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Serum biomarker discovery in renal cancer using 2‐DE and prefractionation by immunodepletion and isoelectric focusing; increasing coverage or more of the same?
Author(s) -
Vasudev Naveen S.,
Ferguson Roisean E.,
Cairns David A.,
Stanley Anthea J.,
Selby Peter J.,
Banks Rosamonde E.
Publication year - 2008
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200800497
Subject(s) - biomarker , isoelectric focusing , gene isoform , chromatography , proteome , biomarker discovery , chemistry , proteomics , cancer , microbiology and biotechnology , biology , biochemistry , enzyme , genetics , gene
As an initial screen for novel markers of renal cancer and to minimise background heterogeneity, we have compared the within‐patient profiles of serum samples from seven patients pre‐ and post‐nephrectomy. Samples were depleted of six of the most abundant proteins using Agilent's multiple affinity removal system (MARS) followed by solution‐phase IEF prior to separation by 2‐DE using narrow range IPG Strips, with a total of 84 gels. The reproducibility of the various steps was demonstrated and an approximate two‐fold increase (from 374 to 779) in the number of protein spots observed in the pH region 4.6–7.0 was obtained. However, the majority of additional proteins seen were further isoforms of existing proteins due to the higher resolution and the majority of protein spots identified were still moderate to highly abundant species. Only one protein spot (as yet unidentified) was found to change significantly in the same direction in at least four patients. Although this powerful prefractionation and analysis strategy allows the visualisation of multiple protein isoforms, it is insufficient to allow detection of lower abundance proteins in serum without the implementation of further strategies.