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ABSTRACT: Human gastric cancer is a big public health problem. Multidrug resistance is a main obstacle to successful chemotherapeutic treatment in gastric cancers and the underlying mechanism is not clear. Glycosylation, one of the most important post translational modifications of proteins, plays a vital role in diverse aspects of tumor progression. In the present study, we applied two multidrug resistance cell lines and their parental drug sensitive gastric cancer cell line to a modified cell surface capturing strategy with triplex labeling to characterize MDR related cell surface glycoproteome. Finally, 56 cell membrane glycoproteins were successfully identified via combination of identification by glycopeptides and quantitation by non-glycopeptides, and 11 of them were found to be differentially expressed with the same trend in both drug resistant cell lines compared with that in sensitive cell line. The further analysis by western blot and in vitro drug sensitivity assay demonstrated that our approach is reliable and accurate and suggested that these glycoproteins may represent as biomarkers for multidrug resistance in gastric cancer. BIOLOGICAL SIGNIFICANCE: In this study, we performed a cell surface glycoproteomics research of multidrug resistance in gastric cancer using a modified CSC approach. Totally we identified and quantified 11 membrane N-glycoproteins which were significantly changed in MDR gastric cancer cells. These glycoproteins are quite possible to be biomarkers for predicting MDR or key regulators for targeted therapy, and are also helpful for better interpreting the sophisticated mechanisms of MDR in gastric cancer. In addition to that, this approach used in this study can be well applied to screen aberrantly glycosylated biomarkers associated with other malignant phenotypes of various kinds of cancers.
Journal of proteomics 03/2013; · 5.07 Impact Factor
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ABSTRACT: Mass spectrometry (MS)-based proteomics has become the preferred tool for the analysis of protein phosphorylation. To be successful at such an endeavor, there is a requirement for an efficient enrichment of phosphopeptides. This is necessary because of the substoichiometric nature of phosphorylation at a given site and the complexity of the cell. Recently, new alternative materials have emerged that allow excellent and robust enrichment of phosphopeptides. These monodisperse microsphere-based immobilized metal ion affinity chromatography (IMAC) resins incorporate a flexible linker terminated with phosphonate groups that chelate either zirconium or titanium ions. The chelated zirconium or titanium ions bind specifically to phosphopeptides, with an affinity that is similar to that of other widely used metal oxide affinity chromatography materials (typically TiO(2)). Here we present a detailed protocol for the preparation of monodisperse microsphere-based Ti(4+)-IMAC adsorbents and the subsequent enrichment process. Furthermore, we discuss general pitfalls and crucial steps in the preparation of phosphoproteomics samples before enrichment and, just as importantly, in the subsequent mass spectrometric analysis. Key points such as lysis, preparation of the chromatographic system for analysis and the most appropriate methods for sequencing phosphopeptides are discussed. Bioinformatics analysis specifically relating to site localization is also addressed. Finally, we demonstrate how the protocols provided are appropriate for both single-protein analysis and the screening of entire phosphoproteomes. It takes ∼2 weeks to complete the protocol: 1 week to prepare the Ti(4+)-IMAC material, 2 d for sample preparation, 3 d for MS analysis of the enriched sample and 2 d for data analysis.
Nature Protocol 02/2013; 8(3):461-80. · 8.36 Impact Factor
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ABSTRACT: Terminal sialylation is very important in cancer biology and has been extensively investigated for the discovery of potential clinical biomarkers of cancers. In this study, we presented a novel approach, by using of Ti(IV)-IMAC, to enrich sialic acid-containing N-glycopeptides for the analysis of terminal sialylation. Compared with conventional method using TiO(2) , this approach obtained 2.5 times more glycopeptides and glycosylation sites. Then, a simple integrated system combining filter aided sample preparation (FASP), acetonitrile improved digestion (AID) and Ti(IV)-IMAC enrichment was established for efficient analysis. In this system, protein digestion, glycopeptide enrichment and deglycosylation were integrated and were performed sequentially in a single filter unit without any need for desalting, lyophilization or sample transfer procedures. As a result, the number of identifications was improved by 1.5 fold and the total processing time was drastically reduced to only 7-8 hours. By using this system, fast and efficient analysis of human serum sialylated N-glycoproteome was achieved. From only 1 μL human serum, 217 unique glycopeptides and 194 glycosylation sites were successfully identified.
