Successful Implementation of Multiple Reaction Monitoring for the Validation of Tuberculosis Biomarkers

摘要

Mycobacterium tuberculosis (Mtb) currently infects one-third of the global population. Rapid determination of active infection versus latent infection is critical to diagnosing, initiating treatment and reducing the spread of tuberculosis. The development of facile, sensitive methods of detection is vital in controlling disease and therefore the identification of relevant biomarkers is urgent. It has been demonstrated that inflammatory processes, including those triggered by infection with Mtb, cause an upregulation of the formation and release of exosomes. Purification of these 30 to 100 nm vesicles from infected cells or animals fluids has enabled us to concentrate and identify bacterial products. Methods: Exosomes were isolated from tissue culture or biological fluids (bronchoalveolar lavage fluid (BALF), urine, sera) using Exoquick (SBI). Exosomes were quantified by microBCA and SDS-PAGE was performed, serving to lyse exosomes, separate protein components and remove residual Exoquick reagent. Proteins were digested with trypsin and extracted from gel. Discovery of mycobacterial proteins was performed using LC-MS/MS and several were confirmed by western blot. From this data, we have developed several multiplexed multiple reaction monitoring (MRM) methods to screen exosomes isolated from clinically relevant fluids (urine and sera) from guinea pigs and validate dozens of candidate protein biomarkers. Optimized methods were then applied to exosomes isolated from human sera and urine specimens. Preliminary Data: Proof of principle experiments utilizing exosomes isolated from Mtb-infected macrophages yielded the identification of over forty mycobacterial proteins (Ag85, KatG & CFP10), majority of which were known or hypothesized to be secreted. Moreover, we verified that the numbers of exosomes and mycobacterial components--isolated from murine BALF--could reflect the progression from infection to active disease. Recently, we have used sera and urine from both Mtb-infected and naive guinea pigs to validate the proteins identified during our discovery experiments. Using multiplexed MRM methods, we have confirmed both previously reported peptides, as well as novel bacterial peptides. At least 2 peptides for the mycobacterial proteins Rv0350, Rv0934, Rv1837, Rv3418c, and Rv3841 have confidently been identified in both guinea pig fluids. Sera and urine are ideal biofluids for translation to a point of care diagnostic test. While MRMs are not a viable option in countries with the highest disease burden, translation of MRM assays to point of care diagnostic platform is essential. We are currently developing an ELISA-based assay to capture and identify exosomes containing mycobacterial proteins.