U.S. Department of Energy

Pacific Northwest National Laboratory

Application of multiplexed ion mobility spectrometry towards the identification of host response protein signatures of treatment of pulmonary tuberculosis

Introduction

Tuberculosis (TB) is one of the leading causes of deaths worldwide. Because of the common delays encountered with cultures of sputa, there has been a call for discovery of new quantitative, non-sputum, non-culture-based TB biomarkers. Liquid Chromatography - Ion Mobility MS (LC-IM-MS) provides a robust tool to analyze larger patient cohorts with necessary sensitivity to capture the global proteome.  In this study, we applied the LC-IM-MS platform to analyze serum proteome of pulmonary TB patients participating in a CDC-conducted Phase 2 TB clinical trial. The goal is to 1) provide an unbiased characterization of the proteome in TB patients comparing baseline to 8 weeks of anti-TB treatment, 2) correlate association of differential proteome with clinical and microbiologic characteristics.  

Methods 

Serum samples from 289 subjects enrolled in the TB Trials Consortium Study 29 were collected, processed and stored at baseline (time of enrollment) and week 8 (40 doses of anti-TB).  Following depletion of the 14 most abundant proteins using an IgY14 LC 10 affinity LC column, bottom-up proteomics was applied and involved digestion of the remaining serum proteins. Sample analysis and data acquisition was performed on an in-house built LC-IM-MS instrument. Multidimensional separations were carried out on a reversed phase nanocapillary LC column followed by ion mobility (IM) separation in a 1-m drift tube combined with a TOF MS mass analyzer. Data interpretation including identification and quantitation was performed using the AMT tag approach.

Preliminary data

Based on a statistical analysis of the interactions of demographic and clinical variables, 198 serum proteins were identified as differentially abundant in the human serum proteome from start to 8-weeks of rifamycin-based intensive treatment.  Significant proteins were characterized into pathways across multiple pathway mapping networks available. Top KEGG pathway, coagulation and compliment cascade, mapped 34 proteins including both up and down regulation. Most proteins were regulated to prevent coagulation or promote dissolution of fibrin clots. Additional examples of proteins increased after 8 weeks of treatment are endothelial protein C receptor (EPCR), serine protease inhibitor (IPSP), and plasminogen (PLMN). Notable proteins decreasing with treatment were fibrinogen alpha chain (FIBA), factor IX (FA9), factor V (FA5), and platelet glycoprotein V (GPV). Examples of other significant protein classes included decreasing acute phase reactants and defense proteins and increases in lipid transport and metabolism. Extensive statistical comparisons helped to clarify these and other proteins in the context of clinical disease severity indicators and patient demographics. The multidimensional LC-IM-MS platform greatly improves upon existing MS technologies in analytical sensitivity and specificity, enhances dynamic range of measurements, and provides reliable identification and quantitation of lower abundance analyte species in highly complex biological matrices. This increase in sensitivity combined with reduced analysis time provides enhanced throughput, thus highlighting the ability of this technology for pursuing large clinical-based studies.

Novel aspect

This is the first study to use multidimensional capabilities of LC-IM-MS to investigate untargeted biomarkers of treatment response for TB.

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