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Identification and relative quantification of phosphopeptides by a combination of multi‐protease digestion and isobaric labeling
Author(s) -
Linke Dennis,
Koudelka Tomas,
Becker Alexander,
Tholey Andreas
Publication year - 2015
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.7185
Subject(s) - chemistry , isobaric labeling , chromatography , trypsin , electrospray ionization , phosphorylation , tandem mass spectrometry , mass spectrometry , chymotrypsin , protease , proteases , bottom up proteomics , biochemistry , protein mass spectrometry , enzyme
Rationale The identification and the determination of the extent of protein phosphorylation are major prerequisites for the comparative analysis of this important posttranslational modification of proteins in different biological situations. High sequence coverages and the availability of straightforward quantification methods are necessary to achieve these goals. Methods Phosphoproteins and non‐phosphorylated analogues separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) were digested using four different proteases (trypsin, chymotrypsin, elastase and GluC) and the digests were isobarically labeled using eight‐plex iTRAQ. The combined labeled digests were subsequently enriched using titanium dioxide and both the phosphorylated and non‐phosphorylated fractions were analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS). The influence of different data analysis algorithms (Percolator or False Discovery Rate) on the outcome of analysis was investigated. Results Almost complete sequence coverage could be achieved upon application of a multi‐protease approach. The formation of peptides of different lengths and physicochemical properties allowed the identification and the mapping of all phosphorylation sites in the investigated model proteins. The introduction of isobaric labels allowed quantification of different peptides of the same phosphorylation site with more than one peptide, leading to significantly improved statistical confidence. Conclusions A workflow for the straightforward comparative analysis of protein phosphorylation in samples of low complexity, e.g. isolated proteins, was developed. The workflow is transferable to other posttranslational modifications. Copyright © 2015 John Wiley & Sons, Ltd.