Double Standards in Quantitative Proteomics

Abstract

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 ( The proteome is generally defined as the total protein complement of a genome present in cells and/or tissue (1). Through splice variation and/or post-translational modifications the proteome is several orders of magnitude more complex than the genome. One extra factor that adds to the relative complexity of the proteome is that protein abundance varies over time, either as a reaction to changes in the environment or during development. To understand these dynamic processes, which may lead to indications why e.g. "healthy" cells are different from "diseased" or "stressed" cells or how cells change during differentiation, it is not only important to identify which proteins are involved but also to measure their differential expression levels. It is especially this latter notion that makes quantitative protein profiling an essential part of proteomics, which requires technologies that accurately, reproducibly, and comprehensively quantify the protein content in biological samples (2-4).Traditionally, and probably still, the most frequently used method to investigate differential protein abundances in large scale proteomic experiments on protein mixtures from cellular extracts or tissue is by two-dimensional (2D) 1 gel electrophoresis (5-9). In such experiments proteins are separated by their pI and molecular weight on a 2D gel and subsequently stained for visualization. ...

Comparison of Full Versus Partial Metabolic Labeling for Quantitative Proteomics Analysis in Arabidopsis thaliana

“…catalase 3), results are generally consistent across replicates. The variability we observe among biological replicates via both full and partial metabolic labeling is comparable to that observed in several previously published studies using metabolic labeling (9,14,24).…”

“…Additionally full 15 N metabolic labeling has been applied for top-down proteomics (12) and has been adapted for use in conjunction with two-dimensional gel electrophoresis (13). Finally as an indication of its reliability, metabolic labeling has served as the standard by which other proposed quantitative proteomics techniques including DIGE (14) and spectral counting (15) have been evaluated.…”

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“…Moreover, with the high sensitivity and dynamic range afforded by these dyes, 2-D DIGE can give more accurate quantitation than silver staining [15]. Consequently, the correlation between quantitation by 2-D DIGE and metabolic stable isotope labeling is exceptionally good [16]. In addition, this method reduces the excessive time and labor costs encountered with standard 2-DE.…”

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“…A good correlation was found between protein samples in a study when quantitative comparisons between DIGE and an isotope labelling technique were conducted. Both LC-and gel-based technologies were more or less complementary, not only in protein identification but also in protein quantification (Kolkman et al, 2005). Both 2-DE and in-solution trypsin digestion of total proteins followed by MudPIT analysis were applied to identify a total of 91 proteins from different groups that are responsible for oil body organelles in rape seed embryonic cells.…”