A fast method for quantitative proteomics based on a combination between two‐dimensional electrophoresis and ¹⁵N‐metabolic labelling

Abstract

We provide a method for accurate protein quantitation that uses two-dimensional (2-D) gel electrophoresis for protein separation, but does not require extensive statistical analysis of staining intensities on gels. Instead, accurate quantitation occurs on the mass spectrometer (MAS) on multiple peptides to provide statistical evidence. In an example study, Sulfolobus solfataricus cells were grown on the carbon sources glucose, fructose and glutamate. The glucose phenotype (reference) was grown on (15)N-enriched medium. Next, the glutamate and the fructose phenotypes are mixed with the reference and two 2-D gels are created. Staining intensities of gel spots in this case are used for initial, semiquantitative assessment of differential expression. On this basis, spots are selected for accurate quantitation on the MAS. A number of differentially expressed proteins were found, for example: a (25.2 +/- 8.2)-fold upregulation of isocitrate lyase and a (7.14 +/- 0.82)-fold downregulation of glucose dehydrogenase on glutamate compared to glucose. With this protocol, intergel and interlaboratory comparisons are facilitated, since the light and heavy versions of a protein are equally affected by variations in sample preparation and buffer composition. Because the statistical evidence is gathered on the MAS, the need to run vast numbers of gels is removed.

Identification of the Missing Links in Prokaryotic Pentose Oxidation Pathways

“…The proteome of S. solfataricus P2 was studied with a combination of two-dimensional gel electrophoresis, 15 N metabolic labeling, and tandem mass spectrometry as previously described (24,25). Two separate growth experiments were set up: 1) S. solfataricus with D-Ara as the carbon source and ( 14 NH 4 ) 2 SO 4 as the nitrogen source; and 2) S. solfataricus with D-Glu as the carbon source and ( 15 NH 4 ) 2 SO 4 as the nitrogen source.…”

Multidimensional liquid phase protein separations in conjunction with stable isotope labelling for quantitative proteomics

“…An alternative workflow was recently suggested which reduces the number of required replicate gels. This method uses stable isotope labelling in conjunction with 2-DE [26]. Staining intensity was still used to select a set of spots that potentially contains differentially expressed proteins; however, accurate quantification was performed on the mass spectrometer.…”

“…In addition, rapid quantitative comparisons were also performed by simply subtracting the 14 N from the 15 N chromatogram using Chromeleon software to highlight the fractions for further LC-MS/MS analysis. Protein quantitation using the stable isotope labelled peptides in the mass spectrometer was performed essentially as previously described [26]. Peptide peak areas in the TOF-MS scans were integrated over time using the LC-MS reconstruct tool in Analyst Software (Applied Biosystems, Foster City, USA).…”

Protein-based stable isotope probing (Protein-SIP) reveals active species within anoxic mixed cultures

“…The metabolic incorporation of stable isotope-labelled substrate into proteins has been used to elucidate the carbon metabolism of the hyperthermophilic Sulfolubus solfataricus (Snijders et al, 2006) using 15 N-containing medium, which was compared to a parallel culture grown on 14 N medium. In another approach, the level of metabolic incorporation was used to determine half-life times of proteins by analyzing the incorporation of [ 13 C 6 ]-glucose in heat-shocked HeLa cells or of 15 NH 4 þ into bacterial proteins in labelling experiments (Cargile et al, 2004;Snijders et al, 2005a).…”