Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology

TLDR

Proteome analysis typically combines two‑dimensional gel electrophoresis with mass spectrometry, yet its ability to capture the entire proteome is still uncertain. The study visualized over 1,500 protein features by silver staining a narrow pH 4.9–5.7 2D gel loaded with 0.5 mg of yeast protein and identified 50 spots from a 4 cm² region by mass spectrometry. Despite high sample load and extended separation, low‑codon‑bias proteins were not detected unless fractionated, showing that the wide dynamic range of protein expression limits 2DE‑MS for medium‑to‑low abundance proteins and thus its overall proteome coverage.

Abstract

Proteome analysis is most commonly accomplished by a combination of two-dimensional gel electrophoresis (2DE) to separate and visualize proteins and mass spectrometry (MS) for protein identification. Although this technique is powerful, mature, and sensitive, questions remain concerning its ability to characterize all of the elements of a proteome. In the current study, more than 1,500 features were visualized by silver staining a narrow pH range (4.9–5.7) 2D gel in which 0.5 mg of total soluble yeast protein was separated. Fifty spots migrating to a region of 4 cm 2 were subjected to MS protein identification. Despite the high sample load and extended electrophoretic separation, proteins from genes with codon bias values of <0.1 (lower abundance proteins) were not found, even though fully one-half of all yeast genes fall into that range. Proteins from genes with codon bias values of <0.1 were found, however, if protein amounts exceeding the capacity of 2DE were fractionated and analyzed. We conclude that the large range of protein expression levels limits the ability of the 2DE-MS approach to analyze proteins of medium to low abundance, and thus the potential of this technique for proteome analysis is likewise limited.