Liquid Chromatography-selected Reaction Monitoring Research Articles

B vitamin quantification in lentil seed tissues using ultra-performance liquid chromatography-selected reaction monitoring mass spectrometry

Lentils are a nutritious food in the human diet. High in protein and with low glycemic index, lentils are also a source of folate and other B vitamins. Understanding variability in B vitamin contents among lentils will allow breeders to select for increased levels. We analyzed 34 cultivated and three wild genotypes for vitamins B1, B2, B3, B5, B6, B7, and B9 in the cotyledons and seed coats. Variation for all B vitamins was observed across the genotypes. Cotyledons had higher concentrations of B1 and B3, while seed coats had higher concentrations of B2, B5, B6, and B9. Wild accessions had the highest concentrations of vitamin B9 and were among the highest for B2. Differential distribution of B vitamins across seed tissues and lentil genotypes has implications for consumption and for breeding. There is useful genetic variability which could be used to increase B vitamin levels in future lentil varieties.

Comparative analysis of heat‐stress‐induced abscisic acid and heat shock protein responses among pea varieties

AbstractHeat shock proteins (HSPs) and abscisic acid (ABA) play important roles in plant heat responses but have not been extensively studied in pea (Pisum sativum L.), whose heat susceptibility is well known. In this study, four pea varieties varying in heat tolerance based on field trials were evaluated. Plants were heat stressed for 3, 6, 12, or 24 h at 38 °C before pollination. Anther and stipule RNA from the same flowering node were sampled for transcriptional profiling of PsHSP18.1 and PsHSP71.2. Additional stipules were sampled for the quantification of ABA concentration and its five key catabolites from the four major ABA catabolic pathways by ultra‐high performance liquid chromatography–selected reaction monitoring mass spectrometry (UHPLC‐SRM/MS). The transcription of both HSP genes was upregulated because of heat stress (HS). In stipules, the upregulation was greatest at 3 h HS, whereas in anthers, the induced transcription was similar among different hours of HS. Likewise, more ABA accumulated in the ABA metabolism pool because of HS, and the ABA response started rapidly after 3 h of treatment. Heat‐tolerant varieties had a higher ABA synthesis and turnover rate at 3 h HS than their respective heat‐susceptible counterparts. This study provides new insights into different heat tolerance among Canadian pea varieties regarding HSP and ABA hormone regulation.

From a non-targeted metabolomics approach to a targeted biomarkers strategy to highlight testosterone abuse in equine. Illustration of a methodological transfer between platforms and laboratories.

In order to overcome the challenge associated with the screening of Anabolic-Androgenic Steroids abuses in animal competitions, a non-targeted liquid chromatography coupled to high resolution mass spectrometry based metabolomics approach was implemented on equine urine samples to highlight potential biomarkers associated with the administration of such compounds, using testosterone esters as model steroids. A statistical model relying on four potential biomarkers intensity could be defined to predict the status of the samples. With a routine application perspective, the monitoring of the highlighted potential biomarkers was first transferred into high-throughput liquid chromatography-selected reaction monitoring (LC-SRM). The model's performances and robustness of the approach were preserved and providing a first demonstration of metabolomics-based biomarkers integration within a targeted workflow using common benchtop MS instrumentation. In addition, with a view to the widespread implementation of such biomarker-based tools, we have transferred the method to a second laboratory with similar instrumentation. This proof of concept allows the development and application of biomarker-based strategies to meet current doping control needs.

Validated B vitamin quantification from lentils by selected reaction monitoring mass spectrometry

A validated method for B vitamin separation and quantification from lentil seeds using ultra high performance liquid chromatography-selected reaction monitoring mass spectrometry (UHPLC-SRM MS) was reported. The use of three enzymes (acid phosphatase, β-glucosidase, and rat serum) with a 4 h incubation was sufficient to convert bound B vitamins into their free forms. Twenty B vitamers were selected and a 5-min UHPLC-SRM MS method was optimized for rapid analysis. This method was applied to quantify B vitamin concentration during lentil seed germination over a 5-day period. Total B vitamins increased up to 1.5-fold on day 5 (from 39.2 µg/g to 60.6 µg/g of dry weight) comparing with dry seeds. Vitamin B5 (pantothenic acid) was the most abundant B vitamin in both dry seeds (34.2%) and in germinated seeds (17.7%-24.5% of total B vitamins); B8 (biotin) and B12 (cyanocobalamin) were not detected in lentil samples.

