Pulmonary disease resulting from non-tuberculous mycobacteria (NTM) infection has emerged as an increasingly prevalent clinical entity in the past two to three decades, but there are no standardized, commercial assays available for the molecular diagnosis of NTM infections from clinical samples. Herein we discuss the development of an assay that employs immunoprecipitation coupled with mass spectrometry (IP-MS) to rapidly and accurately discriminate prevalent slow-growing mycobacterial species (i.e., M. avium and M. intracellulare, M. kansasii, M. gordonae, M. marinum and M. tuberculosis) during early growth in mycobacterial growth indicator tube (MGIT) cultures. This IP-MS assay employs antibodies specific for conserved tryptic peptides of M. tuberculosis EsxN (AQAASLEAEHQAIVR) and CFP-10 (TQIDQVESTAGSLQGQWR) to capture and identify specific mycobacterial species and allows species-specific mycobacterium identification at the first sign of MGIT culture positivity.

In this study, PRM was employed to identify and quantify peptides generated by tryptic digestion of mycobacterial secreted proteins. To select the precursor ions for PRM analysis, we imported eight protein sequences downloaded in April 2020 from Uniprot database (Uniprot accessions: P9WNJ3, P9WNI7, U5WP40, H8IMX9, B5TV82, P9WNK5, B5TV81 and X8B9F3) into the Skyline software and predict their theoretical tryptic peptides and precursor m/z by setting trypsin digestion unless the site is before a proline residue, no miss cleavage, peptide length in 8-25 amino acids and cysteine cabamidomethylation. The eight proteins were selected based on the identified protein lists in our discovery experiments of the culture filtrates from five mycobacteria species and the available proteomes of M. marinum in the Uniprot database. All monitored precursor ions in the PRM analysis are listed in Table 1 with their m/z at charge 2 status. PRM analysis of peptides was performed using an QExactive HF-X Orbitrap mass spectrometer coupled with an UltiMate 3000 ultrahigh-pressure liquid chromatography (UHPLC) system (Thermo Scientific). Peptides were loaded onto an Acclaim PepMap100 C18 trap column (300 μm ID × 5 mm, 5 μm, Thermo Fisher Scientific; Cat. # 160454) and then separated on a PepMap RSLC C18 analytical column (75 μm ID×15 cm, 3 μm, Thermo Fisher Scientific, Cat. # 164568). Peptides were eluted with a 300 nL/min gradient generated by mixing buffer A (0.1% formic acid in water) and buffer B (0.1% formic acid in acetonitrile) as follows: a 5 min wash with 5% buffer B, a 17 min 5–38% buffer B gradient, a 2 min 38–95% buffer B gradient, a 0.1 min 95-5% buffer gradient, and a 1 min 5% buffer B wash. The full mass scan range was 500-1,200 m/z. The analysis used a 200 ms maximum injection time (IT) and 3e6 automatic gain control (AGC) for full mass scan, 100 ms maximum IT and 2e5 AGC for tandem mass scan, 0.7 m/z isolation window, a 12 loop count setting, a 1.8 kV spray voltage and a 275°C capillary temperature. For each peptide, a 1 mg crude powder of its internal standard labeled with stable isotope arginine (13C615N4) was purchased from New England Peptide (Gardner, MA). Their purity was determined by HPLC as >75%. Their masses were analyzed by an amaZon SL ion trap mass spectrometer (Bruker, MA) and the mass error was within 0.1% compared to the theoretical molecular weight. All internal standards were dissolved with 2% acetonitrile, 0.1% trifluoracetic acid at a concentration of 50 µg/mL each to generate a peptide mixture. A 1 µL aliquot of the internal standard mix solution was analyzed by LC-PRM-MS to obtain the retention times and tandem MS spectra for each peptide. To identify interferences that are caused by co-elution of them with the targeted peptides during LC separation, a spectral library was built based on the synthetic internal standard of each peptide targeted, and their tandem MS spectra were searched against the eight protein sequences together with 236 common protein contaminants via Peaks Studio software (version, ). The identified peptide spectra were exported as a mzxml file and imported into the Skyline software to build a spectral library. The dotp, rdotp and peak area ratio (endogenous version/stable isotope labeled heavy version) of each peptide peak in MGIT samples were calculated by Skyline if applicable. A peak is defined as true identification of a peptide target if its dotp and rdotp is not less than 0.9, and its retention time shift compared with the pre-defined retention time of its isotope labeled version (Table 1) is within 1 min. Prior to target peptide immunoprecipitation with antibody conjugated beads, samples were spiked with 40 µL of diluted internal standard solution (50 ng/mL) to permit accurate quantification of the isolated target peptides.

Respiratory specimens were decontaminated using the N-acetyl-L-cysteine/sodium hydroxide method, concentrated by 15 min centrifugation at 3,000 × g, and pellets were suspended in 0.8 ml of sterile phosphate buffer saline (PBS). Microscopic examination for the presence of acid-fast bacilli (AFB) was performed using auramine-rhodamine staining (Becton Dickinson, Sparks, MD). All patient samples were inoculated into Bactec 960 MGIT tubes containing the manufacturer's growth supplement (PANTA) and Middlebrook 7H11 agar (Remel, Lenexa, KS). MGIT samples that were positive for growth were stained with auramine-rhodamine stain to detect the presence of AFB and/or contaminants and sub-cultured on solid media for species identification, and 3 ml of positive MGIT culture was filtered sterilized by passage through a 0.22 μm filter and stored at -80 ˚C for subsequent LC-MS/MS analysis.