Use of in‐sample calibration curve approach for quantification of peptides with high‐resolution mass spectrometry
Authors: Kandi Soumya , Savaryn John P. , Ji Qin C. , Jenkins Gary J. 
Published: August 2022

RationaleThe in‐sample calibration curve (ISCC) approach of quantification utilizes the response of isotopologue ions from spiked‐in stable isotope labeled internal standard (SIL‐IS) to build a standard curve. The quantitative analysis of the study sample is achieved based on the response of selected monoisotopic analyte ion against the calibration curve. Although this methodology has been demonstrated to be feasible by unit and high‐resolution mass spectrometers, quantitation on high‐resolution mass spectrometer with product ions has not been tested. We tested the feasibility of this approach using product ions on an high‐resolution mass spectrometer equipped with an Orbitrap detector.MethodsUsing a proteomics workflow for sample preparation, two surrogate peptides were quantified from a complex matrix of protein digest from human peripheral blood mononuclear cells (hPBMCs). SIL‐IS was spiked in at different levels to construct calibration curves in a traditional manner. ISCCs were prepared using extracted ion chromatograms from isotopically resolved mass spectra and compared with traditional calibration curves.ResultsA linear response was observed with ISCC approach for at least two to three orders of magnitude in MS1 as well as targeted MS2 (tMS2). From protein digests, isobaric interferences were observed for endogenous peptides on the MS1 level; this was circumvented with product‐ion‐based quantitation where for one peptide, %CV for endogenous levels was more than 20% with ISCC but higher with the traditional calibration curve approach. For the second peptide, endogenous levels could not be determined in the traditional approach as calibrant levels did not bracket the lower end, and with the ISCC approach, isotopologues at abundances lower than the endogenous level allowed for quantitative assessments.ConclusionsISCC demonstrated improved precision across surrogate peptides from endogenous protein digests. In samples where endogenous analyte concentrations were low in abundance, ISCC rescued what would have been a non‐reportable result in a traditional bioanalytical assay as calibrant levels were not prepared at adequately low levels to bracket unknowns. ISCC using high‐resolution mass spectrometer is feasible and ideal compared to unit resolution mass spectrometers. High‐resolution mass spectrometer allows for isotopic resolution for analytes with > + 2 charge state and provides flexibility in quantification using multiple product ions. ISCC using high‐resolution mass spectrometer allows for simultaneous assaying of low abundance isotopologues, the signal acquisition of which is not constrained by limits in data acquisition or calibrant preparation as with other approaches but rather limited by platform sensitivity. In contrast to unit resolution mass spectrometers, these features offered by high‐resolution mass spectrometer could be especially useful for the drug discovery assay support where there is less lead time for assay development than for the assays to support the drug development studies.