Abstract
Strategies for removal of high abundance proteins have been increasingly utilized in proteomic studies of serum/plasma and other body fluids to enhance the detection of low abundance proteins and achieve broader proteome coverage; however, both the reproducibility and specificity of the high abundance protein depletion process still represent common concerns. Here we report a detailed evaluation of immunoaffinity subtraction performed applying the ProteomeLab IgY-12 system that is commonly used in human serum/plasma proteome characterization in combination with high resolution LC-MS/MS. Plasma samples were repeatedly processed using this approach, and the resulting flow-through fractions and bound fractions were individually analyzed for comparison. The removal of target proteins by the immunoaffinity subtraction system and the overall process was highly reproducible. Non-target proteins, including one spiked protein standard (rabbit glyceraldehyde-3-phosphate dehydrogenase), were also observed to bind to the column at different levels but also in a reproducible manner. The results suggest that multiprotein immunoaffinity subtraction systems can be readily integrated into quantitative strategies to enhance detection of low abundance proteins in biomarker discovery studies.
Highlights
Strategies for removal of high abundance proteins have been increasingly utilized in proteomic studies of serum/ plasma and other body fluids to enhance the detection of low abundance proteins and achieve broader proteome coverage; both the reproducibility and specificity of the high abundance protein depletion process still represent common concerns
We evaluated the reproducibility of the IgY-12 column using a plasma sample spiked with five protein standards and high resolution capillary LC coupled to linear ion trap tandem MS (LC-MS/ MS), a technique capable of identifying thousands of peptides in a single analysis
In the LC-MS/MS analyses of complex peptide mixtures, the selection of ions for MS/MS is not totally randomized but rather dependent on the width of the chromatographic peaks or the concentration of peptides delivered to the mass spectrometer
Summary
IgY, immunoglobulin yolk; 2D, twodimensional; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; SPE, solid-phase extraction; ⌬Cn, delta correlation; Xcorr, crosscorrelation score; MARS, multiple affinity removal system; D, distribution. Evaluation of Immunoaffinity Subtraction rating high abundance proteins. Our results indicate that high abundance protein separation using the IgY-12 system is highly reproducible, and the low level binding of non-target proteins in this system occurred in a reproducible fashion
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