Detection of Mycobacterium tuberculosis peptides in the exosomes of patients with active and latent M. tuberculosis infection using MRM-MS

Abstract

The identification of easily measured, accurate diagnostic biomarkers for active tuberculosis (TB) will have a significant impact on global TB control efforts. Because of the host and pathogen complexities involved in TB pathogenesis, identifying a single biomarker that is adequately sensitive and specific continues to be a major hurdle. Our previous studies in models of TB demonstrated that exosomes, such as those released from infected macrophages, contain mycobacterial products, including many Mtb proteins. In this report, we describe the development of targeted proteomics assays employing multiplexed multiple reaction monitoring mass spectrometry (MRM-MS) in order to allow us to follow those proteins previously identified by western blot or shotgun mass spectrometry, and enhance biomarker discovery to include detection of Mtb proteins in human serum exosomes. Targeted MRM-MS assays were applied to exosomes isolated from human serum samples obtained from culture-confirmed active TB patients to detect 76 peptides representing 33 unique Mtb proteins. Our studies revealed the first identification of bacteria-derived biomarker candidates of active TB in exosomes from human serum. Twenty of the 33 proteins targeted for detection were found in the exosomes of TB patients, and included multiple peptides from 8 proteins (Antigen 85B, Antigen 85C, Apa, BfrB, GlcB, HspX, KatG, and Mpt64). Interestingly, all of these proteins are known mycobacterial adhesins and/or proteins that contribute to the intracellular survival of Mtb. These proteins will be included as target analytes in future validation studies as they may serve as markers for persistent active and latent Mtb infection. In summary, this work is the first step in identifying a unique and specific panel of Mtb peptide biomarkers encapsulated in exosomes and reveals complex biomarker patterns across a spectrum of TB disease states.

Figure 1. Flow diagram outlining the rational progression from the discovery phase of Mtb biomarkers using LC-MS/MS through the development of targeted MS assays for the identification of Mtb peptides in human biological fluids.

Figure 2. Summary of Peptides Detected by MRM. Colored boxes signify the presence of a peptide above the positive cut-off in samples from EPTB (blue), LTBI (purple), TB− (green) and PTB (orange) categories.

Presence of peptides was determined using a binary metric and those peptides which failed to yield a signal above background are left blank. Peptides moving ahead to validation assay are indicated by *.

Figure 3. Scatter plot showing the total number of peptides identified in each sample class broken down by TB and HIV status.

P-values from unpaired t-tests are represented as follows: **p<0.01, ***p<0.001, ****p<0.0001.