Application for proteomic techniques in studying osteoarthritis: a review

Abstract

After the genomic era, proteomic corresponds to a wide variety of techniques that study the protein content of cells, tissue, or organism and that allow the isolation of protein of interest. It offers the choice between gel-based and gel-free methods or shotgun proteomics. Applications of proteomic technology may concern three principal objectives in several biomedical or clinical domains of research as in osteoarthritis: (i) to understand the physiopathology or underlying mechanisms leading to a disease or associated with a particular model, (ii), to find disease-specific biomarker, and (iii) to identify new therapeutic targets. This review aimed at gathering most of the data regarding the proteomic techniques and their applications to osteoarthritis research. It also reported technical limitations and solutions, as for example for sample preparation. Proteomics open wide perspectives in biochemical research but many technical matters still remain to be solved.

Keywords: osteoarthritis; proteomic.

Figure 1

Proteomic strategies to quantify and identify biomarkers of osteoarthritis from different kind of samples. Proteins extracted from samples could be separated by electrophoresis in one (1DE) or two (2DE) dimensions. Proteins are labeled before or after gel migration to perform quantification. After in-gel digestion proteins are identified by mass spectrometry. Gel-free methods involve the separation of digested peptides which could be quantified directly by mass spectrometry analysis. 1DE, one-dimensional electrophoresis; 2DE, two-dimensional electrophoresis; 2D-DIGE, two-dimensional-differential in-gel electrophoresis; LC, liquid chromatography; MS, mass spectrometry; MRM, multiple-reaction monitoring; SILAC, stable isotope labeling by amino acid; ICAT, Isotope-coded affinity tag; iTRAQ, isobaric tag for relative and absolute quantitation.

Figure 2

Overview of the proteomic workflow using 2D-DIGE (A) or gel-free (B) approaches for differential analysis. (A) 2D-DIGE approach (1) Proteins are extracted from sample which could be optimized for complexity reduction or particular protein identification; (2) Three fluorescent dyes were used. Samples A and B were fluorescently labeled with either Cy3 or Cy5, and a pooled internal standard is labeled with Cy2; (3) Samples are mixed and resolved in the same 2DE gel. Protein spot pattern could be visualized for each dye by selection of specific wavelength. 2D images are analyzed by specific software and internal standard is used for normalization; (4) Differentially expressed protein spots are excised from a preparative gel and identified by mass spectrometry. (B) Gel-free approach. Proteins or peptides could be labeled at different stages of sample preparation depending on SILAC, ICAT, or iTRAQ technology. Light and heavy forms of isotopic analogs are resolved by 2D-LC and then quantified and identified by mass spectrometry. SILAC: stable isotope labeling by amino acid in cell culture; ICAT: isotope-coded affinity tag; iTRAQ: Isobaric tag for relative and absolute quantitation.