High-Throughput Large-Scale Targeted Proteomics Assays for Quantifying Pathway Proteins in Pseudomonas putida KT2440

Targeted proteomics is a mass spectrometry-based protein quantification technique with high sensitivity, accuracy, and reproducibility. As a key component in the multi-omics toolbox of systems biology, targeted liquid chromatography-selected reaction monitoring (LC-SRM) measurements are critical for enzyme and pathway identification and design in metabolic engineering. To fulfill the increasing need for analyzing large sample sets with faster turnaround time in systems biology, high-throughput LC-SRM is greatly needed. Even though nanoflow LC-SRM has better sensitivity, it lacks the speed offered by microflow LC-SRM. Recent advancements in mass spectrometry instrumentation significantly enhance the scan speed and sensitivity of LC-SRM, thereby creating opportunities for applying the high speed of microflow LC-SRM without losing peptide multiplexing power or sacrificing sensitivity. Here, we studied the performance of microflow LC-SRM relative to nanoflow LC-SRM by monitoring 339 peptides representing 132 enzymes in Pseudomonas putida KT2440 grown on various carbon sources. The results from the two LC-SRM platforms are highly correlated. In addition, the response curve study of 248 peptides demonstrates that microflow LC-SRM has comparable sensitivity for the majority of detected peptides and better mass spectrometry signal and chromatography stability than nanoflow LC-SRM.

Targeted quantification of B vitamins using ultra-performance liquid chromatography-selected reaction monitoring mass spectrometry in faba bean seeds

Ultra-performance liquid chromatography-selected reaction monitoring mass spectrometry (UPLC-SRM MS) was used for targeted quantification of water-soluble B vitamins from seeds of 30 genotypes of faba bean with contrasting anti-nutritional profiles. Sixteen vitamers from seven B vitamins were quantified from whole seeds, seed coats and cotyledons. A single-step extraction approach with the use of four enzymes (acid phosphatase, β-glucosidase, protease and rat serum) and 2 h incubation at 37 °C was used. The total B vitamin concentrations ranged from 23.08 to 39.26 μg/g in whole seeds, 11.51 to 30.31 μg/g in seed coats and 23.56 to 32.66 μg/g in cotyledons. Significant genotype by environment interaction effects were observed for all B vitamins. Our results indicated low-tannin genotypes had high B5 content, whereas tannin-containing genotypes had high B1, B2, B6, and B9 content, particularly in seed coats. The analysis of a large number of faba bean germplasm here suggests that its seeds are a rich source of vitamins B1, B2, B3 and B5. The increased consumption of faba bean seeds and ingredients in the human diet can provide enhanced dietary B vitamins.

Verification of a Blood-Based Targeted Proteomics Signature for Malignant Pleural Mesothelioma.

We have verified a mass spectrometry (MS)-based targeted proteomics signature for the detection of malignant pleural mesothelioma (MPM) from the blood. A seven-peptide biomarker MPM signature by targeted proteomics in serum was identified in a previous independent study. Here, we have verified the predictive accuracy of a reduced version of that signature, now composed of six-peptide biomarkers. We have applied liquid chromatography-selected reaction monitoring (LC-SRM), also known as multiple-reaction monitoring (MRM), for the investigation of 402 serum samples from 213 patients with MPM and 189 cancer-free asbestos-exposed donors from the United States, Australia, and Europe. Each of the biomarkers composing the signature was independently informative, with no apparent functional or physical relation to each other. The multiplexing possibility offered by MS proteomics allowed their integration into a single signature with a higher discriminating capacity than that of the single biomarkers alone. The strategy allowed in this way to increase their potential utility for clinical decisions. The signature discriminated patients with MPM and asbestos-exposed donors with AUC of 0.738. For early-stage MPM, AUC was 0.765. This signature was also prognostic, and Kaplan-Meier analysis showed a significant difference between high- and low-risk groups with an HR of 1.659 (95% CI, 1.075-2.562; P = 0.021). Targeted proteomics allowed the development of a multianalyte signature with diagnostic and prognostic potential for MPM from the blood. The proteomic signature represents an additional diagnostic approach for informing clinical decisions for patients at risk for MPM.

Development and validation of a methotrexate adherence assay

BackgroundThe first-line therapy for rheumatoid arthritis (RA) is weekly oral methotrexate (MTX) at low dosages (7.5–25 mg/week). However, ~40% of patients are non-adherent which may explain why some do not...

