Multidimensional protein fractionation of blood proteins coupled to data-independent nanoLC-MS/MS analysis.
In order to exploit human blood as a source of protein disease biomarkers, robust analytical methods are needed to overcome the inherent molecular complexity of this bio-fluid. We present the coupling of label-free SAX chromatography and IMAC to a data-independent nanoLC-MS/MS (nanoLC-MS(E)) platform for analysis of blood plasma and serum proteins. The methods were evaluated using protein standards added at different concentrations to two groups of samples. The results demonstrate that both techniques enable accurate protein quantitation using low sample volumes and a minimal number of fractions. Combining both methods, 883 unique proteins were identified, of which 423 proteins showed high reproducibility. The two approaches resulted in identification of unique molecular signatures with an overlap of approximately 30%, thus providing complimentary information on sub-proteomes. These methods are potentially useful for systems biology, biomarker discovery, and investigation of phosphoproteins in blood.
Levin
Yishai
Y
Cambridge Centre for Neuropsychiatric Research, Institute of Biotechnology, Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, CB 2 1QT Cambridge, United Kingdom.
Jaros
Julian A J
JA
Schwarz
Emanuel
E
Bahn
Sabine
S
D000818:Animals; D001774:Blood Chemical Analysis / Q000295:instrumentation / Q000379:methods; D001798:Blood Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D002417:Cattle; D005591:Chemical Fractionation / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods; D016000:Cluster Analysis; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D006736:Horses; D006801:Humans; D036103:Nanotechnology / Q000379:methods; D040901:Proteomics / Q000379:methods; D013552:Swine; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
D001798:Blood Proteins