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.