Multi-dimensional liquid chromatography in proteomics--a review

Abstract

Proteomics is the large-scale study of proteins, particularly their expression, structures and functions. This still-emerging combination of technologies aims to describe and characterize all expressed proteins in a biological system. Because of upper limits on mass detection of mass spectrometers, proteins are usually digested into peptides and the peptides are then separated, identified and quantified from this complex enzymatic digest. The problem in digesting proteins first and then analyzing the peptide cleavage fragments by mass spectrometry is that huge numbers of peptides are generated that overwhelm direct mass spectral analyses. The objective in the liquid chromatography approach to proteomics is to fractionate peptide mixtures to enable and maximize identification and quantification of the component peptides by mass spectrometry. This review will focus on existing multidimensional liquid chromatographic (MDLC) platforms developed for proteomics and their application in combination with other techniques such as stable isotope labeling. We also provide some perspectives on likely future developments.

Figure 1

Typical bottom-up proteomics experimental workflow. Proteins are isolated from biological samples and enzymatically digested into peptides. Each protein generates many peptides (30-50 or more), which significantly increases the sample complexity. Peptides are separated using multidimensional liquid chromatography (MDLC) prior to mass spectrometry (MS) analyses. Various bioinformatics tools, such as database search algorithms, are employed for protein identification.

Figure 2

Current strategies for quantitative proteomics. In the label-free quantification approach, each sample (s1, etc.) is experimentally analyzed separately. The molecular information extracted from each sample is integrated during data analysis to obtain protein quantities (e.g., spectral counting or area under the curve calculation). Arrows in mass spectra denote differentially expressed peptides. With chemical labeling approaches, samples are labeled with various reagents either as proteins (typical for ICAT; upper box in middle panel) or as proteolytic peptides (as is typical with iTRAQ; lower box in middle panel), and mixed together prior to quantitative analysis by MS. Arrows in MS spectrum indicate an identical but differentially labeled peptide from s1 and s2. The different peak heights reflect differential levels of the parent protein. Metabolic labeling is possible with cultured cells that can incorporate labeled amino acids into proteins during growth in culture (box in right panel). Metabolically labeled samples are mixed together prior to protein isolation and further processed and analyzed by MDLC MS; quantification is again achieved via comparison of isotopically labeled peptides (as for chemical labeling approaches).