Multiple Reaction Monitoring of mTRAQ-Labeled Peptides Enables Absolute Quantification of Endogenous Levels of a Potential Cancer Marker in Cancerous and Normal Endometrial Tissues
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Bibliographic record
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.
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Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from it