Disulfide proteomics for identification of extracellular or secreted proteins.
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.
Biochemistry & Proteomics Group, Department of Chemistry & Biomolecular Science, Clarkson University, Potsdam, NY 13699-5810, USA.
Izabela
I
Sokolowska
Mary Ann
MA
Gawinowicz
Armand G
AG
Ngounou Wetie
Costel C
CC
Darie
D015415:Biomarkers / Q000097:blood / Q000737:chemistry; D001798:Blood Proteins / Q000032:analysis / Q000737:chemistry*; D002853:Chromatography, Liquid; D004220:Disulfides / Q000097:blood / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D009369:Neoplasms / Q000097:blood; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D020543:Proteome / Q000032:analysis / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D012394:Rosaniline Dyes
D015415:Biomarkers; D001798:Blood Proteins; D004220:Disulfides; D020543:Proteome; D012394:Rosaniline Dyes; C048139:Coomassie blue