Proteomics 01/2013; · 4.43 Impact Factor
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ABSTRACT: Large-scale protein phosphorylation analysis by MS is emerging as a powerful tool in plant signal transduction research. However, our current understanding of the phosphorylation regulatory network in plants is still very limited. Here, we report on a proteome-wide profiling of phosphopeptides in nine-day-old Arabidopsis (Arabidopsis thaliana) seedlings by using an enrichment method combining the titanium (Ti(4+))-based IMAC and the RP-strong cation exchange (RP-SCX) biphasic trap column-based online RPLC. Through the duplicated RPLC-MS/MS analyses, we identified 5,348 unique phosphopeptides for 2,552 unique proteins. Among the phosphoproteins identified, 41% of them were first-time identified. Further evolutionary conservation and phosphorylation motif analyses of the phosphorylation sites discovered 100 highly conserved phosphorylation residues and identified 17 known and 14 novel motifs specific for Ser/Thr protein kinases. Gene ontology and pathway analyses revealed that many of the new identified phosphoproteins are important regulatory proteins that are involved in diverse biological processes, particularly in central metabolisms and cell signaling. Taken together, our results provided not only new insights into the complex phosphoregulatory network in plants but also important resources for future functional studies of protein phosphorylation in plant growth and development.
Journal of proteomics 10/2012; · 5.07 Impact Factor
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ABSTRACT: As human serum is an important source for early diagnosis of many serious diseases, analysis of serum proteome and peptidome has been extensively performed. However, the serum phosphopeptidome was less explored probably because the effective method for database searching is lacking. Conventional database searching strategy always uses the whole proteome database, which is very time-consuming for phosphopeptidome search due to the huge searching space resulted from the high redundancy of the database and the setting of dynamic modifications during searching. In this work, a focused database searching strategy using an in-house collected human serum pro-peptidome target/decoy database (HuSPep) was established. It was found that the searching time was significantly decreased without compromising the identification sensitivity. By combining size-selective Ti (IV)-MCM-41 enrichment, RP-RP off-line separation, and complementary CID and ETD fragmentation with the new searching strategy, 143 unique endogenous phosphopeptides and 133 phosphorylation sites (109 novel sites) were identified from human serum with high reliability.
Journal of proteomics 10/2012; · 5.07 Impact Factor
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Zhiwei Zhu,
Sufang Zhang,
Hongwei Liu,
Hongwei Shen,
Xinping Lin,
Fan Yang,
Yongjin J Zhou,
Guojie Jin, Mingliang Ye,
Hanfan Zou,
Zongbao K Zhao
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ABSTRACT: Triacylglycerols are among the most attractive alternative raw materials for biofuel development. Current oil plant-based technologies are limited in terms of triacylglycerol production capacity and rate. These limitations may be circumvented by biotransformation of carbohydrates into lipids; however, our understanding of microbial oleaginicity remains limited. Here we present the results of a multi-omic analysis of Rhodosporidium toruloides, a robust triacylglycerol-producing fungus. The assembly of genome and transcriptome sequencing data reveals a genome of 20.2 Mb containing 8,171 protein-coding genes, the majority of which have multiple introns. Genes including a novel fatty acid synthase are predicted to participate in metabolic pathways absent in non-oleaginous yeasts. Transcriptomic and proteomic data suggest that lipid accumulation under nitrogen-limited conditions correlates with the induction of lipogenesis, nitrogenous compound recycling, macromolecule metabolism and autophagy. The multi-omic map of R. toruloides therefore provides a valuable resource for efforts to rationally engineer lipid-production pathways.
