Clinical Chemistry
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Clinical Chemistry 53: 1915-1920, 2007. First published September 21, 2007; 10.1373/clinchem.2007.091736
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53/11/1915    most recent
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(Clinical Chemistry. 2007;53:1915-1920.)
© 2007 American Association for Clinical Chemistry, Inc.

Proteomics and Protein Markers

Quality Control of Serum Albumin Depletion for Proteomic Analysis

Nitin Seam1,a, Denise A. Gonzales1, Steven J. Kern1, Glen L. Hortin2, Gerard T. Hoehn1 and Anthony F. Suffredini1

1 Critical Care Medicine Department and 2 Department of Laboratory Medicine, Clinical Center, National Institutes of Health, Bethesda, MD.

aAddress correspondence to this author at: National Institutes of Health, Department of Critical Care Medicine, Bldg. 10, Rm. 2C145, 10 Center Dr., Bethesda, MD 20892-1662. Fax (301) 402-1213; e-mail nseam{at}cc.nih.gov.

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 albumin-depleted than unfractionated serum at both high and low m/z.

Conclusions: Sporadic and systematic variation in efficiency of albumin depletion by spin columns may contribute significant preanalytical bias to proteomic approaches of biomarker discovery. This variation requires ongoing QC of the albumin depletion process by quantification of albumin concentration to assess depletion efficiency.




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 Clin. Chem.Home page
G. L. Hortin, D. Sviridov, and N. L. Anderson
High-Abundance Polypeptides of the Human Plasma Proteome Comprising the Top 4 Logs of Polypeptide Abundance
Clin. Chem., October 1, 2008; 54(10): 1608 - 1616.
[Abstract] [Full Text] [PDF]


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