Variation in Seed Allergen Content From Three Varieties of Soybean Cultivated in Nine Different Locations in Iowa, Illinois, and Indiana.

Soybean (Glycine max) is an important food stock, and also considered an allergenic food with at least eight well characterized allergens. However, it is a less prevalent allergen source than many other foods and is rarely life-threatening. Soybean is incorporated into commonly consumed foods, and therefore, the allergens pose a potential concern for individuals already sensitized. The protein profile of soybean can be affected by several factors including genetic and environmental. To investigate how soybean allergen content may be affected by genetics and/or environment, nine soy allergens were quantified from three commercial soybean varieties grown at nine locations in three states within a single climate zone in North America; Iowa, Illinois, and Indiana, United States. Quantitation was achieved using liquid chromatography-selected reaction monitoring (LC-SRM) tandem mass spectrometry with AQUA peptide standards specific to the nine target allergens. Quantitation of allergen concentration indicated that both genetics and location affected specific allergen content. Seven of the nine allergens were significantly influenced by genetics, with the exceptions of glycinin G4 and KTI 3. The allergens P34, Gly m Bd 28k, glycinin G3, and KTI 1 showed statistically significant impact from location as well, but at a lower threshold of significance compared with genetics (cultivar/variety). This dataset contributes to our understanding of the natural variation of endogenous allergens, as it represents a sampling of soybeans grown in a controlled, distributed plot design under agronomic conditions common for commercial soybean food and feed production. The aim was to build upon our recent understanding of how allergens are expressed as part of the overall soybean proteome.

Accurate Measurement of Formaldehyde-Induced DNA-Protein Cross-Links by High-Resolution Orbitrap Mass Spectrometry.

Genomic instability caused by DNA-protein cross-link (DPCs)-induced DNA damage is implicated in disease pathogenesis, aging, and cancer development. The covalent linkages between DNA and protein are induced by chemical reactions catalyzed by the endogenous metabolic intermediates and exogenous agents, such as aldehydes, chemotherapeutic agents, and ionizing radiation. Formaldehyde has been classified as a genotoxic carcinogen. In addition, endogenous formaldehyde-induced DPCs may increase the risks of bone marrow toxicity and leukemia. There is a need to develop an effective detection method for DPC analysis, including the structural differentiation of endogenous and exogenous formaldehyde-induced DPCs. To this end, our group previously reported a useful liquid chromatography-selected reaction monitoring (LC-SRM) approach coupled with stable isotope labeling and low mass resolution-triple quadrupole mass spectrometry. In the present work, we further demonstrate an accurate quantification method using a high-resolution, accurate-mass Orbitrap mass spectrometer for the measurement of the covalent linkage between 2'-deoxyguanosine (dG) and cysteine (Cys), specifically termed dG-Me-Cys, one kind of linkages derived from the formaldehyde-induced DPCs. This quantification method with a wide dynamic range of at least 3 orders generates an interference-free spectrum for unbiased and unambiguous quantification, resulting in good intra- and interday precisions and accuracies with less than 10% variations. The endogenous and exogenous amounts of dG-Me-Cys in a human cell line treated with formaldehyde are analyzed by our new methodology. The quantification strategy demonstrated in this study can be widely applied to characterize and quantify other DPC linkages induced by formaldehyde or other chemical agents.

Manual method of visually identifying candidate signals for a targeted peptide

The purpose of this study is to improve peptide signal identification in groups of extracted ion chromatograms (XICs) obtained with the liquid chromatography-selected reaction monitoring (LC-SRM) technique and a triple quadrupole mass spectrometer (QqQ) operating in one of the supported multiple reaction monitoring (MRM) modes. The imperfection of quadrupole mass analyzers causes ion interference, which impedes the identification of peptide signals as chromatographic peak groups in relevant retention time intervals. To investigate this problem in depth, the QqQ conversion of the eluate into XIC groups was considered as the consecutive transformations of the particles' abundances as the corresponding functions of retention time. In this study, the hypothesis that, during this conversion, the same chromatographic profile should be preserved as an implicit sign in each chromatographic peak of the signal was confirmed for peptides.To examine chromatographic profiles, continuous transformations of XIC groups were derived and implemented in srm2prot Express software (s2pe, http://msr.ibmc.msk.ru/s2pe). Because of ion interference, several peptide-like signals may appear in one XIC group. Therefore, these signals must be considered candidates for a targeted peptide's signal and should be resolved after identification. The theoretical investigation of intensity functions as XICs that are not distorted by noise produced three rules for Identifying Candidate Signals for a targeted Peptide (ICSP, http://msr.ibmc.msk.ru/ICSP) that constitute the proposed manual visual method. We theoretically and experimentally compared this method with the conventional semiempirical intuitive technique and found that the former significantly streamlines peptide signal identification and avoids typical errors.

