Quantitative Analysis Of The AKR Abundances Using MTRAQ/MRM

Aldo-Keto Reductase (AKR) is a protein family whose members are able to reduce aldehydes and ketones and are present in a variety of organisms. In AKR family, a number of the members share with similar 3D structure and high homolog of amino acid sequences, and catalyze the same substrates in some reactions. It is reported by many laboratories that AKRs participate in many physiological processes, however, the deep investigation is not easily carried out due to lack of specific and accurate quantitative methods. Multiple reaction monitoring (MRM) upon mass spectrometry is an accurate quantitative method for proteomics study in recent years. In this study, we established an absolute quantitative MRM assay to measure human AKR proteins, and also applied it to analyze the AKR abundance in different cancer cell lines.Based the shotgun and MRM triggered MS2 results of the digested recombinant AKRs, we selected the unique peptides with the strongest MS signals. In order to improve the detection sensitivity, we enriched AKRs by SDS-PAGE, evaluated the experimental errors caused by enrichment, and confirmed that the additional treatment did not impact quantitative accuracy. We introduced the approach of mTRAQ triplex into the AKR quantitation, which served establishment of the universal calibration curves for absolute quantification to avoid the repeated calibrations for many samples. The components of mTRAQ triplex have the same chemical structure but contain three different isotopes. Triple quadrupole mass spectrometers can differentiate the peptides labeled by different isotope mTRAQs. We labeled mTRAQ reagent with three targets, the synthetic AKR peptides for calibration curves, internal standards and the samples, respectively. With the labeling strategy, the interference of the endogenous target proteins in the cells could be effectively eliminated, and normalization of isotopic internal standards could make the quantitative results based on the calibration curves acquired from the mixed cellular background very consistant with the ones from the single cellular background. Finally, we established the mTRAQ/MRM approach to measure the absolute abundances of 6 AKRs, AKR1A1, AKR1B1, AKR1B10, AKR1C1/C2, AKR1C3 and AKR1C4, respectively (more than 80% of data CV< 5%).We chose 7 different human tumor cell lines, hepatocellular carcinoma (Huh7), lung adenocarcinoma (A549), lung squamous carcinoma (H157), bladder carcinoma (5637), colon adenocarcinoma (SW480), stomach adenocarcinoma (BGC823), and kidney adenocarcinoma (786-0). We overall quantified the AKRs in these cell lines by our mTRAQ/MRM approach.The quantitative results revealed that,1) among all the AKR members, the abundance of AKR1A1 was relatively stable in all the seven cell lines,2) the AKR1C abundances were quite diverse in these cells with inconsistent abundant patterns, and 3) AKR1C4 as a liver-specific protein was only detected in Huh7. We selected two unique peptides from three AKRs, AKR1 Al, AKR1B1 and AKR1B10, and made a comparison of the absolute quantitative data derived from single or two unique peptides per AKR. The absolute quantitation of AKR upon a single unique peptide was different from that upon the two unique peptides for some AKRs, however, the clustering analysis suggested that the abundance distributions for all AKRs in these tumor cell lines were not impacted by the number of selected peptides for MRM.Under extreme oxidative stress, we observed that a MRM signal corresponding to a peptide of AKR1C1, LWCNSHRPELVRPALER, dropped down significantly in Huh7 cells treated with hydrogen peroxide. As no report indicates AKR1C proteins sensitive to oxidative stress yet, the fact prompted us to deeply explore the relationship between the oxidation modification of cysteine in AKR1C1 and its activity. We took IAA to modify recombinant AKR1C1 following monitoring the changes of AKR1C1 catalytic activity, and found AKR1C1 decreased gradually after incubation with IAA. We further modified AKR1C1 with IAA and NEM before and after denaturation respectively, and identified the accessible cysteine sites in AKR1C1 using mass spectrometry. The identification results indicated that Cys87 was an active cysteine easily attacked by the alkylating reagent. We generated the mutant recombinant AKR1C1, AKR1C1-C87S, and made a comparison of the catalytic activity between wild and mutated AKR1C1. The activity assay clearly demonstrated that the catalytic activity of AKR1C1-C87S was not affected by IAA modification. Therefore, for the first time, we claimed that there is a cysteine-active site in AKR1C1. We further hypothesize the reduced status of Cys87 in AKR1C1 is a crucial factor in controlling its detoxification function.