Double standards in quantitative proteomics: direct comparative assessment of difference in gel electrophoresis and metabolic stable isotope labeling.
Quantitative protein expression profiling is a crucial part of proteomics and requires methods that are able to efficiently provide accurate and reproducible differential expression values for proteins in two or more biological samples. In this report we evaluate in a direct comparative assessment two state-of-the-art quantitative proteomic approaches, namely difference in gel electrophoresis (DiGE) and metabolic stable isotope labeling. Therefore, Saccharomyces cerevisiae was grown under well defined experimental conditions in chemostats under two single nutrient-limited growth conditions using (14)N- or (15)N-labeled ammonium sulfate as the single nitrogen source. Following lysis and protein extraction from the two yeast samples, the proteins were fluorescently labeled using different fluorescent CyDyes. Subsequently, the yeast samples were mixed, and the proteins were separated by two-dimensional gel electrophoresis. Following in-gel digestion, the resulting peptides were analyzed by mass spectrometry using a MALDI-TOF mass spectrometer. Relative ratios in protein expression between these two yeast samples were determined using both DiGE and metabolic stable isotope labeling. Focusing on a small, albeit representative, set of proteins covering the whole gel range, including some protein isoforms and ranging from low to high abundance, we observe that the correlation between these two methods of quantification is good with the differential ratios determined following the equation R(Met.Lab.) = 0.98R(DiGE) with r(2) = 0.89. Although the correlation between DiGE and metabolic stable isotope labeling is exceptionally good, we do observe and discuss (dis)advantages of both methods as well as in relation to other (quantitative) approaches.
Kolkman
Annemieke
A
Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.
Dirksen
Eef H C
EH
Slijper
Monique
M
Heck
Albert J R
AJ
D015180:Electrophoresis, Gel, Two-Dimensional; D005456:Fluorescent Dyes; D007553:Isotope Labeling; D009587:Nitrogen Isotopes; D020543:Proteome / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005456:Fluorescent Dyes; D009587:Nitrogen Isotopes; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins