Background
Post-translational modifications and genetic variations give rise to protein variants that significantly increase the complexity of the human proteome. Modified proteins also play an important role in biological processes. While sandwich immunoassays are routinely used to determine protein concentrations, they are oblivious to protein variants that may serve as biomarkers with better sensitivity and specificity than their wild-type proteins. Mass spectrometry, coupled to immunoaffinity separations, can provide an efficient mean for simultaneous detection and quantification of protein variants.
Conclusions
The mass spectrometric immunoassays can find use in quantifying specific protein modifications, either as a part of a specific protein biomarker discovery/rediscovery effort to delineate the role of these variants in the onset of the disease, progression, and response to therapy, or in a more systematic study to delineate and understand human protein diversity.
Results
Presented here is a mass spectrometric immunoassay method for targeted quantitative proteomics analysis of protein modifications. Cystatin C, a cysteine proteinase inhibitor and a potential marker for several pathological processes, was used as a target analyte. An internal reference standard was incorporated into the assay, serving as a normalization point for cystatin C quantification. The precision, linearity, and recovery characteristics of the assay were established. The new assay was also benchmarked against existing cystatin C ELISA. In application, the assay was utilized to determine the individual concentration of several cystatin C variants across a cohort of samples, demonstrating the ability to fully quantify individual forms of post-translationally modified proteins. Conclusions: The mass spectrometric immunoassays can find use in quantifying specific protein modifications, either as a part of a specific protein biomarker discovery/rediscovery effort to delineate the role of these variants in the onset of the disease, progression, and response to therapy, or in a more systematic study to delineate and understand human protein diversity.