Abstract
The use of stable isotope-labeled standards (SIS) is an analytically valid means of quantifying proteins in biological samples. The nature of the labeled standards and their point of insertion in a bottom-up proteomic workflow can vary, with quantification methods utilizing curves in analytically sound practices. A promising quantification strategy for low sample amounts is external standard addition (ExSTA). In ExSTA, multipoint calibration curves are generated in buffer using serially diluted natural (NAT) peptides and a fixed concentration of SIS peptides. Equal concentrations of SIS peptides are spiked into experimental sample digests, with all digests (control and experimental) subjected to solid-phase extraction prior to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Endogenous peptide concentrations are then determined using the regression equation of the standard curves. Given the benefits of ExSTA in large-scale analysis, a detailed protocol is provided herein for quantifying a multiplexed panel of 125 high-to-moderate abundance proteins in undepleted and non-enriched human plasma samples. The procedural details and recommendations for successfully executing all phases of this quantification approach are described. As the proteins have been putatively correlated with various noncommunicable diseases, quantifying these by ExSTA in large-scale studies should help rapidly and precisely assess their true biomarker efficacy.
Highlights
The quantitative proteomics field has advanced tremendously during the past decade
The reproducibility of the methodologies and techniques has been enhanced, as has its utility in interlaboratory studies [2, 4, 5]. These merits are collectively needed for the widespread assessment of protein disease biomarkers toward clinical diagnostic implementation [6]
The peptide concentrations in the endogenous sample can be determined by applying the experimental response ratios (i.e., NAT vs. spiked-in stable isotope-labeled standards (SIS)) to the regression equation of their peptide-specific standard curves. This strategy has been demonstrated to be a robust means for precisely quantifying endogenous proteins in human plasma samples
Summary
The quantitative proteomics field has advanced tremendously during the past decade. This includes the method workflows, automation schemes, analysis regimens, and scope of applications. The labeled standards are designed to resemble the chemical structure of its endogenous (natural or NAT) analogue Their quantitative effectiveness has been evaluated and compared/contrasted in different sample matrices (e.g., plasma, cerebrospinal fluid, urine), using both protein [22] and peptide [23] standards. These studies have demonstrated the utility of labeled standards, with the selection guided by experimental design and quantitative application. The peptide concentrations in the endogenous sample can be determined by applying the experimental response ratios (i.e., NAT vs spiked-in SIS) to the regression equation of their peptide-specific standard curves (plot of NAT/SIS peptide response vs NAT peptide concentration) This strategy has been demonstrated to be a robust means for precisely quantifying endogenous proteins in human plasma samples. Performing the procedures described should produce precise quantitative results for putative biomarker analysis (at the discovery or verification stage) while opening the door for expanded target panels and sample sizes
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