Multiplexed MRM-Based Protein Quantitation Using Two Different Stable Isotope-Labeled Peptide Isotopologues for Calibration

Abstract

When quantifying endogenous plasma proteins for fundamental and biomedical research - as well as for clinical applications - precise, reproducible, and robust assays are required. Targeted detection of peptides in a bottom-up strategy is the most common and precise mass spectrometry-based quantitation approach when combined with the use of stable isotope-labeled peptides. However, when measuring protein in plasma, the unknown endogenous levels prevent the implementation of the best calibration strategies, since no blank matrix is available. Consequently, several alternative calibration strategies are employed by different laboratories. In this study, these methods were compared to a new approach using two different stable isotope-labeled standard (SIS) peptide isotopologues for each endogenous peptide to be quantified, enabling an external calibration curve as well as the quality control samples to be prepared in pooled human plasma without interference from endogenous peptides. This strategy improves the analytical performance of the assay and enables the accuracy of the assay to be monitored, which can also facilitate method development and validation.

Absolute Quantification of Apolipoproteins Following Treatment with Omega-3 Carboxylic Acids and Fenofibrate Using a High Precision Stable Isotope-labeled Recombinant Protein Fragments Based SRM Assay

“…AM-96-PCR-RD; Union City, CA) and subsequently transferred directly to the autosampler for analysis. We used single-point calibration, which is acceptable as long as the concentration lies within the assay linear range (51). The masterbatch was further adjusted to reach as close as possible to a 1:1 ratio between concentrations of the endogenous target proteins and their corresponding SIS PrESTs (Table II).…”

Quantification of Cardiovascular Disease Biomarkers in Human Platelets by Targeted Mass Spectrometry

“…Of the large panel of peptides and proteins mentioned before, we quantified a subset of 99 proteins and 133 peptides via an internal calibration curve in one platelet reference sample. Using calibration curves is a common procedure for an absolute quantification, although the curve preparation can vary [ 37 , 38 , 39 ]. As a reference sample we used a mix of platelets derived and pooled from five healthy donors.…”

Evaluation of a ketogenic diet for improvement of neurological recovery in individuals with acute spinal cord injury: study protocol for a randomized controlled trial

“…Synthetic peptides will be used to obtain quantitative data. To obtain absolute quantitative data on the selected peptide(s), 13 C/ 15 N-isotopically labeled versions of the peptide(s) will be prepared [34].…”