Disulfide proteomics for identification of extracellular or secreted proteins

• Published in: Electrophoresis Vol. 33; no. 16; pp. 2527 - 2536
• Main Authors: Sokolowska, Izabela, Gawinowicz, Mary Ann, Wetie, Armand G. Ngounou, Darie, Costel C.
• Format: Journal Article
• Language: English
• Published: Germany Blackwell Publishing Ltd 01-08-2012
• Subjects: Biomarkers - blood; Biomarkers - chemistry; Blood Proteins - analysis; Blood Proteins - chemistry; Chromatography, Liquid; Disulfide proteomics; Disulfides - blood; Disulfides - chemistry; Electrophoresis, Polyacrylamide Gel; Fractionation; Humans; Mass spectrometry; Mass Spectrometry - methods; Neoplasms - blood; Oxidation-Reduction; Protein Denaturation; Proteome - analysis; Proteome - chemistry; Proteomics - methods; Rosaniline Dyes; Serum biomarkers
• ISSN: 0173-0835; 1522-2683
• DOI: 10.1002/elps.201200182

The combination of SDS‐PAGE and MS is one of the most powerful and perhaps most frequently used gel‐based proteomics approaches in protein identification. However, one drawback of this method is that separation takes place under denaturing and reducing (R) conditions and as a consequence, all proteins with identical apparent molecular mass (Mr) will run together. Therefore, low‐abundant proteins may not be easily identified. Another way of investigating proteins by proteomics is by analyzing subproteomes from a total proteome such as phosphoproteomics, glycoproteomics, or disulfide proteomics. Here, we took advantage of the property of secreted proteins to form disulfide bridges and investigated disulfide‐linked proteins, using SDS‐PAGE under nonreducing (NR) conditions. We separated sera from normal subjects and from patients with various diseases by SDS‐PAGE (NR) and (R) conditions, followed by LC‐MS/MS analysis. Although we did not see any detectable difference between the sera separated by SDS‐PAGE(R), we could easily identify the disulfide‐linked proteins separated by SDS‐PAGE (NR). LC‐MS/MS analysis of the disulfide‐linked proteins correctly identified haptoglobin (Hp), a disulfide‐linked protein usually found as a heterotetramer or as a disulfide‐linked heteropolymer. Western blotting under NR and R conditions using anti‐Hp antibodies confirmed the LC‐MS/MS experiments and further confirmed that upon reduction, the disulfide‐linked Hp heterotetramers and polymers were no longer disulfide‐linked polymers. These data suggest that simply by separating samples on SDS‐PAGEunder NR conditions, a different, new proteomics subset can be revealed and then identified.