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Identification of protein phosphorylation sites by combination of elastase digestion, immobilized metal affinity chromatography, and quadrupole‐time of flight tandem mass spectrometry
Author(s) -
Schlosser Andreas,
Bodem Jochen,
Bossemeyer Dirk,
Grummt Ingrid,
Lehmann Wolf Dieter
Publication year - 2002
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/1615-9861(200207)2:7<911::aid-prot911>3.0.co;2-k
Subject(s) - chemistry , phosphoprotein , chromatography , phosphorylation , mass spectrometry , tandem mass spectrometry , protein phosphorylation , affinity chromatography , electrospray , protein kinase a , biochemistry , enzyme
Using the combination of in‐gel elastase digestion, immobilized metal affinity chromatography and high resolution electrospray tandem mass spectrometry, the phosphorylation sites of two phosphoproteins were determined. Complete coverage of all phosphorylation sites (Ser10, Ser139, Thr197, Ser338) of the model phosphoprotein protein kinase A C α ‐subunit could be achieved by this strategy in the low picomole range. In addition, three previously unknown phosphorylation sites of the human transcription initiation factor TIF‐IA (Ser44, Ser170, Ser172) were determined in this way. Both phosphoproteins could be identified in a protein database on the basis of their elastase generated phosphopeptides alone. The data of seven phosphopeptides were used for identification of protein kinase A, and those of two phosphopeptides for TIF‐IA, respectively. The accurate mass data of the electrospray mass spectra recorded at high resolution are extremely useful for sequencing of the elastase generated phosphopeptides and for protein identification by database searching.