Accuracy and Reproducibility in Quantification of Plasma Protein Concentrations by Mass Spectrometry without the Use of Isotopic Standards.

ABSTRACT

BACKGROUND: Quantitative proteomic analysis with mass spectrometry holds great promise for simultaneously quantifying proteins in various biosamples, such as human plasma. Thus far, studies addressing the reproducible measurement of endogenous protein concentrations in human plasma have focussed on targeted analyses employing isotopically labelled standards. Non-targeted proteomics, on the other hand, has been less employed to this end, even though it has been instrumental in discovery proteomics, generating large datasets in multiple fields of research. RESULTS: Using a non-targeted mass spectrometric assay (LCMSE), we quantified abundant plasma proteins (43 mg/mL-40 ug/mL range) in human blood plasma specimens from 30 healthy volunteers and one blood serum sample (ProteomeXchange PXD000347). Quantitative results were obtained by label-free mass spectrometry using a single internal standard to estimate protein concentrations. This approach resulted in quantitative results for 59 proteins (cut off ≥11 samples quantified) of which 41 proteins were quantified in all 31 samples and 23 of these with an inter-assay variability of ≤ 20%. Results for 7 apolipoproteins were compared with those obtained using isotope-labelled standards, while 12 proteins were compared to routine immunoassays. Comparison of quantitative data obtained by LCMSE and immunoassays showed good to excellent correlations in relative protein abundance (r = 0.72-0.96) and comparable median concentrations for 8 out of 12 proteins tested. Plasma concentrations of 56 proteins determined by LCMSE were of similar accuracy as those reported by targeted studies and 7 apolipoproteins quantified by isotope-labelled standards, when compared to reference concentrations from literature. CONCLUSIONS: This study shows that LCMSE offers good quantification of relative abundance as well as reasonable estimations of concentrations of abundant plasma proteins.