Enrichment of low molecular weight fraction of serum for MS analysis of peptides associated with hepatocellular carcinoma

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Abstract
A challenging aspect of biomarker discovery in serum is the interference of abundant proteins with identification of disease‐related proteins and peptides. This study describes enrichment of serum by denaturing ultrafiltration, which enables an efficient profiling and identification of peptides up to 5 kDa. We consistently detect several hundred peptide‐peaks in MALDI‐TOF and SELDI‐TOF spectra of enriched serum. The sample preparation is fast and reproducible with an average CV for all 276 peaks in the MALDI‐TOF spectrum of 11%. Compared to unenriched serum, the number of peaks in enriched spectra is 4 times higher at an S/N ratio of 5 and 20 times higher at an S/N ratio of 10. To demonstrate utility of the methods, we compared 20 enriched sera of patients with hepatocellular carcinoma (HCC) and 20 age‐matched controls using MALDI‐TOF. The comparison of 332 peaks at p < 0.001 identified 45 differentially abundant peaks that classified HCC with 90% accuracy in this small pilot study. Direct TOF/TOF sequencing of the most abundant peptide matches with high probability des‐Ala‐fibrinopeptide A. This study shows that enrichment of the low molecular weight fraction of serum facilitates an efficient discovery of peptides that could serve as biomarkers for detection of HCC as well as other diseases.