Proteomic Analysis in Cardiovascular Diseases
The complete sequencing of the human genome in 2001 showed that fewer than 3000 genes encode more than a million proteins. One gene can encode not only a single protein but several proteins, depending on the number of transcriptional (mRNA splicing) and post-translational (glycosylation) modifications. Currently the Swiss-Prot protein database contains 276 256 annotated proteins in UniProtKB/Swiss-Prot (release 54.0 of 4 July 2007) and 4 672 908 proteins translated from the European Molecular Biology Laboratory (EMBL) nucleotide sequence in UniProtKB/TrEMBL (release 37.0 of 4 July 2007). These data suggest that the genome does not reflect the organism’s functional complexity, which is inversely correlated with its biological complexity.Classical strategies have measured mRNA transcripts or proteins according to a priori hypothesis based on literature reviews. Because of the need for global approaches without any a priori hypothesis, techniques known as ‘OMICS’ have been developed for use in analysing genes (genomics), mRNA (transcriptomics), proteins (proteomics) and metabolites (metabolomics). These techniques should make it possible to elucidate the functional role of several genes or gene products and thus better understand phenotypes linked to various types of disease status. The importance of measuring proteins has become clear, as mRNA transcripts cannot be directly correlated with protein expression 3 and post-translational modifications, such as phosphorylation and glycosylation are known to produce several proteins with different functions from a single gene. it summarizes the most common of the more than 200 post-translational modifications that have so far been described. These modifications can be seen only at the protein level and they play a role in protein abundance and in cellular localization. The notion of cartography for protein level expression is an old one, dating back to the publication of a technique to separate proteins simultaneously in 2D electrophoresis gel. The term ‘proteome’, however, was first used in 1994 by Wilkins and defined as all proteins expressed by a genome, cell or tissue.
Proteomics is the analysis of the proteome to characterize qualitatively and quantitatively all of the proteins presented in a biological sample obtained in defined conditions. Differential proteomics is the comparison of protein profiles from various samples obtained in different conditions to identify proteins differentially expressed without any a priori hypothesis.
Proteomic analysis strategies
Several strategies, summarized, are often used in differential proteomic analysis. Samples can be analysed with or without preliminary treatment. In some conditions, the presence of major proteins detection can make it difficult to detect rare proteins or peptides. These major proteins can be either discarded or equalized to enrich the samples in minor proteins.
List of abbreviations:
| 2 DE | bi-dimensional electrophoresis | pI | isoelectric point |
| IPG | immobilized pH gradient | SELDI-TOF | Surface-Enhanced Laser Desorption/Ionization Time of Flight |
Proteomic analysis can be divided in four main steps: (i) processing samples, which has been described above; (ii) separation of proteins and peptides; (iii) detection and quantification of proteins and peptides; and (iv) purification and identification of the corresponding proteins by mass spectrometry (MS) and bioinformatic analysis.
Proteins can be separated either by electrophoresis (mono- or two-dimensional) or directly by MS techniques such as Surface-Enhanced Laser Desorption/Ionization Time of Flight (SELDI-TOF) or liquid chromatography-MS. For direct MS analysis, quantification relies on the relative intensity of peaks. Bioinformatic tools make it possible to target the spots or peaks of interest that should be identified. Spots are directly cut into the 2D gel, and for SELDI-TOF purified peaks are cut into 1D gel, for trypsin digestion and MS analysis. Other differential proteomics strategies allow direct quantification of peptides by MS (ICAT: isotope-coded affinity tag) or MS (–MS) (Isobaric Tagging Reagents for Quantitative Proteomic Analysis (iTRAQ)) after labelling, trypsin digestion and MS (–MS) analysis. This multiplex approach mixes many samples after labelling with different tags that allow them to be differentiated in MS analysis or 2D-differential gel electrophoresis (DIGE). The latter uses protein labelling of each sample by a specific fluorophore before mixing and electrophoresis. In most cases, MS that combines peptide mapping and MS-MS data finally identifies the selected proteins.
Two-dimensional gel electrophoresis
Two-dimensional electrophoresis (2DE) is a two-step technique that was developed 30 years ago that makes it possible to separate proteins: (i) by their isoelectric point (pI), a step called isoelectrofocusing and (ii) by their molecular weight, using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). Reliable proteomic analysis requires a reproducible technology of protein separation that uses immobilized pH gradient (IPG), to improve reproducibility and resolution.
After 2DE, the proteins can be visualized with either universal or specific stains. Stain properties that should be considered are sensitivity, linear dynamic range, reproducibility and compatibility for protein identification by MS. Standard universal stains are silver nitrate, coomassie blue and more recently fluorescent probes (SYPRO Ruby, FLAMINGO). Specific stains allow the visualization of, for example, phosphorylated proteins (ProQDiamond). This staining technique provides quantitative screening to analyse essentially hydrophilic proteins in a molecular mass range between 15 and 100 kDa and a pH range of 3–8. Protein detection then uses non-specific labelling such as coomassie blue, silver nitrate or fluorescent probes. Quantification is carried out directly by densitometry using an appropriate scanner and assumes that spot intensity is directly proportional to spot abundance.
The development of 2D bioinformatic software (Progenesis Samespots, Platinium, etc.) allows the detection of specific polypeptidic spots and their intensities as well as a comparative analysis of several 2D gels. These statistical tools make it possible to select spots whose intensity is modulated between several experimental conditions.
SELDI-TOF analysis
This technology has some advantages over 2DE for proteins that are hydrophobic, very basic or of low molecular mass (< 20 kDa). 7 The key elements are ProteinChips arrays of various chemical (anionic (Q10) or cationic (CM10)), hydrophobic (H50) and hydrophilic (NP20) surfaces that can divide the proteome by capturing proteins with affinity for the surface before the direct MS analysis. Spectra obtained with a ProteinChip system time-of-flight mass spectrometer can be analysed by three types of software (Biomarker Wizard, Biomarker Pattern Software and Ciphergen Express). The intensity of peaks obtained in the spectra under different experimental conditions are compared to find potential markers.
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