Quantitative protein profiling using two-dimensional gel electrophoresis, isotope-coded affinity tag labeling, and mass spectrometry.
Quantitative protein profiling is an essential part of proteomics and requires new technologies that accurately, reproducibly, and comprehensively identify and quantify the proteins contained in biological samples. We describe a new strategy for quantitative protein profiling that is based on the separation of proteins labeled with isotope-coded affinity tag reagents by two-dimensional gel electrophoresis and their identification and quantification by mass spectrometry. The method is based on the observation that proteins labeled with isotopically different isotope-coded affinity tag reagents precisely co-migrate during two-dimensional gel electrophoresis and that therefore two or more isotopically encoded samples can be separated concurrently in the same gel. By analyzing changes in the proteome of yeast (Saccharomyces cerevisiae) induced by a metabolic shift we show that this simple method accurately quantifies changes in protein abundance even in cases in which multiple proteins migrate to the same gel coordinates. The method is particularly useful for the quantitative analysis and structural characterization of differentially processed or post-translationally modified forms of a protein and is therefore expected to find wide application in proteomics research.
Smolka
Marcus
M
Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, Sao Paulo 13083-970, Brazil.
Zhou
Huilin
H
Aebersold
Ruedi
R
D000345:Affinity Labels / Q000032:analysis* / Q000138:chemical synthesis / Q000378:metabolism; D000418:Albumins / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002645:Chickens; D003545:Cysteine / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D007553:Isotope Labeling; D007768:Lactalbumin / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008970:Molecular Weight; D010047:Ovalbumin / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism
D000345:Affinity Labels; D000418:Albumins; D010446:Peptide Fragments; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D010047:Ovalbumin; D007768:Lactalbumin; D014357:Trypsin; D003545:Cysteine