Nature Communications 10/2012; 3:1112. · 7.40 Impact Factor
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ABSTRACT: Incorporation of isotopic tag onto peptides via chemical labeling is a popular approach for quantitative proteomics. Chemical labeling via solution based methods usually lead to a tedious process and sample loss because several sample preparation steps including buffer exchange and desalting are performed. In this study, a solid phase based labeling approach by integration of glycopeptide enrichment and stable isotope labeling on hydrazide beads was developed for relative quantification of protein glycosylation, by which enrichment, washing, labeling, and release of the glycopeptides were all performed on the hydrazide beads sequentially. This approach was proved to be accurate in quantitative glycoproteome analysis and have good linearity range with 2 orders of magnitude for quantification of glycopeptides. Compared with dimethyl labeling conventionally performed in solution, the developed approach has better enrichment recovery (10-330% improvement) and high detection sensitivity in which 42% of annotated glycosites (vs 26%) still can be quantified using only 10 μg of four standard glycoprotein mixtures and 400 μg of bovine serum album interference as starting sample. The applicability of the approach for quantitative glycopeptide profiling was also explored by differential analysis of glycoproteome between human normal serum and liver cancer serum.
Analytical Chemistry 09/2012; 84(20):8452-6. · 5.86 Impact Factor
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ABSTRACT: The Ser/Thr protein kinases fall into three major subgroups, pro-directed, basophilic, and acidophilic, on the basis of the types of substrate sequences that they preferred. Despite many phosphoproteomics efforts that have been taken for global profiling of phosphopeptides, methodologies focusing on analyzing a particular type of kinase substrates have seldom been reported. Selective enrichment of phosphopeptides from basophilic kinase substrates is difficult because basophilic motifs are cleaved by trypsin during digestion. In this study, we develop a negative enrichment strategy to enhance the identification of basophilic kinase substrates. This method is based on an observation that high pH strong anion exchange (SAX) chromatography can separate tryptic phosphopeptides according to the number of acidic amino acidic residues that they have. Thus, SAX was applied to deplete acidic phosphopeptides from the phosphopeptide mixture, which improved the coverage for the detection of basophilic kinase substrates. The SAX depletion approach was further combined with online SCX-RP separation for large-scale analysis of mouse liver phosphoproteome, which resulted in the identification of 6944 phosphorylated sites. It was found that motifs associated with basophilic kinases prevail for these identified phosphorylated sites.
Journal of Proteome Research 08/2012; 11(9):4673-81. · 5.11 Impact Factor
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ABSTRACT: Strong cation exchange (SCX) chromatography is one of the most important separation modes in liquid chromatography and SCX column is widely applied in high resolution separation or fractionation of various samples. In this work, a sulfonate SCX hybrid monolithic column was successfully prepared by "one-pot" strategy and the hybrid monolith is well optimized to obtain homogenous structure. It was observed that this sulfonate SCX hybrid monolithic column had ∼7 times permeability (in water) and ∼3 times sample loading capacity (tested by dipeptide Gly-Tyr) comparing to particulate SCX column packed with commercial available material. Then, it was used as trap column for fast sample loading of the enriched phosphopeptides. Comparing to phosphate SCX polymer monolithic column, the number of identified phosphopeptides increased ∼19% due to the sulfonate group has higher retention strength than phosphate group for peptide cations. And the coverage of phosphoproteome obtained by sulfonate SCX hybrid monolithic column is similar to particulate packed SCX column, because they had identical sulfonate group to retain the peptide cations. Finally, the sulfonate SCX hybrid monolithic column was used as enzyme reactor for online protein digestion. Comparing to particulate SCX packed column, the identified peptides number increased 40% and the protein coverage increased 10%. This might be ascribed to the high porous structure and relative high surface area that elevated the digestion efficiency.
Journal of chromatography. A 07/2012; 1256:136-43. · 4.19 Impact Factor
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ABSTRACT: The human serum proteome is closely associated with the state of the body. Endogenous peptides derived from proteolytic enzymes cleaving on serum proteins are widely studied due to their potential application in disease-specific marker discovery. However, the reproducibility of peptidome analysis of endogenous peptides is significantly influenced by the proteolytic enzymes within body fluids, thereby limiting the clinical use of the endogenous peptides. We comprehensively investigated the N and C terminus of endogenous peptides using peptidomics. The cleavage site patterns of the N and C terminus and adjacent sites from all the identified endogenous peptides were highly conserved under different sample preparation conditions, including long-term incubation at 37°C and pretreatment with repeated freeze-thaw cycles. Furthermore, a distinguishable cleavage site pattern was obtained when a different disease serum was analyzed. The conserved cleavage site pattern derived from proteolytic enzymes holds potential in highly specific disease diagnosis.
