Phospho-proteomics: evaluation of the use of enzymatic de-phosphorylation and differential mass spectrometric peptide mass mapping for site specific phosphorylation assignment in proteins separated by gel electrophoresis.
Detection of phosphorylated proteins as well as assignment of the phosphorylated sites in such proteins is a major challenge in proteomics. In the present study we evaluate the use of enzymatic de-phosphorylation in combination with differential peptide mass mapping for identification of phosphorylated peptides in peptide mixtures derived from in-gel digested phospho-proteins. Phospho-peptides could be identified provided that improved sample preparation methods prior to mass spectrometric analysis were used. An attempt to identify the proteins visualized by [32P] autoradiography in a proteomics study and their phosphorylation sites, demonstrated that protein identification was possible whereas reliable identification of the phospho-peptides requires more protein than normally available in our proteomics studies.
Larsen
M R
MR
Department of Biochemistry and Molecular Biology, Center for Proteome Analysis, University of Southern Denmark, Denmark.
Sørensen
G L
GL
Fey
S J
SJ
Larsen
P M
PM
Roepstorff
P
P
D000469:Alkaline Phosphatase; D000818:Animals; D001665:Binding Sites; D002364:Caseins / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007633:Keratins / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D010047:Ovalbumin / Q000737:chemistry; D020648:Peptide Elongation Factor 1 / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping / Q000379:methods*; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D020543:Proteome / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin
D002364:Caseins; D020648:Peptide Elongation Factor 1; D010446:Peptide Fragments; D010750:Phosphoproteins; D020543:Proteome; D007633:Keratins; D010047:Ovalbumin; D000469:Alkaline Phosphatase; D014357:Trypsin