Differential expression profiling of serum proteins and metabolites for biomarker discovery

A liquid chromatography-mass spectrometry (LC-MS) proteomics and metabolomics platform is presented for quantitative differential expression analysis. Proteome profiles obtained from 1.5 μL of human serum show ∼5000 de-isotoped and quantifiable molecular ions. Approximately 1500 metabolites are observed from 100 μL of serum. Quantification is based on reproducible sample preparation and linear signal intensity as a function of concentration. The platform is validated using human serum, but is generally applicable to all biological fluids and tissues. The median coefficient of variation (CV) for ∼5000 proteomic and ∼1500 metabolomic molecular ions is approximately 25%. For the case of C-reactive protein, results agree with quantification by immunoassay. The independent contributions of two sources of variance, namely sample preparation and LC-MS analysis, are respectively quantified as 20.4 and 15.1% for the proteome, and 19.5 and 13.5% for the metabolome, for median CV values. Furthermore, biological diversity for ∼20 healthy individuals is estimated by measuring the variance of ∼6500 proteomic and metabolomic molecular ions in sera for each sample; the median CV is 22.3% for the proteome and 16.7% for the metabolome. Finally, quantitative differential expression profiling is applied to a clinical study comparing healthy individuals and rheumatoid arthritis (RA) patients.