This review focuses on techniques for quantification and identification in proteomics by stable isotope coding. Methods are examined for analyzing expression, post-translational modifications, protein:protein interactions, single amino acid polymorphism, and absolute quantification. The bulk of the quantification literature in proteomics focuses on expression analysis, where a wide variety of methods targeting different features of proteins are described. Methods for the analysis of post-translational modification (PTM) focus primarily on phosphorylation and glycosylation, where quantification is achieved in two ways, either by substitution or tagging of the PTM with an isotopically coded derivatizing agent in a single process or by coding and selecting PTM modified peptides in separate operations. Absolute quantification has been achieved by age-old internal standard methods, in which an isotopically labeled isoform of an analyte is synthesized and added to a mixture at a known concentration. One of the surprises is that isotope coding can be a valuable aid in the examination of intermolecular association of proteins through stimulus:response studies. Preliminary efforts to recognize single amino acid polymorphism are also described. The review ends with the conclusion that (1) isotope ratio analysis of protein concentration between samples does not necessarily relate directly to protein expression and rate of PTM and (2) that multiple new methods must be developed and applied simultaneously to make existing stable isotope quantification methods more meaningful. Although stable isotope coding is a powerful, wonderful new technique, multiple analytical issues must be solved for the technique to reach its full potential as a tool to study biological systems.

A new strategy for specifically targeting cysteine-containing peptides in a tryptic digest is described. The method is based on quantitatively derivatizing cysteine residues with a quaternary amine tag (QAT). Tags were introduced into proteins following reduction of disulfide bonds through derivatization of cysteine residues with (3-acrylamidopropyl)trimethylammonium chloride. After trypsin digestion, derivatized cysteine-containing peptides were enriched by strong cation exchange chromatography. The method was validated using model peptides and a protein. The QAT strategy has several advantages over other methods for the selection of cysteine-containing peptides. One is that it increases the ionization efficiency of cysteine-containing peptides. The other is that chromatographic selection is achieved with simple, robust cation exchange chromatography columns. As a result, this new strategy provides a simple way to facilitate enrichment of cysteine-containing peptides, thereby reducing sample complexity in bottom-up proteomics.

Various isotope coding strategies are being used today in the field of comparative proteomics. This article specifically reviews the strengths and limitations of various N-termini-directing strategies. N-termini-directed coding strategy allows for use of different chromatographic enrichment techniques. Since N-termini-directed coding strategies are global in nature, they can be utilized in studying PTMs as well as protein expression. Using different N-termini-directed coding strategies, both relative and absolute quantification of proteins can be achieved either in the MS mode or in the MS/MS mode. The review ends with the conclusion that significant improvements have been made in the last decade. Among various issues, a need still exists for a better understanding of the kinetic issues in proteomics, relative protein pool sizes for different proteins and the issue of stimulus-induced changes in protein aggregation. Another critical issue that needs to be addressed in great detail is the role of PTMs in regulation.

Stable isotope coding continues to be a powerful approach in comparative proteomics. This review focuses on recent developments in stable isotope coding-based strategies targeted towards protein expression, protein interactions with other biomolecules, post-translational modifications and absolute quantification. The focus of the bulk of proteomics studies is still on protein expression. An important recent application of isotope coding has been in organelle proteomics. The review ends with the conclusion that isotope coding remains an integral part of quantitative proteomics. There is, however, a need to develop coding strategies which can differentiate changes in protein expression and post-translational modification, address issues of protein dynamic range and facilitate real-time detection of proteins which show a statistically significant change after stimulus.

Soybean (Glycine max) is considered a major allergenic food. Gly m 4 is one of several soybean allergens that has been identified to cause an allergic reaction, typically the symptoms are localized effects including the skin, gastrointestinal tract, or respiratory tract. Soybean allergens are considered a complete food allergen in that they are capable of inducing specific IgE as well as eliciting a range of severity from mild rashes up to anaphylaxis. In this study, we have isolated, purified, and characterized an endogenous Gly m 4 protein. The endogenous protein has 88.0% sequence homology with the theoretically predicted Gly m 4 sequence. Following detailed characterization, an assay was developed for quantification of endogenous Gly m 4 using two-dimensional liquid chromatography with ultraviolet and mass spectrometric detection (2DLC-UV/MS). A linear relationship (R(2) > 0.99) was observed over the concentration range of 12.5-531.7 μg/mL. Over the linear range, the assay recoveries (percent relative error, % RE) ranged from -1.5 to 10.8%. The assay precision (percent coefficient of variation, % CV) was measured at three different Gly m 4 levels on each of the 4 days and did not exceed 11.2%. The developed method was successfully applied to quantify Gly m 4 level in 10 commercial soybean lines. To the best of our knowledge, this represents the first quantitative assay for an intact endogenous Gly m 4 protein.