Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

Abstract

Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist.

Comparative proteomic analysis of the contractile-protein-depleted fraction from normal versus dystrophic skeletal muscle

“…Disorder-specific protein markers play a central diagnostic, prognostic, and therapeutic role in skeletal muscle pathology and the systematic application of proteomics has greatly expanded the range of biomarkers for neuromuscular disorders [6]. Proteome-wide studies combine protein separation methods, such as high-resolution two-dimensional gel electrophoresis [7][8][9] and liquid chromatography [10], with sophisticated mass spectrometric techniques to determine potential changes in protein concentration, isoform expression patterns, protein-protein interactions and posttranslational modifications [11][12][13].…”

Coomassie Blue as a Near-infrared Fluorescent Stain: A Systematic Comparison With Sypro Ruby for In-gel Protein Detection

“…As the original top-down approach to proteomic analyses, among its many attributes the high resolution achievable by two dimensional gel-electrophoresis (2DE) 1 ensures that it remains an effective analytical technology despite the appearance of alternatives. However, in-gel detection remains a limiting factor for gel-based analyses; available technology generally permits the detection and quantification of only relatively abundant proteins (35). Many critical components in normal physiology and also disease may be several orders of magnitude less abundant and thus below the detection threshold of in-gel stains, or indeed most techniques.…”

Advances in purification and separation of posttranslationally modified proteins

“…The detection limit is reportedly 25-100 ng for free carbohydrates, but the reported sensitivity for glycoproteins is in the range 1-10 Pg [171]. Sensitivity has been slightly improved by combining the PAS reaction with alcian blue or dansyl chloride staining, or by tagging periodate-oxidized aldehydes with biotin hydrazide and detecting the biotin (by streptavidin-bound peroxidase for instance), but these labeling procedures are seldom utilized in practice [171][172][173]. The fluorescent dye Pro-Q Emerald (Life Technologies) also reacts with periodic acid-oxidized carbohydrate groups, but the protocols are more rapid than previously mentioned PAS-based labeling procedures and reportedly at least 50-fold more sensitive [174].…”

“…The fluorescent dye Pro-Q Emerald (Life Technologies) also reacts with periodic acid-oxidized carbohydrate groups, but the protocols are more rapid than previously mentioned PAS-based labeling procedures and reportedly at least 50-fold more sensitive [174]. Similar alternatives have been produced, for example by Sigma (Glycoprofile III fluorescent kit; discontinued) and Pierce (Krypton Glycoprotein staining kit) [172]. Specific protein glycosylation PTMs can be detected after electrophoretic separation by Western blotting using glycan-specific antibodies, or overlays with radio-labeled or enzymeconjugated lectins.…”