Identification of protein phosphorylation sites by combination of elastase digestion, immobilized metal affinity chromatography, and quadrupole-time of flight tandem mass spectrometry.
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(alpha)-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.
Schlosser
Andreas
A
Central Spectroscopy, German Cancer Reserch Center, Germany.
Bodem
Jochen
J
Bossemeyer
Dirk
D
Grummt
Ingrid
I
Lehmann
Wolf Dieter
WD
D002846:Chromatography, Affinity / Q000379:methods*; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism; D030562:Databases, Protein; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010196:Pancreatic Elastase / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D035463:Pol1 Transcription Initiation Complex Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism
D007527:Isoenzymes; D010750:Phosphoproteins; D035463:Pol1 Transcription Initiation Complex Proteins; C428423:RRN3 protein, human; D011994:Recombinant Proteins; D014157:Transcription Factors; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; D017868:Cyclic AMP-Dependent Protein Kinases; C463959:protein kinase A Calpha; D010196:Pancreatic Elastase