Liquid chromatography-mass spectrometry-based quantitative proteomics

Abstract

LC-MS-based quantitative proteomics has become increasingly applied to a wide range of biological applications due to growing capabilities for broad proteome coverage and good accuracy and precision in quantification. Herein, we review the current LC-MS-based quantification methods with respect to their advantages and limitations and highlight their potential applications.

FIGURE 1.

General workflow for LC-MS-based global proteomics. Proteins in complex biological samples are first converted into peptides by proteolytic digestion (e.g. tryptic digestion). The resulting peptide mixture is then separated by LC and ionized by electrospray before entering the mass spectrometer. In a typical data-dependent acquisition operation mode, a full MS spectrum is acquired for the peptides that are eluting from the LC column at any given time; one of the most intensive ion species (i.e. peptides) is then isolated and fragmented to obtain the MS pattern of its fragments (i.e. MS/MS spectrum). Because peptide bonds are prone to fragmentation under collision-induced dissociation conditions in the MS/MS analysis and produce predominantly b- or y-type ions (N- or C-terminal fragments carrying charge, respectively), the peptide sequence can be readily deduced from the MS/MS spectrum. This process is fully automated by searching the MS/MS spectra against protein sequence databases. Possible post-translational modifications can also be identified by including dynamic modification on certain amino acid residues (e.g. Ser, Thr, or Tyr for phosphorylation) in the database search. Quantification of each peptide is typically performed at the extracted ion chromatogram level; however, for isobaric tagging approaches (e.g. iTRAQ), quantification is carried out at the MS/MS spectrum level. PTMs, post-translational modifications.

FIGURE 2.

Schematic diagrams of three major strategies in quantitative proteomics using stable isotope labeling. A, pairwise comparison is used to compare two samples; ¹⁸O labeling, SILAC, and ICAT fall into this category. B, multiple comparison is used to compare up to four, six, or eight samples depending the isobaric tags used (i.e. 4-plex iTRAQ, 6-plex TMT, or 8-plex iTRAQ). C, large-scale comparison employs an internal standard: ¹⁸O-labeled universal reference, super-SILAC mixture, or synthetic isotope-labeled peptides. AQUA, absolute quantification.