Analysis of human serum phosphopeptidome by a focused database searching strategy.
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.
CAS Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
Jun
J
Zhu
Fangjun
F
Wang
Kai
K
Cheng
Chunxia
C
Song
Hongqiang
H
Qin
Lianghai
L
Hu
Daniel
D
Figeys
Mingliang
M
Ye
Hanfa
H
Zou
D030562:Databases, Protein; D005260:Female; D006801:Humans; D008297:Male; D010750:Phosphoproteins / Q000097:blood* / Q000235:genetics; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics
D010750:Phosphoproteins; D020543:Proteome