Quantification of Vitamin B12-Related Proteins in Marine Microbial Systems Using Selected Reaction Monitoring Mass Spectrometry.

Mass spectrometry-based proteomic approaches to studying microbial systems enable assessment of taxonomically resolved functional capacity. A subset of these proteomic approaches are absolutely quantitative, enabling comparisons of protein expression patterns between different studies and across environments. This chapter outlines a method for applying quantitative assays in marine microbial communities, using proteins involved in vitamin B12 (cobalamin) utilization and production as specific examples. This approach involves identifying important protein targets, determining taxonomic resolution of the required assays, identifying suitable peptides, developing and optimizing liquid chromatography-selected reaction monitoring mass spectrometry assays (LC-SRM-MS), and processing the resulting data. Implementing the method outlined here results in measurements (fmol diagnostic peptide per μg of total bulk protein) that, in this case, define the nutritional status of microbial community members with respect to vitamin B12, and are comparable across and between marine microbial systems.

Method for Continuous Monitoring of Electrospray Ion Formation.

A method for continuously monitoring the performance of electrospray ionization without the addition of hardware or chemistry to the system is demonstrated. In the method, which we refer to as SprayDx, cluster ions with solvent vapor natively formed by electrospray are followed throughout the collection of liquid chromatography-selected reaction monitoring data. The cluster ion extracted ion chromatograms report on the consistency of the ion formation and detection system. The data collected by the SprayDx method resemble the data collected for postcolumn infusion of analyte. The response of the cluster ions monitored reports on changes in the physical parameters of the ion source such as voltage and gas flow. SprayDx is also observed to report on ion suppression in a fashion very similar to a postcolumn infusion of analyte. We anticipate the method finding utility as a continuous readout on the performance of electrospray and other atmospheric pressure ionization processes. Graphical Abstract ᅟ.

Large-Scale SRM Screen of Urothelial Bladder Cancer Candidate Biomarkers in Urine

Urothelial bladder cancer is a condition associated with high recurrence and substantial morbidity and mortality. Noninvasive urinary tests that would detect bladder cancer and tumor recurrence are required to significantly improve patient care. Over the past decade, numerous bladder cancer candidate biomarkers have been identified in the context of extensive proteomics or transcriptomics studies. To translate these findings in clinically useful biomarkers, the systematic evaluation of these candidates remains the bottleneck. Such evaluation involves large-scale quantitative LC-SRM (liquid chromatography-selected reaction monitoring) measurements, targeting hundreds of signature peptides by monitoring thousands of transitions in a single analysis. The design of highly multiplexed SRM analyses is driven by several factors: throughput, robustness, selectivity and sensitivity. Because of the complexity of the samples to be analyzed, some measurements (transitions) can be interfered by coeluting isobaric species resulting in biased or inconsistent estimated peptide/protein levels. Thus the assessment of the quality of SRM data is critical to allow flagging these inconsistent data. We describe an efficient and robust method to process large SRM data sets, including the processing of the raw data, the detection of low-quality measurements, the normalization of the signals for each protein, and the estimation of protein levels. Using this methodology, a variety of proteins previously associated with bladder cancer have been assessed through the analysis of urine samples from a large cohort of cancer patients and corresponding controls in an effort to establish a priority list of most promising candidates to guide subsequent clinical validation studies.

Quantitative proteomics of breast tumors: Tissue quality assessment to clinical biomarkers.

Liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM) is not only a proven tool for clinical chemistry, but also a versatile method to enhance the capability to quantify biomarkers for tumor biology research. As the treatment of cancer continues to evolve, the ability to assess multiple biomarkers to assign cancer phenotypes based on the genetic background and the signaling of the individual tumor becomes paramount to our ability to treat the patient. In breast cancer, the American Society of Clinical Oncology has defined biomarkers for patient assessment to guide selection of therapy: estrogen receptor, progesterone receptor, and the HER2/Neu receptor tyrosine kinase; therefore, these proteins were selected for LC-SRM assay development. Detailed molecular characterization of these proteins is necessary for patient treatment, so expression and phosphorylation assays have been developed and applied. In addition, other LC-SRM assays were developed to further evaluate tumor biology (e.g. Ki-67 for proliferation and vimentin for tumor aggressiveness related to the epithelial-to-mesenchymal transition). These measurements combined with biomarkers for tissue quality and histological content are implemented in a three-tier multiplexed assay platform, which is translated from cell line models into frozen tumor tissues banked from breast cancer patients.

