We aimed to determine whether shotgun proteomic approaches could be used to identify tuberculosis (TB)-specific biomarkers in the urine of well-characterised patients with active TB versus no TB.Patients with suspected TB (n563) were classified as: definite TB (Mycobacterium tuberculosis positive culture, n521); presumed latent-TB infection (LTBI) (M. tuberculosis negative culture, no radiological features of active TB, a positive QuantiFERON-TB Gold In-Tube (QFT-IT) test and a positive T-SPOT.TB test, n524); and presumed non-TB/non-LTBI (M. tuberculosis negative culture, no radiological features of active TB, a negative QFT-IT test and a negative T-SPOT.TB test, n518). Urine proteins, in the range of 3-50 kDa, were collected, separated by a one-dimensional SDS-PAGE gel and digested using trypsin, after which high-performance liquid chromatography-tandem mass spectrometry was used to identify the urinary proteome.10 mycobacterial proteins were observed exclusively in the urine of definite TB patients, while six mycobacterial proteins were found exclusively in the urine of presumed LTBI patients. In addition, a gene ontology enrichment analysis identified a panel of 20 human proteins that were significant discriminators (p,0.05) for TB disease compared to no TB disease. Furthermore, seven common human proteins were differentially over-or under-expressed in the TB versus the non-TB group.These biomarkers hold promise for the development of new point-of-care diagnostics for TB. @ERSpublicationsThe application of proteomics for the identification of novel urinary tuberculosis biomarkers

A new mass spectrometric method has been developed for the analysis of low molecular weight polyethylene (PE). Laser-induced acoustic desorption (LIAD), combined with chemical ionization by the cyclopentadienyl cobalt radical cation (CpCo.+) in a Fourier transform ion cyclotron resonance mass spectrometer, produces predominantly a quasimolecular ion, (R + CpCo - 2H2).+, for each PE oligomer (R). An examination of artificial alkane mixtures revealed no mass bias for alkanes of differing molecular weights. However, the success of the LIAD/CpCo.+ CI technique depends greatly upon the LIAD sample preparation method used. Several sample preparation methods were evaluated, and pneumatically assisted spin coating was concluded to provide the best mass spectra as a result of its ability to provide uniform PE coverage on the LIAD foils. The molecular weight distributions measured for several low molecular weight PE samples (200-655) were found to be in good agreement with manufacturers' values as determined by gel permeation chromatography.

Despite affecting up to 70% of HIV-positive patients and being the leading cause of dementia in patients under 40 years, the molecular mechanisms involved in the onset of HIV-associated neurocognitive disorders (HAND) are not well understood. To address this, we performed SILAC-based quantitative proteomic analysis on HIV-Tat treated SH-SY5Y neuroblastoma cells. Isolated protein was fractionated by SDS-PAGE and analyzed by nLC-MS/MS on an Orbitrap Velos. Using MaxQuant, we identified and quantified 3077 unique protein groups, of which 407 were differentially regulated. After applying an additional standard deviation-based cutoff, 29 of these were identified as highly significantly and stably dysregulated. GO term analysis shows dysregulation in both protein translation machinery as well as cytoskeletal regulation that have both been implicated in other dementias. In addition, several key cytoskeletal regulatory proteins such as ARHGEF17, the Rho GTPase, SHROOM3, and CMRP1 are downregulated. Together, these data demonstrate that HIV-Tat can dysregulate neuronal cytoskeletal regulatory proteins that could lead to the major HAND clinical manifestation-synapse loss.

The reactivity of ClMn(H(2)O)(+) towards small organic compounds (L) was examined in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. The organic compounds studied are aliphatic and aromatic alcohols, aliphatic amines, ketones, an epoxide, an ether, a thiol and a phosphine. All the reactions lead to the formation of the ClMn(H(2)O)(L)(+) complex, which dissociates by loss of the H(2)O molecule. In general, the reactions were found to occur with high efficiencies (>85%), indicating them to be exothermic. Electron transfer was also observed between ClMn(H(2)O)(+) and compounds with low ionization energies (IE), to form the molecular ion (L(+•)) of the analyte. Based on these observations, the IE of ClMn(H(2)O)(+) is approximated to be 8.1 ± 0.1 eV. Thus, the utility of ClMn(H(2)O)(+) as a chemical ionization reagent in mass spectrometry is expected to be limited to organic compounds with IEs greater than 8 eV.

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