Mass spectrometric-based approaches in quantitative proteomics.
Classically, experiments aimed at studying changes in protein expression have always followed a small set of proteins. This focused approach was necessary since tools to efficiently analyze large numbers of proteins were simply not available. Large-scale quantitative proteomics promises to produce reams of data that previously would have taken decades to measure with classical methods. Mass spectrometry is already a well-established protein identification tool and recent methodological developments indicate that it can also be successfully applied to extract quantitative data of protein abundance. From the first reports 4 years ago, numerous schemes to take advantage of stable isotope nuclei incorporation in proteins and peptides have been developed. Here we review the benefits and pitfalls of some of the most commonly used protocols, focusing on a procedure now being used extensively in our laboratory, stable isotope labeling with amino acids in cell culture (SILAC). The basic theory, application, and data analysis of a SILAC experiment are discussed. The emerging nature of these techniques and the rapid pace of technological development make forecasting the directions of the field difficult but we speculate that SILAC will soon be a key tool of quantitative proteomics.
Ong
Shao-En
SE
Center for Experimental Bioinformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, M 5230 Odense, Denmark.
Foster
Leonard J
LJ
Mann
Matthias
M
D000596:Amino Acids / Q000032:analysis / Q000737:chemistry; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D005591:Chemical Fractionation; D006801:Humans; D007553:Isotope Labeling; D007554:Isotopes / Q000032:analysis; D013058:Mass Spectrometry / Q000379:methods* / Q000639:trends; D040901:Proteomics / Q000379:methods* / Q000639:trends; D012680:Sensitivity and Specificity
D000596:Amino Acids; D007554:Isotopes