Quantification of Breast Cancer Protein Biomarkers at Different Expression Levels in Human Tumors.

Liquid chromatography-selected reaction monitoring (LC-SRM) mass spectrometry has developed into a versatile tool for quantification of proteins with a wide range of applications in basic science, translational research, and clinical patient assessment. This strategy uniquely complements traditional pathology approaches, like hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). The multiplexing capabilities offered by mass spectrometry are currently unmatched by other techniques. However, quantification of biomarkers in tissue specimens without the other data obtained from H&E-stained slides or IHC, including tumor cellularity or percentage of positively stained cells inter alia, may not provide as much information that is needed to fully understand tumor biology or properly assess the patient. Therefore, additional characterization of the tissue proteome is needed, which in turn requires the ability to assess protein markers across a wide range of expression levels from a single sample. This protocol provides an example of multiplexed analysis in breast tumor tissue quantifying specific biomarkers, specifically estrogen receptor, progesterone receptor, and the HER2 receptor tyrosine kinase, in combination with other proteins that can report on tissue content and other aspects of tumor biology.

Proteoform-Specific Protein Binding of Small Molecules in Complex Matrices.

Characterizing the specific binding between protein targets and small molecules is critically important for drug discovery. Conventional assays require isolation and purification of small molecules from complex matrices through multistep chromatographic fractionation, which may alter their original bioactivity. Most proteins undergo posttranslational modification, and only certain proteoforms have the right conformation with accessible domains and available residues for small molecule binding. We developed a top-down mass spectrometry (MS) centric workflow for rapid evaluation of the bioactivity of crude botanical extracts after a one-step reaction. Our assay distinguished covalent from noncovalent binding and mapped the residue for covalent binding between bioactive constituents and specific proteoforms of the target protein. We augmented our approach with a nanoflow liquid chromatography-selected reaction monitoring (SRM)-MS assay for simultaneous identification and label-free multiplex quantitation of small molecules in the crude botanical extracts. Our assay was validated for various proteoforms of human serum albumin, which plays a key role in pharmacokinetics of small molecules in vivo. We demonstrated the utility of our proteoform-specific assay for evaluating thymoquinone in crude botanical extracts, studying its pharmacokinetics in human blood, and interpreting its toxicity to human breast cancer cells in tissue culture.

Multiplex and accurate quantification of acute kidney injury biomarker candidates in urine using Protein Standard Absolute Quantification (PSAQ) and targeted proteomics

There is a need for multiplex, specific and quantitative methods to speed-up the development of acute kidney injury biomarkers and allow a more specific diagnosis. Targeted proteomic analysis combined with stable isotope dilution has recently emerged as a powerful option for the parallelized evaluation of candidate biomarkers. This article presents the development of a targeted proteomic assay to quantify 4 acute kidney injury biomarker candidates in urine samples. The proteins included in the assessed panel consisted of myo-inositol oxygenase (MIOX), phosphoenolpyruvate carboxykinase 1 (PCK1), neutrophil gelatinase-associated lipocalin (NGAL) and liver fatty acid-binding protein (L-FABP). The proteomic assay combined an antibody-free sample preparation and a liquid chromatography-selected reaction monitoring (LC-SRM) analysis pipeline. For accurate quantification of the selected candidates, we used PSAQ (Protein Standard Absolute Quantification) standards which are isotopically labeled versions of the target proteins. When added directly to the biological samples, these standards improve detection specificity and quantification accuracy. The multiplexed assay developed for the 4 biomarker candidates showed excellent analytical performance, in line with the recommendations of health authorities. Tests on urine from two small patient cohorts and a group of healthy donors confirmed the relevance of NGAL and L-FABP as biomarkers for AKI diagnosis. The assay is readily adaptable to other biomarker candidates and should be very useful for the simultaneous and accurate quantification of multiple biomarkers.

