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

Abstract

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.

Proteomics, Personalized Medicine and Cancer

“…Targeted proteomics approaches facilitate the development of high throughput sensitive, reproducible and quantitative assays for the measurement and validation of potential biomarkers and biomarker panels. Targeted approaches have been reported to be 5 - 10 fold more sensitive than DDA [ 53 ] and sensitivity can be further increased using immuno-enrichment [ 54 , 55 ]. It is worth noting that for immuno-MS the specificity requirements for the antibodies used are less stringent than for ELISA as the ultimate specificity is obtained from the fragmentation patterns of the proteotypic peptides used.…”

Precise Quantitation of PTEN by Immuno-MRM: A Tool To Resolve the Breast Cancer Biomarker Controversy

“…Samples were subjected to reduction, alkylation, digestion, and immunoenrichment for NNIDDVVR analyzed by LC-MRM, as described above. Endogenous PTEN concentrations were determined using our 2-PIC strategy, 44 and the LC-MRM PTEN results of the in-house cell lines were compared to the Western blot data for PTEN and pHER2.…”

“…Samples were analyzed by LC-MRM as described above. Endogenous PTEN concentrations were determined using our 2-PIC strategy 44 and are reported in fmol per 10 μg of protein, which can be more precisely determined than, for instance, actual tumor tissue volume or weight.…”

“…The inclusion of our recent two-point internal calibration (2-PIC) internal calibration strategy greatly facilitates the robust quantitation of clinical specimens. 44 We demonstrate the generic utility of our PTEN−iMRM assay by quantifying endogenous PTEN protein levels in different BC cell lines, fresh frozen tissues, and FFPE tissues. Due to its robustness and performance, we believe that our PTEN− iMRM assay will finally enable the important and urgent study of PTEN's role as a BC biomarker.…”

“…Here, we present and analytically validate an iMRM method that allows the quantitation of PTEN with high sensitivity and precision. The inclusion of our recent two-point internal calibration (2-PIC) internal calibration strategy greatly facilitates the robust quantitation of clinical specimens . We demonstrate the generic utility of our PTEN–iMRM assay by quantifying endogenous PTEN protein levels in different BC cell lines, fresh frozen tissues, and FFPE tissues.…”