Proteomics: moving from a discovery to a quality assurance tool

Abstract

Proteomics is an evolving field that encompasses an array of techniques and instruments used to analyze proteins on a grand scale. At the most basic level, proteomic methods are used to identify proteins (qualitative proteomics) or to determine how much of a particular protein is present (quantitative proteomics). More sophisticated proteomic techniques are available to characterize proteins that are altered through posttranslational modification or to understand how proteins interact with one another within complex biologic networks.1 In this issue of TRANSFUSION, Thon and colleagues2 used several distinct proteomics techniques to study changes in the platelet (PLT) “proteome” that occur when PLTs are stored under standard conditions for up to 7 days. The proteomics methods employed included both gel-based (two-dimensional gel electrophoresis–differential gel electrophoresis [DIGE]) and solution-phase techniques (isotope-codes affinity tagging [ICAT] and isotope tagging for relative and absolute quantitation [iTRAQ]). The latter are termed “peptide-centric” by the authors because only small pieces of the protein (<20 amino acids long) are analyzed across experimental samples. The “protein-centric” gel-based methods separate proteins by differential mobility in their native state. Although gel-based methods are straightforward, they remain technically challenging when used to study complex protein mixtures. Thon and colleagues utilized proteomic techniques that still have limitations despite being reasonably mature. As might be expected, gel-based techniques were not particularly sensitive to changes in the PLT proteome with storage, identifying only a small number of protein differences between fresh and stored PLTs. Even with optimized storage conditions, we might expect more PLT proteins to be altered after 7 days on an agitator. Overall, only 93 proteins were identified by gel-based methods, which is comparable to other studies when redundant proteins are removed.3 The reliability of two-dimensional (2D) gels has been improved for quantitative purposes with the advent of DIGE, where samples are differentially labeled with fluorescent dyes (e.g., Cy3, Cy5) and simultaneously analyzed in a single gel. DIGE is a major advance in quantitative gel-based proteomics; however, it still suffers from the limitations of any gel-based system including gel-to-gel reproducibility. Despite better standardization of gels and robotics to pick and digest protein spots, such protein-centric techniques remain cumbersome and not easily applied to large-volume screening. Other quantitative proteomic approaches are being used more frequently by investigators wishing to compare protein expression levels between samples. These methods vary in their ability to simultaneously identify and quantify peptides. In addition to DIGE, Thon and coworkers used two other techniques including ICAT and iTRAQ. Based on the PLT storage data, it appears that iTRAQ is one of the most promising technologies, as suggested by others who have compared these methods.4 ICAT reagents, which were commercially available before the iTRAQ variety, have been successfully utilized in similar settings. These reagents have limitations that hinder their resolving power when dealing with complex samples, but several of the major shortcomings of ICAT have been addressed with iTRAQ reagents. Both ICAT and iTRAQ do require sophisticated and expensive instruments at this time, along with specialized analysis software that facilitates quantifying the differentially labeled peptides. To comprehensively study a proteome, it is well accepted that complementary techniques must be utilized as emphasized by Thon's group. The choice of techniques will depend on the ultimate goals of the study. The earliest “fishing” stages of proteomic experiments determine the universe of proteins that might be altered by a process, in this case, PLT storage. This strategy will produce a list of potential “bi