Peptide mass fingerprint sequence coverage from differently stained proteins on two-dimensional electrophoresis patterns by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS).
Identification of proteins separated by two-dimensional electrophoresis (2-DE) is a necessary task to overcome the purely descriptive character of 2-DE and a prerequisite to the construction of 2-DE databases in proteome projects. Matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has a sensitivity for peptide detection in the lower fmol range, which should be sufficient for an analysis of even weakly silver-stained protein spots by peptide mass fingerprinting. Unfortunately, proteins are modified by the silver staining procedure, leading to low sequence coverage. Omission of glutaraldehyde increased the sequence coverage, but this improved sequence coverage is still clearly below the sequence coverage starting with Coomassie Brilliant Blue (CBB) R-250-stained spots. Other factors additionally seem to modify proteins during silver staining. By decreasing the protein amount, the advantage of very sensitive detection on the gel is lost during identification, because the resulting low sequence coverage is not sufficient for secure identification. Low-quantity proteins can be identified better starting with CBB G-250 or Zn-imidazol-stained proteins. In contrast, for high-quantity CBB R-250-stained spots, a sequence coverage of up to 90% can be obtained by using only one cleaving enzyme, and up to 80% was reached for medium-quantity spots after combination of tryptic digest with Asp-N- and Glu-C digest.
Scheler
C
C
Technical University Berlin, Max-Volmer-Institute, Germany.
Lamer
S
S
Pan
Z
Z
Li
X P
XP
Salnikow
J
J
Jungblut
P
P
D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D007093:Imidazoles; D009124:Muscle Proteins / Q000032:analysis; D009206:Myocardium / Q000737:chemistry / Q000201:enzymology; D010449:Peptide Mapping / Q000295:instrumentation* / Q000379:methods*; D012394:Rosaniline Dyes / Q000378:metabolism; D017421:Sequence Analysis / Q000379:methods; D016622:Silver Staining; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013194:Staining and Labeling; D015032:Zinc
D007093:Imidazoles; D009124:Muscle Proteins; D012394:Rosaniline Dyes; C024757:Coomassie brilliant blue R; D015032:Zinc