High MS-compatibility of silver nitrate-stained protein spots from 2-DE gels using ZipPlates and AnchorChips for successful protein identification.
The availability of easy-to-handle, sensitive, and cost-effective protein staining protocols for 2-DE, in conjunction with a high compatibility for subsequent MS analysis, is still a prerequisite for successful proteome research. In this article we describe a quick and easy-to-use methodological protocol based on sensitive, homogeneous, and MS-compatible silver nitrate protein staining, in combination with an in-gel digestion, employing the Millipore 96-well ZipPlate system for peptide preparation. The improved quality and MS compatibility of the generated protein digests, as compared to the otherwise weakly MS-compatible silver nitrate staining, were evaluated on real tissue samples by analyzing 192 Coomassie-stained protein spots against their counterparts from a silver-stained 2-DE gel. Furthermore, the applicability of the experimental setup was evaluated and demonstrated by the analysis of a large-scale MALDI-TOF MS experiment, in which we analyzed an additional ~1000 protein spots from 2-DE gels from mouse liver and mouse brain tissue.
Nebrich
Grit
G
Institute for Human Genetics Humboldt-University Berlin, Charité, Berlin, Germany.
Herrmann
Marion
M
Sagi
Dijana
D
Klose
Joachim
J
Giavalisco
Patrick
P
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D007202:Indicators and Reagents / Q000737:chemistry; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011506:Proteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis; D015203:Reproducibility of Results; D012394:Rosaniline Dyes / Q000737:chemistry*; D012680:Sensitivity and Specificity; D017421:Sequence Analysis / Q000379:methods; D012835:Silver Nitrate / Q000737:chemistry*; D016622:Silver Staining; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D007202:Indicators and Reagents; D010446:Peptide Fragments; D011506:Proteins; D020543:Proteome; D012394:Rosaniline Dyes; C024757:Coomassie brilliant blue R; D012835:Silver Nitrate; D014357:Trypsin