Assessment of the partitioning capacity of high abundant proteins in human cerebrospinal fluid using affinity and immunoaffinity subtraction spin columns.
Artikel i vetenskaplig tidskrift, 2010
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%.
Adolescent
Adult
methods
Matrix-Assisted Laser Desorption-Ionization
analysis
Affinity
Chromatography
Middle Aged
Humans
Polyacrylamide Gel
methods
Isotope Labeling
methods
Spectrometry
Reproducibility of Results
Computational Biology
Electrophoresis
Aged
Mass
Immunosorbent Techniques
Cerebrospinal Fluid Proteins
Författare
Magnus Wetterhall
Aida Zuberovic
Jörg Hanrieder
Göteborgs universitet
Jonas Bergquist
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
1873-376X (ISSN)
Vol. 878 19 1519-30Ämneskategorier (SSIF 2011)
Analytisk kemi
Neurovetenskaper
DOI
10.1016/j.jchromb.2010.04.003
PubMed
20444656