Application of thiophilic chromatography to deplete serum immunoglobulins in sample preparation for bidimensional electrophoresis.
Serum is a typical sample for non-invasive studies in clinical research. Its proteome characterization is challenging, since requires extensive protein depletion. Methods used nowadays for removal of high-abundance proteins are expensive or show quite often a low loading capacity, which has strong repercussions on the number of samples and replicates per analysis. In order to deplete immunoglobulins (Igs) and albumin (HSA) from 1 mL serum samples, we have developed a protocol based on a combination of thiophilic chromatography, not previously used in clinical proteomics, and a HSA-specific resin. Ig/HSA-depleted samples, immunoglobulinome and albuminone were analyzed by 2-DE. Thiophilic chromatography, coupled with HSA-depletion, allows a good 2-DE resolution as well as the visualization of new spots. Moreover, it yields enough protein to evaluate technical variability and facilitate subsequent protein identification. To validate the protocol, we carried out a preliminary comparative study between triplicate Igs/HSA-depleted serum samples from healthy control individuals and recently diagnosed/untreated rheumatoid arthritis (RA) patients. RA patients showed several acute phase proteins, as well as additional serum proteins, differentially and significantly regulated. Therefore, thiophilic chromatography can be used as an efficient and economical method in 2-DE to deplete immunoglobulins from large human serum samples before a more extensive fractioning.
Salgado
Francisco J
FJ
Department of Biochemistry and Molecular Biology, Biological Research Centre of University of Santiago/CIBUS, University of Santiago de Compostela, Lope Gómez de Marzoa, 15782 Santiago de Compostela, A Coruña, Spain. franciscojavier.salgado@usc.es
Vázquez
Sara
S
Iglesias
Alba
A
Pérez-Díaz
Amparo
A
Mera-Varela
Antonio
A
Arias
Pilar
P
Nogueira
Montserrat
M
D001172:Arthritis, Rheumatoid / Q000175:diagnosis; D001798:Blood Proteins / Q000032:analysis / Q000737:chemistry*; D002850:Chromatography, Gel; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D007136:Immunoglobulins / Q000302:isolation & purification; D012709:Serum Albumin / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D014357:Trypsin / Q000378:metabolism
D001798:Blood Proteins; D007136:Immunoglobulins; D012709:Serum Albumin; D013438:Sulfhydryl Compounds; D014357:Trypsin