Protein & Cell 07/2012; 3(9):669-74.
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Chunxia Song, Mingliang Ye,
Zexian Liu,
Han Cheng,
Xinning Jiang,
Guanghui Han,
Zhou Songyang,
Yexiong Tan,
Hongyang Wang,
Jian Ren,
Yu Xue,
Hanfa Zou
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ABSTRACT: In eukaryotes, hundreds of protein kinases (PKs) specifically and precisely modify thousands of substrates at specific amino acid residues to faithfully orchestrate numerous biological processes, and reversibly determine the cellular dynamics and plasticity. Although over 100,000 phosphorylation sites (p-sites) have been experimentally identified from phosphoproteomic studies, the regulatory PKs for most of these sites still remain to be characterized. Here, we present a novel software package of iGPS for the prediction of in vivo site-specific kinase-substrate relations mainly from the phosphoproteomic data. By critical evaluations and comparisons, the performance of iGPS is satisfying and better than other existed tools. Based on the prediction results, we modeled protein phosphorylation networks and observed that the eukaryotic phospho-regulation is poorly conserved at the site and substrate levels. With an integrative procedure, we conducted a large-scale phosphorylation analysis of human liver and experimentally identified 9719 p-sites in 2998 proteins. Using iGPS, we predicted a human liver protein phosphorylation networks containing 12,819 potential site-specific kinase-substrate relations among 350 PKs and 962 substrates for 2633 p-sites. Further statistical analysis and comparison revealed that 127 PKs significantly modify more or fewer p-sites in the liver protein phosphorylation networks against the whole human protein phosphorylation network. The largest data set of the human liver phosphoproteome together with computational analyses can be useful for further experimental consideration. This work contributes to the understanding of phosphorylation mechanisms at the systemic level, and provides a powerful methodology for the general analysis of in vivo post-translational modifications regulating sub-proteomes.
Molecular & Cellular Proteomics 07/2012; 11(10):1070-83. · 7.40 Impact Factor
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ABSTRACT: Highly site-selective dimethyl labeling of N-terminus of peptides has been obtained by adjusting the acidic strength of the reaction solution. And this selective labeling strategy combined with the use of different isotope formaldehyde reagents has been successfully used in the proteome quantification by using the isobaric peptide cross-sequence labeling method, which would play increasingly important roles in the clinical diagnosis, especially in the discovery of biomarkers for diseases.
Chemical Communications 05/2012; 48(50):6265-7. · 6.17 Impact Factor
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ABSTRACT: Sample handling procedures including protein digestion, glycopeptide enrichment, and deglycosylation have significant impact on the performance of glycoproteome analysis. Several glycoproteomic analysis systems were developed to integrate some of these sample preparation procedures. However, no microsystem integrates all of above three procedures together. In this work, we developed a glycoproteomic microreactor enabling seamless integration of all these procedures. In this reactor, trypsin digestion was accelerated by adding acetonitrile to 80%, and after acidification of protein digest by trifluoroacetic acid (TFA), the following hydrophilic interaction chromatography (HILIC) enrichment and deglycosylation were sequentially performed without any desalting, lyophilization, or buffer exchange steps. The total processing time could be as short as 1.5 h. The detection limit of human IgG as low as 30 fmol was also achieved. When applied to human serum glycoproteome analysis, a total number of 92, 178, and 221 unique N-glycosylation sites were identified from three replicate analyses of 10 nL, 100 nL, and 1 μL of human serum, respectively. It was demonstrated that the glycoproteomic microreactor based method had very high sensitivity and was well suited for glycoproteome analysis of minute protein samples.
Analytical Chemistry 05/2012; 84(11):5146-53. · 5.86 Impact Factor
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ABSTRACT: Peptidomics draws more and more attention in discovering useful biomarkers for early diagnosis of disease. However, there
is lack of efficient quantification strategy in peptidome analysis. In this study, a strategy with label-free quantification
of the targeted endogenous peptides based on peak intensity using μUPLC-Q-TOF-MS/MS was developed for quantitative peptidome
analysis of human serum. Different amounts of standard BSA tryptic digesting peptides were added into the same serum extracts
for evaluation of the developed strategy, and it was observed that the average relative error of the targeted peptides was
6.42%, which was superior to the result obtained directly by commercially available software PLGS. It was also demonstrated
that this quantification strategy could obviously increase the detection sensitivity of the peptide by DDA analysis. Then,
this strategy was applied to comparatively analyze the peptides extracted from the serum of HCC or breast cancer patients
and healthy individuals, respectively. Peptides with charge states up to 5 and molecular weight over 4000 can be reliably
identified and quantified. This quantitative analysis method based on μUPLC-Q-TOF-MS/MS exhibited superior sensitivity than
that by MALDI-TOF-MS commonly used in peptidome analysis. Finally, some interesting endogenous peptides related to corresponding
diseases were successfully obtained.
