Assessment approach for evaluating high abundance protein depletion methods for cerebrospinal fluid (CSF) proteomic analysis.
Optimal proteomic analysis of human cerebrospinal fluid (CSF) requires depletion of high-abundance proteins to facilitate observation of low-abundance proteins. The performance of two immunodepletion (MARS, Agilent Technologies and ProteoSeek, Pierce Biotechnology) and one ultrafiltration (50 kDa molecular weight cutoff filter, Millipore Corporation) methods for depletion of abundant CSF proteins were compared using a graphical method to access the depth of analysis using "marker proteins" with known normal concentration ranges. Two-dimensional LC/MS/MS analysis of each depleted sample yielded 171 and 163 unique protein identifications using the MARS and ProteoSeek immunodepletion methods, respectively, while only 46 unique proteins were identified using a 50 kDa molecular weight cutoff filter. The relative abundance of the identified proteins was estimated using total spectrum counting and compared to the concentrations of 45 known proteins in CSF as markers of the analysis depth. Results of this work suggest a clear need for methodology designed specifically for depletion of high-abundance proteins in CSF, as depletion methods designed to deplete high-abundance serum proteins showed little improvement in analysis depth compared to analysis without depletion. The marker protein method should be generally useful for assessing depth of analysis in the comparison of proteomic analysis methods.
Shores
Kevin S
KS
Department of Pharmacology and MUSC Proteomics Center, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Knapp
Daniel R
DR
D015415:Biomarkers / Q000378:metabolism; D002021:Buffers; D002412:Cations; D002555:Cerebrospinal Fluid / Q000737:chemistry*; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods*
D015415:Biomarkers; D002021:Buffers; D002412:Cations; D007074:Immunoglobulin G; D010455:Peptides; D011506:Proteins; D020543:Proteome