Development of Phage-Based Antibody Fragment Reagents for Affinity Enrichment of Bacterial Immunoglobulin G Binding Proteins.

Disease and death caused by bacterial infections are global health problems. Effective bacterial strategies are required to promote survival and proliferation within a human host, and it is important to explore how this adaption occurs. However, the detection and quantification of bacterial virulence factors in complex biological samples are technically demanding challenges. These can be addressed by combining targeted affinity enrichment of antibodies with the sensitivity of liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS). However, many virulence factors have evolved properties that make specific detection by conventional antibodies difficult. We here present an antibody format that is particularly well suited for detection and analysis of immunoglobulin G (IgG)-binding virulence factors. As proof of concept, we have generated single chain fragment variable (scFv) antibodies that specifically target the IgG-binding surface proteins M1 and H of Streptococcus pyogenes. The binding ability of the developed scFv is demonstrated against both recombinant soluble protein M1 and H as well as the intact surface proteins on a wild-type S. pyogenes strain. Additionally, the capacity of the developed scFv antibodies to enrich their target proteins from both simple and complex backgrounds, thereby allowing for detection and quantification with LC-SRM MS, was demonstrated. We have established a workflow that allows for affinity enrichment of bacterial virulence factors.

Abstract 3450: Resistance to P450c17 inhibitors in castration-resistant prostate cancer may result from the DHEA-S depot that remains and can be used by AKR1C3 for intratumoral androgen biosynthesis

Abstract Localized intermediate and high-risk prostate cancer can be treated with androgen deprivation therapy (ADT). Initially, patients undergo remission but inevitably relapse due to the emergence of castration resistant prostate cancer (CRPC). Newer agents such as the P450c17 inhibitor, abiraterone acetate (AA), have gained approval for the treatment of CRPC and increased median survival by 4 months. We have developed a novel and validated stable isotope dilution liquid chromatography selected reaction monitoring mass spectrometry (SID-LC/ESI/SRM/MS) method for the quantification of conjugated and unconjugated keto-androgens in human serum (J Steroid Biochem Mol Biol (2013) 138:281). This method was applied to human serum from patients enrolled in a neoadjuvant AA clinical trial (J Clin Oncol (2014) 32:3705). We found that testosterone (T) and 5α-dihydrotestosterone (DHT) did not always decrease in tandem, which suggests that pathways that bypass T may lead to DHT. These may include the alternative pathway (Δ4-androstene-3,17-dione -> 5α-androstane-3,17-dione -> DHT) or the backdoor pathway (androsterone -> 5α-androstane-3α,17β-diol -> DHT). Second, despite achieving >90% inhibition of P450c17, the level of dehydroepiandrosterone sulfate (DHEA-S) that remains (20,000 ng/dL) is 4,000-fold higher than castrate levels of T achieved in the trial. We hypothesize that the DHEA-S depot that remains is sufficient to feed intratumoral androgen biosynthesis and that this could account for the clinical failure of P450c17 inhibitors in CRPC. Adaptive intratumoral androgen biosynthesis may be facilitated by the up-regulation of AKR1C3 (type 5 17β-hydroxysteroid dehydrogenase). In order to test our hypothesis, we have developed a panel of isogenic prostate cancer cell lines that either express AKR1C3 or are AKR1C3 null. These cell lines are then challenged with post-AA levels of circulating androgens (DHEA-S, DHEA and Δ4-androstene-3,17-dione) to determine whether they can still make sufficient androgens to activate the androgen receptor (AR), where androgen metabolism is measured using SID-LC/ESI/SRM/MS. Using LNCaP and LNCaP-AKR1C3 cells, we find that the level of T detected as T-17β-glucuronide in the AKR1C3 expressing cells could be sufficient to activate the AR. These results would suggest that AKR1C3 inhibitors in combination with AA could benefit patients who might otherwise fail AA treatment. [Supported by grants: R25-CA101871 to DT; P30-ES-13508 to TMP; P01-CA-163337 to SPB, EM, TMP and PSN)] Citation Format: Daniel Tamae, Elahe Mostaghel, Bruce Montgomery, Peter S. Nelson, Steven P. Balk, Philip W. Kantoff, Mary-Ellen Taplin, Trevor M. Penning. Resistance to P450c17 inhibitors in castration-resistant prostate cancer may result from the DHEA-S depot that remains and can be used by AKR1C3 for intratumoral androgen biosynthesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3450. doi:10.1158/1538-7445.AM2015-3450