Multiple Reaction Monitoring of mTRAQ-Labeled Peptides Enables Absolute Quantification of Endogenous Levels of a Potential Cancer Marker in Cancerous and Normal Endometrial Tissues

Abstract

While iTRAQ analyses have proved invaluable for the discovery of potential cancer markers, two outstanding issues that remained were its ineffectiveness to consistently detect specific proteins of interest in a complex sample and to determine the absolute abundance of those proteins. These have been addressed by availability of the mTRAQ reagents (Applied Biosystems, Inc., Foster City, CA) a nonisobaric variant of iTRAQ. We have applied this newly emerging technique to quantify one of our potential markers for endometrial cancer, viz. pyruvate kinase M1/M2. The mTRAQ methodolgy relies on multiple reaction monitoring (MRM) to target tryptic peptides from the protein of interest, thus, ensuring maximal opportunity for detection, while the nonisobaric tags enable specific quantification of each version of the labeled peptides through unique MRM transitions conferred by the labels. Known amounts of synthetic peptides tagged with one of the two available mTRAQ labels, when used as quantification standards in a mixture with the oppositely labeled tryptically digested sample, permit determination of the absolute amounts of the corresponding protein in the sample. The ability to label the sample and reference peptides with either one of the two possible combinations is an inherent advantage of this method, as it provides a means for verification of the reported ratios. In this study, we determined that the amount of pyruvate kinase present in the homogenate from a biopsied EmCa tissue sample was 85 nmol/g of total proteins, while the equivalent concentration in the nonmalignant controls was 21-26 nmol/g of total proteins. This approximately 4-fold higher amount of pyruvate kinase in the cancer sample was further confirmed not only by a direct comparison between the cancer sample and one of the nonmalignant controls, but also independently by an enzyme-linked immunosorbant assay (ELISA). Additionally, the 4-fold higher level of pyruvate kinase amount in the cancer homogenate reported in this study is considerably higher than the 2-fold higher ratio reported across 20 cancer samples in the discovery phase with the iTRAQ technique, suggesting that there exists a possibility that the dynamic range of ratios determined by the iTRAQ technique may have been compressed.

Current affairs in quantitative targeted proteomics: multiple reaction monitoring-mass spectrometry

“…Computing a ratio of heavy/light for all peptides of interest in the dataset enables relative quantitative comparison. Recently a new labeling reagent is available, the mTRAQ reagent [13]. mTRAQ is derived from the original 4plex iTRAQ reagents.…”

iTRAQ Labeling is Superior to mTRAQ for Quantitative Global Proteomics and Phosphoproteomics

“…Because most peptides in an experiment are present at 1:1:1:1 ratios between multiplexed samples, all ratios in the experiment tend to be dampened toward unity when cofragmentation occurs. This inaccuracy led to the development of mTRAQ labels to facilitate accurate precursor-based quantification of proteins initially identified in iTRAQ discovery experiments with targeted assays, such as multiple reaction monitoring (MRM) (8). Although iTRAQ has been widely used in discovery-based proteomics studies, mTRAQ has only appeared in a small number of studies thus far (8).…”

Applications of targeted proteomics in systems biology and translational medicine

“…Much technology development has gone into tissue preparation and quantitation of candidate biomarkers addressing the dual challenges of using a limited amount of material in which the candidate biomarker may also be low abundant. DeSouza and coworkers quantified a marker of endometrial cancer, pyruvate kinase M1/M2, in biopsied tissue at 85 nmol/g compared to 21–26 nmol/g in nonmalignant tissue using the mTRAQ labeling method, compared to only a 2x elevation initially determined by discovery iTRAQ proteomics, suggesting that the dynamic range for quantitation may be compressed in discovery scans 122. Chen and coworkers developed quantitative assays for 22 proteins in the Wnt/beta‐catenin signaling pathway in colon cancer cell lines and applied them to frozen colon tissue sections and laser capture microdissected tumor cells 123.…”