Podium Presentation in Room 5 on Wednesday at 11:40 (Chair: Chris Shuford)
Authors: Junyan Shi (1,2), Meng Wang (1,2), Mari L. DeMarco (1,2,3)
BACKGROUND: In the design of quantitative protein LC-MS/MS methods employing in vitro proteolytic digestion, multi-point external calibration curves are commonly utilized. The selection of the preferred molecular calibrant has generally been regarded as the intact form of the measurand; however, using an intact protein-based calibrant is not always practical or possible.
OBJECTIVE: Explore approaches to calibration using surrogate peptides versus the intact protein measurand, and assess analytical performance.
METHODS & RESULTS: In one example, we explore calibration of a quantitative LC-MS/MS assay for apolipoprotein A1 in human plasma. Patient samples were prepared as follows: 5 μL of EDTA human plasma was diluted in 45 μL of PBS. A volume of 10 μL of the PBS-diluted plasma and 10 μL of the internal standard solution were added to 490 μL of the digestion buffer. After addition of the internal standard, all samples (i.e., QCs, calibrators, and patient samples) were denatured by heating at 99 °C for 10 min with shaking. After denaturation, samples were left to cool at room temperature, and 30 μL of 5 μg/μl trypsin was added. Samples were incubated at 37 °C for 20 min. A volume of 12 μL of the quenched digest solution was injected into the LC-MS/MS (Phenomenex Aeris Peptide 3.6-μm XB-C18 column, Shimadzu LC 20AD, and SCIEX Triple Quad 5500) and run on a 10-min gradient at a flow rate of 0.25 mL/min. A method comparison was performed against the Siemens BNII assay (n= 61 human EDTA plasma samples). Three general calibration schemes were attempted: (i) protein-based, (ii) peptide-based, and (iii) a hybrid protein/peptide approach. For the protein-based scheme, BCR-393 Human Apoliporotein AI certified reference material was utilized as the calibrant. For the peptide-based approach, the surrogate peptide THLAPYSDELR was utilized and assigned using the peptide manufacturer’s stated mass and purity. In the hybrid approach, EDTA plasma specimens measured by immunonephelometry were used to assign the peptide calibrators [Shi et al. Clin Chem. 2018;64(12):1782-4].
CONCLUSION: A limitation of peptide-based calibrators and internal standards is the potential for bias associated with incomplete protein digestion and peptide decay; however, by developing a reproducible and rapid digestion process and assigning peptide calibrators against protein standards, a hybrid protein/peptide approach can yield results analytically superior to pure protein-based calibration schemes.
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