Protocol for the purification of proteins from biological extracts for identification by mass spectrometry

• Published in: Nature protocols Vol. 2; no. 4; pp. 831 - 837
• Main Authors: Boschetti, Egisto, Guerrier, Luc
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 01.04.2007
• Subjects: Analysis; Biomarkers; Biomarkers - analysis; Blood Proteins - analysis; Blood Proteins - isolation & purification; Chromatography; Chromatography, Liquid - methods; Electrophoresis; Experimental data; Humans; Laboratories; Mass spectrometry; Mass Spectrometry - methods; Methods; Peptide Fragments - analysis; Peptide Fragments - isolation & purification; Peptides; Proteins; Prothrombin - analysis; Prothrombin - isolation & purification; Protocol; Scientific imaging; Separation; Sorbents; Thyroxine-Binding Proteins - analysis; Thyroxine-Binding Proteins - isolation & purification; Tissue Extracts - chemistry; France
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2007.114

When a protein signal is selected by mass spectrometry as being a potential biomarker, it is necessary to formally identify it. This process involves separation of the protein in question and its identification by either peptide fingerprinting or tandem mass spectrometry sequencing. In the following pages, a simple and rapid protocol is described. Basically, the protocol consists of an initial rational selection of a few sorbents followed by alignment of these as a series of columns to obtain the purified target protein. This preparation is then submitted to electrophoresis, the band is excised and the trypsin digest is analyzed by either mass spectrometry (mass fingerprinting approach) or by LC-MS/MS (sequencing). The development of the process takes only a few days. Experimental data for the isolation and identification of proteins are discussed and two examples are shown.