Linke, Dennis, Koudelka, Tomas, Becker, Alexander and Tholey, Andreas (2015) Identification and relative quantification of phosphopeptides by a combination of multi-protease digestion and isobaric labeling. Rapid Communications in Mass Spectrometry, 29 (10). pp. 919-926. DOI 10.1002/rcm.7185.

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Abstract

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.

Document Type:	Article
Research affiliation:	Kiel University > Kiel Marine Science
Refereed:	Yes
Open Access Journal?:	Yes
Date Deposited:	01 Feb 2018 12:54
Last Modified:	01 Feb 2018 12:54
URI:	https://oceanrep.geomar.de/id/eprint/41794

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