Abstract
BackgroundPost-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.ResultsPresented 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.ConclusionsThe 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.
Highlights
Post-translational modifications and genetic variations give rise to protein variants that significantly increase the complexity of the human proteome
The secondary antibody used in sandwich immunoassays cannot discriminate between structural protein modifications and the resulting quantitative signal is the sum of signals from all variants for a given protein captured by the primary antibody
Internal reference standard In the past we have utilized homologous proteins from other animal species that are recognized by the same antibody that binds the human antigen, so that upon spiking of the internal reference standard (IRS) into the analytical sample, the human protein and its animal homologue are retrieved by the single antibody
Summary
Post-translational modifications and genetic variations give rise to protein variants that significantly increase the complexity of the human proteome. Protein modifications play important roles in biological process, and can serve as diagnostic indicators of pathological events [1]. Their detection and quantification is not straightforward because oftentimes these modified forms are only slightly chemically different from their wild-type proteins. Immunoaffinity separation offers simple and targeted isolation of proteins and their variants in preparation for MS detection, by using antibodies toward invariable epitopes in the protein sequence. Presence of signals in the mass spectra that do not correspond to the one empirically calculated from the native protein sequence is the first indication of protein variants existence. These signals can be initially assigned to specific variants and modifications by accurate measurements of the observed mass shifts and knowledge of the protein sequence and possible modifications, and are further verified using proteolytic digestion and mapping experiments [4,5,6]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