Keywordspeptidomics-label-free quantification-hepatocellular carcinoma-breast cancer-human serum
Science China-Chemistry 04/2012; 53(4):759-767. · 1.02 Impact Factor
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ABSTRACT: Complete coverage of all phosphorylation sites in a proteome is the ultimate goal for large-scale phosphoproteome analysis. However, only making use of one protease trypsin for protein digestion cannot cover all phosphorylation sites, because not all tryptic phosphopeptides are detectable in MS. To further increase the phosphoproteomics coverage of HeLa cells, we proposed a tandem digestion approach by using two different proteases. By combining the data set of the first Glu-C digestion and the second trypsin digestion, the tandem digestion approach resulted in the identification of 8062 unique phosphopeptides and 8507 phosphorylation sites in HeLa cells. The conventional trypsin digestion approach resulted in the identification of 3891 unique phosphopeptides and 4647 phosphorylation sites. It was found that the phosphorylation sites identified from the above two approaches were highly complementary. By combining above two data sets, in total we identified 10899 unique phosphopeptides and 11262 phosphorylation sites, corresponding to 3437 unique phosphoproteins with FDR < 1% at peptide level. We also compared the kinase motifs extracted from trypsin, Glu-C, or a second trypsin digestion data sets. It was observed that basophilic motifs were more frequently found in the trypsin and the second trypsin digestion data sets, and the acidic motifs were more frequently found in the Glu-C digestion data set. These results demonstrated that our tandem digestion approach is a good complement to the conventional trypsin digestion approach for improving the phosphoproteomics analysis coverage of HeLa cells.
Journal of Proteome Research 04/2012; 11(5):2828-37. · 5.11 Impact Factor
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Yong Zhao,
Zhaoshun Jiang,
Tingbao Zhao, Mingliang Ye,
Chengjin Hu,
Zhaohui Yin,
Heng Li,
Ye Zhang,
Yalin Diao,
Yunxiang Li,
Yingjian Chen,
Xiaoming Sun,
Mary Beth Fisk,
Randal Skidgel,
Mark Holterman,
Bellur Prabhakar,
Theodore Mazzone
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ABSTRACT: Inability to control autoimmunity is the primary barrier to developing a cure for type 1 diabetes (T1D). Evidence that human cord blood-derived multipotent stem cells (CB-SCs) can control autoimmune responses by altering regulatory T cells (Tregs) and human islet β cell-specific T cell clones offers promise for a new approach to overcome the autoimmunity underlying T1D.
We developed a procedure for Stem Cell Educator therapy in which a patient's blood is circulated through a closed-loop system that separates lymphocytes from the whole blood and briefly co-cultures them with adherent CB-SCs before returning them to the patient's circulation. In an open-label, phase1/phase 2 study, patients (n=15) with T1D received one treatment with the Stem Cell Educator. Median age was 29 years (range: 15 to 41), and median diabetic history was 8 years (range: 1 to 21).
Stem Cell Educator therapy was well tolerated in all participants with minimal pain from two venipunctures and no adverse events. Stem Cell Educator therapy can markedly improve C-peptide levels, reduce the median glycated hemoglobin A1C (HbA1C) values, and decrease the median daily dose of insulin in patients with some residual β cell function (n=6) and patients with no residual pancreatic islet β cell function (n=6). Treatment also produced an increase in basal and glucose-stimulated C-peptide levels through 40 weeks. However, participants in the Control Group (n=3) did not exhibit significant change at any follow-up. Individuals who received Stem Cell Educator therapy exhibited increased expression of co-stimulating molecules (specifically, CD28 and ICOS), increases in the number of CD4+CD25+Foxp3+ Tregs, and restoration of Th1/Th2/Th3 cytokine balance.
