Analysis of Human Proteome Organization Plasma Proteome Project (HUPO PPP) reference specimens using surface enhanced laser desorption/ionization‐time of flight (SELDI‐TOF) mass spectrometry: Multi‐institution correlation of spectra and identification of biomarkers

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Abstract
We report on a multicenter analysis of HUPO reference specimens using SELDI‐TOF MS. Eight sites submitted data obtained from serum and plasma reference specimen analysis. Spectra from five sites passed preliminary quality assurance tests and were subjected to further analysis. Intralaboratory CVs varied from 15 to 43%. A correlation coefficient matrix generated using data from these five sites demonstrated high level of correlation, with values >0.7 on 37 of 42 spectra. More than 50 peaks were differentially present among the various sample types, as observed on three chip surfaces. Additionally, peaks at ∼9200 and ∼15 950 m/z were present only in select reference specimens. Chromatographic fractionation using anion‐exchange, membrane cutoff, and reverse phase chromatography, was employed for protein purification of the ∼9200 m/z peak. It was identified as the haptoglobin alpha subunit after peptide mass fingerprinting and high‐resolution MS/MS analysis. The differential expression of this protein was confirmed by Western blot analysis. These pilot studies demonstrate the potential of the SELDI platform for reproducible and consistent analysis of serum/plasma across multiple sites and also for targeted biomarker discovery and protein identification. This approach could be exploited for population‐based studies in all phases of the HUPO PPP.