Second generation multiple reaction monitoring assays for enhanced detection of ultra-low abundance Mycobacterium tuberculosis peptides in human serum

• Published in: Clinical proteomics Vol. 14; no. 1; p. 21
• Main Authors: Mehaffy, Carolina, Dobos, Karen M, Nahid, Payam, Kruh-Garcia, Nicole A
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 05.06.2017; BioMed Central; BMC
• Subjects: Acquired immune deficiency syndrome; AIDS; Analytical chemistry; Antigens; Bacteria; Biomarker; Biomarkers; Biopsy; Candidates; Data processing; Deoxyribonucleic acid; Diagnosis; DNA; Drug therapy; Enrichment; Exosomes; Filters; Genetic aspects; Geography; HIV; Human immunodeficiency virus; Infections; Infectious diseases; Mass Spectrometry; Microscopy; MRM (Multiple Reaction Monitoring); Mycobacterium infections; Mycobacterium tuberculosis; Patients; Peptides; Polymerase chain reaction; Product development; Proteins; Proteomics; Serum albumin; Serum proteins; Sputum; Tuberculosis; Urine; MRM (Multiple Reaction Monitoring); Biomarker; Tuberculosis; Mycobacterium tuberculosis; Exosomes; Mass Spectrometry
• ISSN: 1542-6416; 1559-0275
• DOI: 10.1186/s12014-017-9156-y

is the causative agent of Tuberculosis (TB), the number one cause of death due to an infectious disease. TB diagnosis is performed by microscopy, culture or PCR amplification of bacterial DNA, all of which require patient sputum or the biopsy of infected tissue. Detection of mycobacterial products in serum, as biomarkers of diagnosis or disease status would provide an improvement over current methods. Due to the low-abundance of mycobacterial products in serum, we have explored exosome enrichment to improve sensitivity. resides intracellularly where its secreted proteins have been shown to be packaged into host exosomes and released into the bloodstream. Exosomes can be readily purified assuring an enrichment of mycobacterial analytes from the complex mix of host serum proteins. Multiple reaction monitoring assays were optimized for the enhanced detection of 41 peptides in exosomes purified from the serum of individuals with TB. Exosomes isolated from the serum of healthy individuals was used to create and validate a unique data analysis algorithm and identify filters to reduce the rate of false positives, attributed to host / interference. The final optimized method was tested in 40 exosome samples from TB positive patients. Our enhanced methods provide limit of detection and quantification averaging in the low femtomolar range for detection of mycobacterial products in serum. At least one mycobacterial peptide was identified in 92.5% of the TB positive patients. Four peptides from the proteins, Cfp2, Mpt32, Mpt64 and BfrB, show normalized total peak areas significantly higher in individuals with active TB as compared to healthy controls; three of the peptides from these proteins have not previously been associated with serum exosomes from individuals with active TB disease. Some of the detected peptides were significantly associated with specific geographical locations, highlighting potential markers that can be linked to the strains circulating within each given region. An enhanced MRM method to detect ultra-low abundance peptides in human serum exosomes is demonstrated, highlighting the potential of this methodology for TB diagnostic biomarker development.