Analysis of human serum phosphopeptidome by a focused database searching strategy

Zhu, Jun; Wang, Fangjun; Cheng, Kai; Song, Chunxia; Qin, Hongqiang; Hu, Lianghai; Figeys, Daniel; Ye, Mingliang; Zou, Hanfa

doi:10.1016/j.jprot.2012.10.006

Cited by 11 publications

(7 citation statements)

References 32 publications

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“…To confirm the enrichment effectiveness for the real biosamples, the Fe 3 O 4 @fTiO 2 affinity microspheres were further applied to capture phosphopeptides from human serum. Human serum contains some endogenous phosphopeptides, which might be used as biomarkers for diagnostic and therapeutic methods. , However, the serum also contains inorganic salts and abundant proteins, as well as a large amount of nonphosphopeptides which seriously interfere with the detection of target phosphopeptides. Figure a shows the mass spectrum of the diluted human serum before treatment using the affinity microspheres.…”

Section: Results and Discussionmentioning

confidence: 99%

Magnetic Affinity Microspheres with Meso-/Macroporous Shells for Selective Enrichment and Fast Separation of Phosphorylated Biomolecules

Cheng

Wang

Liu

et al. 2013

ACS Appl. Mater. Interfaces

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The flowerlike multifunctional affinity microspheres prepared by a facile solvothermal synthesis and subsequent calcination process consist of magnetic cores and hierarchical meso-/macroporous TiO2 shells. The hierarchical porous structure of the flowerlike affinity microspheres is constructed by the macroporous shell from the stacked mesoporous nanopetals which are assembled by small crystallites. The affinity microspheres have a relatively large specific surface area of 50.45 m(2) g(-1) and superparamagnetism with a saturation magnetization (Ms) value of 30.1 emu g(-1). We further demonstrate that they can be applied for rapid and effective purification of phosphoproteins, in virtue of their selective affinity, porous structure, and strong magnetism. In addition, the affinity microspheres can also be used for enrichment of phosphopeptides, and the selectivity is greatly improved due to the increase of mass transport and prevention of the possible "shadow effect" resulting from the smaller and deeper pores by taking advantage of the unique porous structure. Overall, this work will be highly beneficial for future applications in the isolation and identification of phosphorylated biomolecules.

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Section: Results and Discussionmentioning

confidence: 99%

Magnetic Affinity Microspheres with Meso-/Macroporous Shells for Selective Enrichment and Fast Separation of Phosphorylated Biomolecules

Cheng

Wang

Liu

et al. 2013

ACS Appl. Mater. Interfaces

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“…In literature, the number of unique endogenous phosphopeptides identified from serum by one-dimensional LC-MS/MS analyze did not exceeded 20 [11]. Much more endogenous phosphopeptides identified in this study may be attribute to two main factors: (i) the de novo-assisted database search strategy allowed the sensitive identification without significantly increasing the number of peptide hypothesis; (ii) both the precursor ions and fragment ions were recorded in high mass accuracy, which facilitated the de novo-assisted database search process.…”

Section: Identification Of Endogenous Phosphopeptides From Serummentioning

confidence: 99%

“…An easy way to tackle the big search space issue is to reduce the database size by removing the redundant protein sequences. Recently, a focused database search strategy using an in‐house collected human serum propeptidome database was applied to the identification of endogenous phosphopeptides . However, the building of low redundant database for endogenous phosphopeptide is often difficult.…”

Section: Introductionmentioning

confidence: 99%

Identification of phosphopeptides with unknown cleavage specificity by a de novo sequencing assisted database search strategy

Dong

Cheng

et al. 2014

Proteomics

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In theory, proteases with broad cleavage specificity could be applied to digest protein samples to improve the phosphoproteomic analysis coverage. However, in practice this approach is seldom employed. This is because the identification of phosphopeptides without enzyme specificity by conventional database search strategy is extremely difficult due to the huge search space. In this study, we investigated the performance of a de novo sequencing assisted database search strategy for the identification of such phosphopeptides. Firstly, we compared the performance of conventional database search strategy and the de novo sequencing assisted database search strategy for the identification of peptides and phosphopeptides without stetting enzyme specificity. It was found that the identification sensitivity dropped significantly for the conventional one while it was only slightly decreased for the new approach. Then, this new search strategy was applied to identify phosphopeptides generated by Proteinase K digestion, which resulted in the identification of 717 phosphopeptides. Finally, this strategy was utilized for the identification of serum endogenous phosphopeptides, which were generated in vivo by different kinds of proteases and kinases, and the identification of 68 unique serum endogenous phosphopepitdes was successfully achieved.

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“…They made an in-house collected human serum pro-peptidome target/decoy database (HuSPep) to accelerate database searching and increase the sensitivity of phosphopeptide identification [40]. By utilizing large-scale phosphoproteome datasets, Li YiXue and his colleagues found that the phosphosites in the vertebrate-specific functional modules are more highly conserved than in the basic functional modules and their flanking regions.…”

Section: Technology Development For Phosphoproteomicsmentioning

confidence: 99%

“…By utilizing large-scale phosphoproteome datasets, Li YiXue and his colleagues found that the phosphosites in the vertebrate-specific functional modules are more highly conserved than in the basic functional modules and their flanking regions. Based on these data, they suggested that phosphorylation may have played an essential role in the evolution of vertebrates [40]. Yao XueBiao and Xue Yu's lab analyzed single nucleotide polymorphism (SNP) and phosphosite information with their kinase-specific phosphorylation site predictor (GPS 2.0), and found that approximately 70% of the reported nonsynonymous SNPs are potential phosSNPs.…”

Section: Technology Development For Phosphoproteomicsmentioning

confidence: 99%

Rapid development of proteomics in China: from the perspective of the Human Liver Proteome Project and technology development

Zhai

et al. 2014

Sci. China Life Sci.

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Proteomics focuses on the systematic identification and quantification of entire proteomes and interpretation of proteins' biological functions. During the last decade, proteomics in China has grown much faster than other research fields in life sciences. At the beginning of the second decade of the 21st century, the rapid development of high-resolution and high-speed mass spectrometry makes proteomics a powerful tool to study the mechanisms underlying physiological/pathological processes in organisms. This article provides a brief overview of proteomics technology development and representative scientific progress of the Human Liver Proteome Project in China over the past three years. proteomics, LC-MS/MS, HLPP, protein-protein interaction, protein posttranslational modification, bioinformaticsCitation:

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Analysis of human serum phosphopeptidome by a focused database searching strategy

Cited by 11 publications

References 32 publications

Magnetic Affinity Microspheres with Meso-/Macroporous Shells for Selective Enrichment and Fast Separation of Phosphorylated Biomolecules

Magnetic Affinity Microspheres with Meso-/Macroporous Shells for Selective Enrichment and Fast Separation of Phosphorylated Biomolecules

Identification of phosphopeptides with unknown cleavage specificity by a de novo sequencing assisted database search strategy

Rapid development of proteomics in China: from the perspective of the Human Liver Proteome Project and technology development

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