Stem Cell Educator therapy is safe, and in individuals with moderate or severe T1D, a single treatment produces lasting improvement in metabolic control. Initial results indicate Stem Cell Educator therapy reverses autoimmunity and promotes regeneration of islet β cells. Successful immune modulation by CB-SCs and the resulting clinical improvement in patient status may have important implications for other autoimmune and inflammation-related diseases without the safety and ethical concerns associated with conventional stem cell-based approaches.
ClinicalTrials.gov number, NCT01350219.
BMC Medicine 01/2012; 10:3. · 6.03 Impact Factor
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ABSTRACT: Direct mass spectrometric analysis of aberrant protein glycosylation is a challenge to the current analytical techniques. Except lectin affinity chromatography, no other glycosylation enrichment techniques are available for analysis of aberrant glycosylation. In this study, we developed a combined chemical and enzymatic strategy as an alternative for the mass spectrometric analysis of aberrant glycosylation. Sialylated glycopeptides were enriched with reverse glycoblotting, cleaved by endoglycosidase F3 and analyzed by mass spectrometry with both neutral loss triggered MS(3) in collision induced dissociation (CID) and electron transfer dissociation (ETD). Interestingly, a great part of resulted glycopeptides were found with fucose attached to the N-acetylglucosamine (N-GlcNAc), which indicated that the aberrant glycosylation that is carrying both terminal sialylation and core fucosylation was identified. Totally, 69 aberrant N-glycosylation sites were identified in sera samples from hepatocellular carcinoma (HCC) patients. Following the identification, quantification of the level of this aberrant glycosylation was also carried out using stable isotope dimethyl labeling and pooled sera sample from liver cirrhosis and HCC was compared. Six glycosylation sites demonstrated elevated level of aberrancy, which demonstrated that our developed strategy was effective in both qualitative and quantitative studies of aberrant glycosylation.
Journal of proteomics 12/2011; 75(5):1666-74. · 5.07 Impact Factor
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ABSTRACT: Ti(4+)-EPO nanoparticles were adopted as the adsorbent for in situ solid phase enrichment and isotope labeling of endogenous phosphopeptides, which has great potential application in high-throughput analyses of biological samples for screening and discovery of disease-specific biomarkers.
Chemical Communications 11/2011; 48(7):961-3. · 6.17 Impact Factor
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ABSTRACT: Deciphering the kinase-substrate relationship is vital for the study of phosphorylation network. The use of immobilized proteins on protein chip as the library for screening of potential kinase substrates is a tried-and-tested method. However, information on phosphorylation sites is lacking and the creation of the library with proteins of whole proteome by recombinant expression is costly and difficult. In this study, a new solid-phase approach by immobilization of proteins from cell lysate onto beads as a protein library for kinase substrate screening was developed. It was found that consensus phosphorylation sites motif for kinase substrates could be accurately determined and hundreds of in vitro kinase substrates and their phosphorylation sites could be identified by using this method.
Proteomics 10/2011; 11(24):4632-7. · 4.43 Impact Factor
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ABSTRACT: Protein phosphorylation is a ubiquitous post-translational modification that regulates almost all cellular processes. The analysis of protein phosphorylation is challenging due to the high dynamic range and low abundance natures of the analyte. Shotgun based proteomics has emerged as a very useful platform to achieve a comprehensive phosphoproteome analysis in considerable depth. In the past few years, significant breakthroughs on the large scale phosphorylation analysis have been witnessed along with the great development of related technologies. The combination of effective enrichment materials, refined analysis workflows, new type of powerful mass spectrometers, and sophisticated bioinformatic tools greatly boost the performance of comprehensive phosphoproteome analysis. In this Perspective, we briefly reviewed recent technological developments on the enrichment materials, prefractionation workflows, and different mass spectrometry fragmentation modes as well as software tools for phosphoproteome identification and quantification. Then, we described the current challenges and potential directions for the future of comprehensive phosphoproteome analysis. We also provide perspectives on how to further improve the performance of related analysis methods and technologies.
Analytical Chemistry 09/2011; 83(21):8078-85. · 5.86 Impact Factor
