A Biologist's Field Guide to Multiplexed Quantitative Proteomics

Abstract

High-throughput genomic and proteomic studies have generated near-comprehensive catalogs of biological constituents within many model systems. Nevertheless, static catalogs are often insufficient to fully describe the dynamic processes that drive biology. Quantitative proteomic techniques address this need by providing insight into closely related biological states such as the stages of a therapeutic response or cellular differentiation. The maturation of quantitative proteomics in recent years has brought about a variety of technologies, each with their own strengths and weaknesses. It can be difficult for those unfamiliar with this evolving landscape to match the experiment at hand with the best tool for the job. Here, we outline quantitative methods for proteomic mass spectrometry and discuss their benefits and weaknesses from the perspective of the biologist aiming to generate meaningful data and address mechanistic questions.

Fig. 1.

Comparison of quantitative proteomic mass spectrometry techniques. The six techniques, more fully described in the text, differ in the methods of incorporation of stable isotopes (represented as filled shapes), the number of samples directly compared, and the method of quantification. In some techniques (SILAC and NeuCode), stable isotopes are metabolically introduced during sample growth, although in others the labels are introduced after proteins have been harvested and proteolytically digested into peptides, either through introduction of a synthetic labeled peptide (AQUA) or via covalent attachment of a molecule containing the label (ICAT, iTRAQ/TMT; ICAT also employs an unlabeled molecule indicated by an open circle). Samples are then combined as indicated and introduced to the mass spectrometer. After separation of different peptide ions by mass (MS¹), all methods identify features from MS² spectra. In most methods, the quantitative data are derived from an extracted ion chromatogram (XIC) formed by monitoring intact peptide ions from MS¹ spectra over the time the feature elutes from liquid chromatography (in NeuCode, ultra-high resolution spectra are necessary to distinguish the near-isobaric masses). iTRAQ/TMT differs in this regard as relative abundances are measured directly from an MS³ spectrum.