanks to the good biocompatibility of CCC-based material, CCC-Ti4+ fibers showed excellent performance in phosphopeptide enrichment from protein-rich human serum. Finally, CCC-Ti4+ fibers were applied for selective capture of phosphopeptides from tryptic digests of rat brain lysate followed by LC-MS/MS analysis. Using the proposed method, we identified 3950 unique phosphosites from 1 mg of rat brain in a single experiment, which is much better than previously reported IMAC-based strategies. Taken together, this efficient method will find broad application in large-scale phosphoproteomics analysis because of the rapid (3 min) convenient procedure and excellent performance.

Sequential enrichment with titania-coated magnetic mesoporous hollow silica microspheres and zirconium arsenate-modified magnetic nanoparticles for the study of phosphoproteome of HL60 cells

As one of the most important types of post-translational modifications, reversible phosphorylation of proteins plays crucial roles in a large number of biological processes. However, owing to the relatively low abundance and dynamic nature of phosphorylation and the presence of the unphosphorylated peptides in large excess, phosphopeptide enrichment is indispensable in large-scale phosphoproteomic analysis. Metal oxides including titanium dioxide have become prominent affinity materials to enrich phosphopeptides prior to their analysis using liquid chromatography-mass spectrometry (LC-MS). In the current study, we established a novel strategy, which encompassed strong cation exchange chromatography, sequential enrichment of phosphopeptides using titania-coated magnetic mesoporous hollow silica microspheres (TiO2/MHMSS) and zirconium arsenate-modified magnetic nanoparticles (ZrAs–Fe3O4@SiO2), and LC-MS/MS analysis, for the proteome-wide identification of phosphosites of proteins in HL60 cells. In total, we were able to identify 11,579 unique phosphorylation sites in 3432 unique proteins. Additionally, our results suggested that TiO2/MHMSS and ZrAs–Fe3O4@SiO2 are complementary in phosphopeptide enrichment, where the two types of materials displayed preferential binding of peptides carrying multiple and single phosphorylation sites, respectively.

Large-scale phosphoproteome analysis in seedling leaves of Brachypodium distachyon L.

BackgroundProtein phosphorylation is one of the most important post-translational modifications involved in the regulation of plant growth and development as well as diverse stress response. As a member of the Poaceae, Brachypodium distachyon L. is a new model plant for wheat and barley as well as several potential biofuel grasses such as switchgrass. Vegetative growth is vital for biomass accumulation of plants, but knowledge regarding the role of protein phosphorylation modification during vegetative growth, especially in biofuel plants, is far from comprehensive.ResultsIn this study, we carried out the first large-scale phosphoproteome analysis of seedling leaves in Brachypodium accession Bd21 using TiO2 microcolumns combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MaxQuant software. A total of 1470 phosphorylation sites in 950 phosphoproteins were identified, and these phosphoproteins were implicated in various molecular functions and basic cellular processes by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Among the 950 phosphoproteins identified, 127 contained 3 to 8 phosphorylation sites. Conservation analysis showed that 93.4% of the 950 phosphoproteins had phosphorylation orthologs in other plant species. Motif-X analysis of the phosphorylation sites identified 13 significantly enriched phosphorylation motifs, of which 3 were novel phosphorylation motifs. Meanwhile, there were 91 phosphoproteins with both multiple phosphorylation sites and multiple phosphorylation motifs. In addition, we identified 58 phosphorylated transcription factors across 21 families and found out 6 significantly over-represented transcription factor families (C3H, Trihelix, CAMTA, TALE, MYB_related and CPP). Eighty-four protein kinases (PKs), 8 protein phosphatases (PPs) and 6 CESAs were recognized as phosphoproteins.ConclusionsThrough a large-scale bioinformatics analysis of the phosphorylation data in seedling leaves, a complicated PKs- and PPs- centered network related to rapid vegetative growth was deciphered in B. distachyon. We revealed a MAPK cascade network that might play the crucial roles during the phosphorylation signal transduction in leaf growth and development. The phosphoproteins and phosphosites identified from our study expanded our knowledge of protein phosphorylation modification in plants, especially in monocots.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-375) contains supplementary material, which is available to authorized users.

Impact of digestion conditions on phosphoproteomics.

In the past few years, the focus of phosphoproteomics has shifted from merely qualitative to quantitative and targeted studies. Tryptic digestion is a critical step that directly affects quantification and that can be impaired by phosphorylation. Therefore, we systematically characterized the digestion efficiency of 19 nonmodified and phosphorylated model peptides. Whereas we quantified a strong reduction of tryptic cleavage within phosphorylated PKA motifs (R)-R-X-pS/pT and also R-X-X-pT sequences, (R)-R-X-pY sequences were almost unaffected. Structural prediction implied the formation of salt bridges between R/K cleavage sites and phosphoamino acids pS/pT as the main reason for impaired tryptic digestion. We evaluated different conditions to optimize the digestion of such "resistant" phosphopeptides, yielding a substantial improvement of digestion efficiency. We performed a quantitative large-scale phosphoproteomic analysis of human platelets to validate our findings in a complex biological sample. Here, increasing trypsin concentrations up to a trypsin to peptide ratio of 1:10 led to a significant gain (i) in the overall number of phosphorylation sites (up to 9%) and (ii) in the intensities of individual phosphopeptides, thereby improving the sensitivity of phosphopeptide quantification. Still, for certain sequences, the negative impact of phosphorylation on digestion efficiency will further complicate the analysis of phosphorylation stoichiometry.