title: Improve the coverage for the analysis of phosphoproteome of HeLa cells by a tandem digestion approach.
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.
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Yangyang
authors.initials: Y
authors.lastname: Bian
authors.forename: Mingliang
authors.initials: M
authors.lastname: Ye
authors.forename: Chunxia
authors.initials: C
authors.lastname: Song
authors.forename: Kai
authors.initials: K
authors.lastname: Cheng
authors.forename: Chunli
authors.initials: C
authors.lastname: Wang
authors.forename: Xiaoluan
authors.initials: X
authors.lastname: Wei
authors.forename: Jun
authors.initials: J
authors.lastname: Zhu
authors.forename: Rui
authors.initials: R
authors.lastname: Chen
authors.forename: Fangjun
authors.initials: F
authors.lastname: Wang
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D018698:Glutamic Acid / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000737:chemistry*; D013211:Staphylococcus aureus / Q000201:enzymology; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry*
chemical_list: D010748:Phosphopeptides; D010750:Phosphoproteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin