Quality control of serum albumin depletion for proteomic analysis.
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.
Seam
Nitin
N
Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD 20892-1662, USA. nseam@cc.nih.gov
Gonzales
Denise A
DA
Kern
Steven J
SJ
Hortin
Glen L
GL
Hoehn
Gerard T
GT
Suffredini
Anthony F
AF
D000328:Adult; D015415:Biomarkers / Q000097:blood; D001800:Blood Specimen Collection; D006801:Humans; D007136:Immunoglobulins; D040901:Proteomics / Q000379:methods*; D011786:Quality Control; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D015415:Biomarkers; C016512:IgY; D007136:Immunoglobulins; D012709:Serum Albumin