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Assessment of the partitioning capacity of high abundant proteins in human cerebrospinal fluid using affinity and immunoaffinity subtraction spin columns.

Journal article
Authors Magnus Wetterhall
Aida Zuberovic
Jörg Hanrieder
Jonas Bergquist
Published in Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
Volume 878
Issue 19
Pages 1519-30
ISSN 1873-376X
Publication year 2010
Published at Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry
Pages 1519-30
Language en
Links dx.doi.org/10.1016/j.jchromb.2010.0...
Keywords Adolescent, Adult, Aged, Cerebrospinal Fluid Proteins, analysis, Chromatography, Affinity, methods, Computational Biology, Electrophoresis, Polyacrylamide Gel, Humans, Immunosorbent Techniques, Isotope Labeling, methods, Middle Aged, Reproducibility of Results, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, methods
Subject categories Analytical Chemistry, Neurochemistry

Abstract

The performance of three different affinity and immunoaffinity subtraction spin columns was investigated for the removal of the most abundant proteins in human cerebrospinal fluid (CSF). A pool of human CSF was processed with the spin columns and both the bound and flow through fractions were compared with each other and with intact CSF using 1D gel electrophoresis and nanoLC-MALDI-TOF/TOF-MS analysis. MASCOT MS/MS ionscores were compared before and after processing with the columns. The non-specific co-removal of proteins bound to the high abundant proteins, so called "sponge effect" was also examined for each spin column. The reproducibility of one of the spin columns, ProteomeLab IgY-12 proteome partitioning spin column, was further investigated by isobaric tags for relative and absolute quantification (iTRAQ) labeling and MS/MS analysis. Overall, 173 unique proteins were identified on a 95% MudPIT confidence scoring level. For all three spin columns, the number of proteins identified and their MASCOT scores were increased up to 10 times. The largest degree of non-specific protein removal was observed for a purely affinity based albumin removal column, where 28 other proteins also were present. The ProteomeLab IgY-12 proteome partitioning spin column showed very high reproducibility when combined with iTRAQ labeling and MS/MS analysis. The combined relative standard deviation (R.S.D.) for the high abundant protein removal, iTRAQ labeling and nanoLC-MALDI-TOF/TOF-MS analysis was less than 17.5%.

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