Identification of Host Proteins Predictive of Early Stage Mycobacterium tuberculosis Infection.

The objective of this study was to identify blood-based protein biomarkers of early stage Mycobacterium tuberculosis (Mtb) infection. We utilized plasma and serum specimens from TB patients and their contacts (age≥12) enrolled in a household contact study in Uganda. In the discovery phase cross-sectional samples from 104 HIV-uninfected persons classified as either active TB, latent Mtb infection (LTBI), tuberculin skin test (TST) converters, or persistent TST-negative were analyzed. Two hundred eighty-nine statistically significant (false discovery rate corrected p<0.05) differentially expressed proteins were identified across all comparisons. Proteins associated with cellular immunity and lipid metabolism were induced early after Mtb infection. One hundred and fifty-nine proteins were selected for a targeted mass spectrometry assay. A set of longitudinal samples from 52 TST-negative subjects who converted to TST-positive or remained TST-negative were analyzed, and multivariate logistic regression was used to identify unique protein panels able to predict TST conversion with cross-validated AUC>0.85. Panel performance was confirmed with an independent validation set of longitudinal samples from 16 subjects. These candidate protein biomarkers may allow for the identification of recently Mtb infected individuals at highest risk for developing active TB and most likely to benefit from preventive therapy.

Modification of peptide and protein cysteine thiol groups by conjugation with a degradation product of ascorbate.

Ascorbate is an important water-soluble antioxidant, which when oxidized by reactive oxygen species is converted into dehydroascorbate (DHA). If not rapidly reduced back to ascorbate, DHA decomposes to a reactive 5-carbon compound (DHA*, +130 Da) that can modify reduced cysteinyl residues in peptides and proteins in vitro. The formation of cysteine adducts by DHA* was characterized by mass spectrometry using reduced insulin B-chain, α-lactalbumin, and hemoglobin. Mass spectrometry of DHA* modified insulin B-chain revealed the presence of one and two DHA* adducts. Enzymatic cleavage and tandem mass spectrometry of modified peptides allowed unambiguous localization of DHA* to the two cysteine residues in positions 7 and 19 of the insulin B-chain. Incubations of DHA with α-lactalbumin revealed that approximately 25% of the protein population was in a reduced state and could be modified by DHA*. The adduct was assigned to the N-terminally located cysteinyl residue in position 6. Incubation of hemoglobin with DHA followed by pepsin digestion and electrospray ionization tandem mass spectrometry (ESI-MSMS) of the peptide mixture allowed for the identification of three modified peptides. Tandem mass spectrometry of the modified peptides, two from the hemoglobin A-chain with identical mass and one from the hemoglobin B-chain, gave a complete series of y-type fragment ions, which were assigned to the cysteine containing peptides (100)LLSHCL(105) (A-chain), (101)LSHCLL(106) (A-chain), and (111)VCVLAHHFGKE(121) (B-chain). Although the DHA* adduct was lost from the peptides derived from α-lactalbumin and hemoglobin before fragmentation of the peptide bond, carbamidomethylation of the proteins prior to incubation with DHA abolished the formation of DHA*-protein adducts and confirmed that the target was indeed the cysteine thiol group. Future studies are focused on the modification of proteins by DHA* in cells and in vivo.