Using Two Peptide Isotopologues as Internal Standards for the Streamlined Quantification of Low-Abundance Proteins by Immuno-MRM and Immuno-MALDI.

Autor:	Ibrahim S; Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montréal, Québec H3T 1E2, Canada., Froehlich BC; University of Victoria-Genome British Columbia Proteomics Centre, University of Victoria, Victoria V8Z 7X8, Canada., Aguilar-Mahecha A; Segal Cancer Center, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, McGill University, Montréal, Québec H3T 1E2, Canada., Aloyz R; Segal Cancer Center, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, McGill University, Montréal, Québec H3T 1E2, Canada.; Division of Experimental Medicine, McGill University, Montréal, Québec H3T 1E2, Canada.; Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montréal, Québec H3T 1E2, Canada., Poetz O; NMI Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen 72770, Germany.; SIGNATOPE GmbH, Reutlingen 72770, Germany., Basik M; Segal Cancer Center, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, McGill University, Montréal, Québec H3T 1E2, Canada.; Division of Experimental Medicine, McGill University, Montréal, Québec H3T 1E2, Canada.; Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montréal, Québec H3T 1E2, Canada., Batist G; Segal Cancer Center, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, McGill University, Montréal, Québec H3T 1E2, Canada.; Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montréal, Québec H3T 1E2, Canada., Zahedi RP; Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montréal, Québec H3T 1E2, Canada.; Center for Computational and Data-Intensive Science and Engineering, Skolkovo Institute of Science and Technology, Moscow 121205, Russia., Borchers CH; Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montréal, Québec H3T 1E2, Canada.; University of Victoria-Genome British Columbia Proteomics Centre, University of Victoria, Victoria V8Z 7X8, Canada.; Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montréal, Québec H3T 1E2, Canada.; Center for Computational and Data-Intensive Science and Engineering, Skolkovo Institute of Science and Technology, Moscow 121205, Russia.
Jazyk:	angličtina
Zdroj:	Analytical chemistry [Anal Chem] 2020 Sep 15; Vol. 92 (18), pp. 12407-12414. Date of Electronic Publication: 2020 Sep 01.
DOI:	10.1021/acs.analchem.0c02157
Abstrakt:	Mass spectrometry (MS), particularly targeted proteomics, is increasingly being used for quantifying specific proteins and peptides in clinical specimens. The coupling of immuno-enrichment of proteotypic peptides with MS [e.g., immuno-multiple reaction monitoring (MRM) and immuno-matrix-assisted laser desorption ionization (MALDI)] enables the development of highly sensitive and specific assays for low-abundance signaling proteins. By incorporating stable isotope-labeled standards, these workflows allow the determination of endogenous protein concentrations. This is typically achieved through external calibration, often using surrogate matrices, which has inherent limitations for the analysis of clinical specimens as there are often substantial variations in the sample matrix, and sample amounts are typically limited. We have previously introduced the use of two peptide isotopologues for generating external calibration curves in plasma. Here, we present a two-point internal calibration (2-PIC) strategy using two isotopologues for immuno-MS assays and demonstrate its flexibility and robustness. Quantification of the tumor suppressor PTEN in Colo-205 cells by immuno-MRM and immuno-MALDI using 2-PIC and external calibration yielded very similar results (relative standard deviation between 2-PIC and external calibration: 4.9% for immuno-MRM; 1.1% for immuno-MALDI), without the need for a surrogate matrix or additional patient material for calibration, while concurrently reducing the instrument time and cost. Although our PTEN immuno-MRM and immuno-MALDI assays can be considered to be orthogonal as they utilized entirely different sample preparation and MS analysis workflows, targeted different PTEN peptides, and were performed in different laboratories, the endogenous Colo-205 PTEN levels determined with 2-PIC showed a good correlation ( r 2 = 0.9966) and good agreement (0.48 ± 0.01 and 0.29 ± 0.02 fmol/μg of total protein) between immuno-MRM and immuno-MALDI.
Databáze:	MEDLINE
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