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.
The use of neural precursor cells (NPCs) represents a promising repair strategy for many neurological disorders. This requires an understanding of the molecular events and biological features that regulate the self-renewal of NPCs and their differentiation into neurons, astrocytes, and oligodendendrocytes. In this study, we have characterized the proteomic changes that occur upon differentiation of these cells using the novel iTRAQ labeling chemistry for quantitative mass spectrometry. In total, 55 distinct proteins underwent expression changes during NPC differentiation. This included 14 proteins that were identified by our previous two-dimensional difference gel electrophoresis (2D-DIGE) analysis of differentiating mouse neurospheres. The importance of the iTRAQ approach was demonstrated by the identification of additional proteins that were not resolved by the 2D-DIGE technology. The proteins identified by the iTRAQ approach included growth factors, signaling molecules, proliferating cell-specific proteins, heat shock proteins, and other proteins involved in the regulation of metabolism and the transcriptional and translational machinery. Further characterization of the identified proteins should provide greater insight into the mechanisms involved in regulation of neurogenesis in the adult central nervous system and potentially that of other proliferating cell types, including peripheral stem cells or cancer cells.
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