Improved mass spectrometry compatibility is afforded by ammoniacal silver staining.
Sequence coverage in MS analysis of protein digestion-derived peptides is a key issue for detailed characterization of proteins or identification at low quantities. In gel-based proteomics studies, the sequence coverage greatly depends on the protein detection method. It is shown here that ammoniacal silver detection methods offer improved sequence coverage over standard silver nitrate methods, while keeping the high sensitivity of silver staining. With the development of 2D-PAGE-based proteomics, another burden is placed on the detection methods used for protein detection on 2-D-gels. Besides the classical requirements of linearity, sensitivity, and homogeneity from one protein to another, detection methods must now take into account another aspect, namely their compatibility with MS. This compatibility is evidenced by two different and complementary aspects, which are (i) the absence of adducts and artefactual modifications on the peptides obtained after protease digestion of a protein detected and digested in - gel, and (ii) the quantitative yield of peptides recovered after digestion and analyzed by the mass spectrometer. While this quantitative yield is not very important per se, it is however a crucial parameter as it strongly influences the S/N of the mass spectrum and thus the number of peptides that can be detected from a given protein input, especially at low protein amounts. This influences in turn the sequence coverage and thus the detail of the analysis provided by the mass spectrometer.
CEA-Laboratoire d'Immunochimie, DRDC/ICH, INSERM U 548, CEA-Grenoble, Grenoble, France.
Mireille
M
Chevallet
Hélène
H
Diemer
Sylvie
S
Luche
Alain
A
van Dorsselaer
Thierry
T
Rabilloud
Emmanuelle
E
Leize-Wagner
D000641:Ammonia / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007525:Isoelectric Focusing; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods*; D015203:Reproducibility of Results; D016622:Silver Staining / Q000379:methods*
D010455:Peptides; D000641:Ammonia