Evaluation of protein depletion methods for the analysis of total-, phospho- and glycoproteins in lumbar cerebrospinal fluid.
A proper sample preparation, in particular, abundant protein removal is crucial in the characterization of low-abundance proteins including those harboring post-translational modifications. In human cerebrospinal fluid (CSF), approximately 80% of proteins originate from serum, and removal of major proteins is necessary to study brain-derived proteins that are present at low concentrations for successful biomarker and therapeutic target discoveries for neurological disorders. In this study, phospho- and glycoprotein specific fluorescent stains and mass spectrometry were used to map proteins from CSF on two-dimensional gel electropherograms after immunoaffinity based protein removal. Two protein removal methods were evaluated: batch mode with avian IgY antibody microbeads using spin filters and HPLC multiple affinity removal column. Six abundant proteins were removed from CSF: human serum albumin (HSA), transferrin, IgG, IgA, IgM, and fibrinogen with batch mode, and HSA, transferrin, IgG, IgA, antitrypsin, and haptoglobin with column chromatography. 2D gels were compared after staining for phospho-, glyco- and total proteins. The column format removed the major proteins more effectively and approximately 50% more spots were visualized when compared to the 2D gel of CSF without protein depletion. After protein depletion, selected phospho- and glycoprotein spots were identified using mass spectrometry in addition to some of the spots that were not visualized previously in nondepleted CSF. Fifty proteins were identified from 66 spots, and among them, 12 proteins (24%) have not been annotated in previously published 2D gels.
Ogata
Yuko
Y
Mayo Proteomics Research Center and Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.
Charlesworth
M Cristine
MC
Muddiman
David C
DC
D000328:Adult; D000368:Aged; D002556:Cerebrospinal Fluid Proteins / Q000032:analysis* / Q000145:classification / Q000302:isolation & purification; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D005456:Fluorescent Dyes; D006023:Glycoproteins / Q000134:cerebrospinal fluid / Q000302:isolation & purification; D006801:Humans; D007136:Immunoglobulins; D008297:Male; D013058:Mass Spectrometry; D008722:Methods; D008863:Microspheres; D008875:Middle Aged; D010750:Phosphoproteins / Q000134:cerebrospinal fluid / Q000302:isolation & purification
D002556:Cerebrospinal Fluid Proteins; D005456:Fluorescent Dyes; D006023:Glycoproteins; C016512:IgY; D007136:Immunoglobulins; D010750:Phosphoproteins