Comprehensive and reliable phosphorylation site mapping of individual phosphoproteins by combination of multiple stage mass spectrometric analysis with a target-decoy database search.
Since the emergence of proteomics, much attention has been paid to the development of new technologies for phosphoproteomcis analysis. Compared with large scale phosphorylation analysis at the proteome level, comprehensive and reliable phosphorylation site mapping of individual phosphoprotein is equally important. Here, we present a modified target-decoy database search strategy for confident phosphorylation site analysis of individual phosphoproteins without manual interpretation of spectra. Instead of using all protein sequences in a proteome database of an organism for the construction of a target-decoy database for phosphoproteome analysis, the composite database constructed for phosphorylation site analysis of individual phosphoproteins only included the sequences of the individual target proteins and a decoy version of a small inhomogeneous protein database. It was found that the confidence of phosphopeptide identifications could be effectively controlled when the acquired MS2 and MS3 spectra were searched against the above composite database followed with data processing. Because of the small size of the composite database, the computation time for the database search is very short, which allows the adoption of low-specificity proteases for protein digestion to increase the coverage of phosphorylation site mapping. The sensitivity and comprehensive phosphorylation site mapping of this approach was demonstrated by using two standard phosphoprotein samples of alpha-casein and beta-casein, and this approach was further applied to analyze the phosphorylation of the cyclic AMP-dependent protein kinase (PKA), which resulted in the identification of 17 phosphorylation sites, including five novel sites on four PKA subunits.
Han
Guanghui
G
CAS 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.
Ye
Mingliang
M
Jiang
Xinning
X
Chen
Rui
R
Ren
Jian
J
Xue
Yu
Y
Wang
Fangjun
F
Song
Chunxia
C
Yao
Xuebiao
X
Zou
Hanfa
H
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002364:Caseins / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism; D030562:Databases, Protein; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors
D002364:Caseins; D010750:Phosphoproteins; D021122:Protein Subunits; D017868:Cyclic AMP-Dependent Protein Kinases; D010447:Peptide Hydrolases