Absolute Quantification of Potential Cancer Markers in Clinical Tissue Homogenates Using Multiple Reaction Monitoring on a Hybrid Triple Quadrupole/Linear Ion Trap Tandem Mass Spectrometer

Abstract

Multidimensional liquid chromatography with tandem mass spectrometry with iTRAQ-labeling typically used for differential expression analysis in biomarker discovery does not always detect peptides from these biomarkers in all samples analyzed. Herein we describe the results of targeted analyses using multiple reaction monitoring (MRM) on a hybrid triple quadrupole/linear ion-trap tandem mass spectrometer. The MRM approach when combined with the newly released mTRAQ reagent, a non-isobaric variant of the iTRAQ tag available in two versions, enables absolute quantification of peptides and proteins via isotope-dilution mass spectrometry. This approach was applied to clinical endometrial tissue homogenates in an effort to quantify two endometrial cancer biomarkers, pyruvate kinase (PK) and polymeric immunoglobulin receptor (PIGR). We successfully demonstrated the feasibility of this approach on 20 individual samples and further verified the differential expressions of these two biomarkers in endometrial carcinoma. PK was determined to be present at an average concentration of 58.33 pmol/mg of total proteins and in the range of 9.13−87.66 pmol/mg in the soluble fraction of the normal proliferative endometrium homogenates. By contrast, the average concentration of PK in the cancer sample homogenates was 237.2 pmol/mg of total proteins and in the range of 66.10−570.9 pmol/mg. PIGR was found to be expressed at an average concentration of 8.85 pmol/mg of total proteins with a range of 1.02−49.61 pmol/mg in the normal proliferative control samples, and an average concentration of 200.2 pmol/mg with a range of 7.63−810.4 pmol/mg in the cancer samples. This study confirmed qualitatively the differential expressions previously observed but also showed that the actual relative differential expressions in these samples were much higher than those reported in the discovery study. These results validated earlier observations of dynamic-range compression in iTRAQ-labeling with hybrid quadrupole/time-of-flight mass spectrometry (DeSouza, L.V. et al. J. Proteome Res. 2008, 7, 3525−3534).