Reproducible protein quantitation of 270 human proteins at increased depth using nanoparticle-based fractionation and multiple reaction monitoring mass spectrometry with stable isotope-labelled internal standards†

Abstract

Here we show that when using a mix of 274 light synthetic peptide standards (NAT) as surrogates for 270 human plasma proteins, as well as stable isotope-labelled standards (SIS) as normalizers (both from MRM Proteomics Inc.) for targeted quantitative analysis by liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM-MS), the Seer Proteograph™ platform allowed for the enrichment and absolute quantitation of up to an additional 62 targets (median) compared to two standard proteomic workflows without enrichment, representing an increase of 44%. The nanoparticle-based fractionation workflow resulted in improved reproducibility compared to a traditional proteomic workflow with no fractionation (median 8.3% vs. 13.1% CV). As expected, the protein concentrations in nanoparticle coronas were higher and had more compressed dynamic range in comparison to the concentrations determined either by a 3-hour Trypsin/LysC or overnight tryptic digestion methods. As the nanoparticle-based fractionation technology gains popularity, additional steps such as establishing technique-specific protein reference ranges across plasma samples and comparisons to well-established protein quantitation methods like enzyme-linked immunosorbent assay (ELISA) and LC/MRM-MS may be explored to enable absolute quantification of plasma proteins based on nanoparticle-based fractionation data.