Quality Control of Serum Albumin Depletion for Proteomic Analysis

Abstract

Background: Prefractionation techniques such as serum albumin depletion are useful precursors to proteomic analysis, but they may introduce preanalytical bias if the depletion is not reproducible. We examined the reproducibility of albumin immunodepletion and describe a method of QC for this process. Methods: Depletion of albumin from pooled serum, performed using IgY immunoaffinity spin columns, was assessed for 21 runs on each of 4 columns. We measured albumin concentrations, after albumin depletion, by use of an immunoturbidimetric assay on the Beckman LX 20 analyzer and assessed mass spectra of albumin-depleted samples by use of SELDI-TOF mass spectrometry. Results: There was substantial run-to-run variation in efficiency of albumin depletion, with systematic decline in efficiency after multiple uses of the columns. Mean depletion efficiency was >95% for 15 of the 1st 17 runs and <90% for runs 18 to 21. We evaluated the 10 highest-intensity peaks present in all spectra from runs 1, 8, 17, and 21 and assessed the effect of albumin depletion on SELDI-TOF mass spectrometry reproducibility. Comparing the %CV of relative intensities for low and high m/z measurements revealed a significant difference of run 21 compared with runs 1, 8, and 17 (P <0.0001). Six-fold more peaks were found in albumindepleted than unfractionated serum at both high and low m/z.

Mass spectrometry of peptides and proteins from human blood

“…Considering the capacity of a typical nano-LC column coupled with LC-MS/MS is on the order of 1 mg or less, the dilute product is still sufficient for direct analysis even after separation of digested peptides. There is evidence of non-specific binding of proteins to these depletion columns (Stempfer et al, 2008) with mixed results regarding the reproducibility of depletion column (Dekker et al, 2007;Seam et al, 2007). There is still a concentration range of 7-8 orders of magnitude after elimination of high abundance proteins (Linke, Doraiswamy, & Harrison, 2007).…”

“…There will be an error associated with every laboratory manipulation, even a binary fractionation like depletion introduces significant error Seam et al, 2007;Stempfer et al, 2008). Moreover, to achieve sensitivity, multiple fractionation methods might multiply the apparent error associated with the measurement (Stalder, Haeberli, & Heller, 2008).…”

High Abundance Proteins: Proteomer’s Thorns in the Flesh?

“…However, as these prefractionation techniques involve and additional step, these could potentially affect the reproducibility of the obtained data, leading to the preanalytical bias [23,24]. Increasing the number of biological and technical replicates per sample can reduce the preanalytical bias.…”

Proteomics - A Powerful Tool to Deepen the Molecular Mechanisms of Aortic Stenosis Disease

“…Nowadays, the most common technique is the immunodepletion, which has been extensively used for the specific removal of the most abundant proteins, based on the action of specific antibodies (Zolotarjova N. et al, 2005 andWang YY et al, 2003). Among these, Multiple Affinity Removal Columns (MARC) is the most effective method because it simultaneously removes multiple abundant proteins (Bjorhall K, et al, 2005 andSeam N et al, 2007). There are several affinity columns as MARS-6 or MARS-14 (Agilent Tecnologies) to deplete the 6 or 14 more abundant proteins and even to deplete the 20 more abundant proteins with the Top-20 column (Sigma).…”