Comparative and Quantitative Global Proteomics Approaches: An Overview

Abstract

Proteomics became a key tool for the study of biological systems. The comparison between two different physiological states allows unravelling the cellular and molecular mechanisms involved in a biological process. Proteomics can confirm the presence of proteins suggested by their mRNA content and provides a direct measure of the quantity present in a cell. Global and targeted proteomics strategies can be applied. Targeted proteomics strategies limit the number of features that will be monitored and then optimise the methods to obtain the highest sensitivity and throughput for a huge amount of samples. The advantage of global proteomics strategies is that no hypothesis is required, other than a measurable difference in one or more protein species between the samples. Global proteomics methods attempt to separate quantify and identify all the proteins from a given sample. This review highlights only the different techniques of separation and quantification of proteins and peptides, in view of a comparative and quantitative global proteomics analysis. The in-gel and off-gel quantification of proteins will be discussed as well as the corresponding mass spectrometry technology. The overview is focused on the widespread techniques while keeping in mind that each approach is modular and often recovers the other.

Keywords: electrophoresis; fluorescent dies; isotope labelling; proteins and peptides; proteomics; proteomics: methods.

Figure 1

Flowchart of the most currently used techniques in view of a comparative and quantitative proteomics approach using a protein-based approach (panel A) or a peptide-based approach (panel B). The proteomic analysis is made up of four steps: (i) sample conditioning (not illustrated); (ii) sample preparation; (iii) separation; and (iv) quantification and identification of the proteins. The separation can be performed on proteins or peptides, by electrophoresis or chromatography. The quantification is possible either in-gel or off-gel, whereas the identification is always performed by MS. MS, mass spectrometry; HPLC: high performance liquid chromatography; IEF: isoelectric focusing; PAGE: polyacrylamide gel electrophoresis; PMF: peptide mass fingerprint; PFF: peptide fragmentation fingerprint.

Figure 2

Comparison of the proteins extracted with Triton X-100 from bovine brain capillary endothelial cells showing limited (Lim. BBB) (A) or re-induced (Re-ind. BBB) BBB functionalities (B). Digital image obtained after 2D-PAGE of the proteins separated according to their pI and MW. The gel was silver nitrate stained. The numbering corresponds to the enriched protein in each condition. Each spot was identified by peptide mass fingerprinting (PMF) and/or peptide fragmentation fingerprinting (PFF) on a Proteineer ^TM workstation (adapted with permission from [45]).

Figure 3

2D-like view illustrating the distribution of the peptides according to their retention time vs. their m/z ratio. (A) print-screen image obtained from Warp-LC Survey Viewer (Bruker Daltonics) after RP-HPLC-MALDI-TOF-MS analysis of tryptic peptides issued from the enzymatic digestion of a sample of proteins extracted from brain capillary endothelial cells; (B) detail of a particular region; (C) selection of an ion; (D) fragmentation and identification of the peptide (unpublished data from the authors’ laboratory).

Figure 4

Differences between label free quantification (A) and quantification by means of stable-isotope labelling (B). The label free quantification consists of two analyses carried out independently before their comparison. The quantification by means of stable-isotope labelling allows the direct comparison of isotope-labelled peptide pairs. LC-MS/MS, liquid chromatography coupled to tandem MS. (Adapted from [69], Creative Common Attribution License CC-BY).