Amino Acid-Coded Mass Tagging for Quantitative Profiling of Differentially Expressed Proteins and Modifications in Cells

Abstract

To date, large-scale protein quantitation mainly relies on high-resolution separation of complex mixtures using two-dimensional gel electrophoresis (2-DE). In comparison with 2-DE-based protein intensity displays for quantitating differentially expressed proteins, mass spectrometry (MS)-based quantitation of protein expression remains a challenging task because a mass spectrometer itself is a poor quantitative analyzer, as a result of the uneven ionization efficiency of different peptides. Recently, several techniques coupling stable isotope labeling (SIL) to MS have emerged as the primary approach for rapid, large-scale protein quantitation (1–4). There are two major strategies to quantitate proteins through SIL—chemically introducing SIL tags either after cell lysis, or in vivo/in vitro during cell growth (5–7). Chemical SIL approaches usually target at a particular residue of tryptic peptides through chemical reactions after cell lysis, thus reducing the complexity of a sample. The most representative methods are isotope-coded affinity tags (ICAT) (1) and mass-coded abundance tagging (MCAT) (4). Disadvantages include the relatively low efficiency of these chemical modification reactions and the limited abundance of target residues. On the other hand, during cell growth, for example, Oda et al. used ¹⁵N uniformly labeled medium to label all nitrogen atoms in the whole proteome, and applied this strategy to quantitate protein expression and to identify modifications (5). Our laboratory originally developed a different isotope-tagging strategy, using SIL amino acids as tag precursors that can be incorporated into cellular proteins in a residue-specific manner. These amino acidcoded mass tags (AACTs) then provide a signature for each individual protein or modification for quantitation and concurrent identification (7–13).