Bedaquiline (BDQ), an ATP synthase inhibitor, is the first drug to be approved for treatment of multidrug-resistant tuberculosis in decades. Though BDQ has shown excellent efficacy in clinical trials, its early bactericidal activity during the first week of chemotherapy is minimal. Here, using microfluidic devices and time-lapse microscopy of Mycobacterium tuberculosis, we confirm the absence of significant bacteriolytic activity during the first 3–4 days of exposure to BDQ. BDQ-induced inhibition of ATP synthesis leads to bacteriostasis within hours after drug addition. Transcriptional and proteomic analyses reveal that M. tuberculosis responds to BDQ by induction of the dormancy regulon and activation of ATP-generating pathways, thereby maintaining bacterial viability during initial drug exposure. BDQ-induced bacterial killing is significantly enhanced when the mycobacteria are grown on non-fermentable energy sources such as lipids (impeding ATP synthesis via glycolysis). Our results show that BDQ exposure triggers a metabolic remodelling in mycobacteria, thereby enabling transient bacterial survival.

In proteomics it is essential to be able to detect proteins separated by gel electrophoresis at high sensitivity. Silver staining is currently the most popular method. Here we present silver staining protocols that are optimized for staining sensitivity, peptide recovery and compatibility with digestion and mass spectrometry.

Molecular and fragment ion data of intact 8-to 43-kDa proteins from electrospray Fourier-transform tandem mass spectrometry are matched against the corresponding data in sequence data bases. Extending the sequence tag concept of Mann and Wilm for matching peptides, a partial amino acid sequence in the unknown is first identified from the mass differences of a series of fragment ions, and the mass position of this sequence is defined from molecular weight and the fragment ion masses. For three studied proteins, a single sequence tag retrieved only the correct protein from the data base; a fourth protein required the input of two sequence tags. However, three of the data base proteins differed by having an extra methionine or by missing an acetyl or heme substitution. The positions of these modifications in the protein examined were greatly restricted by the mass differences of its molecular and fragment ions versus those of the data base. To characterize the primary structure of an unknown represented in the data base, this method is fast and specific and does not require prior enzymatic or chemical degradation.With the new genomic data bases of model species (1-3), such as Esherichia coli, Saccharomyces cerevisae, mouse, and human, the sequences of many proteins of biological interest will in principle be known, and the problem of characterizing a protein primary structure will be reduced to identifying it in the data base and localizing differences between the putative data base sequence and the actual protein. Analysis at the protein level should be especially relevant for characterization of the products of genes expressed in a given cell line (4) and of proteins with variable mRNA processing or with posttranslational modifications, variations that cannot be identified at the DNA level. Such characterization will be important for correlation of genes with protein function and elucidation of biosynthetic and cell signaling pathways.Mass spectrometry (MS) has recently become increasingly relevant to the analysis of biomolecules. Ionization techniques such as matrix-assisted laser desorption (5, 6) and electrospray ionization (ESI) (7, 8) have greatly extended the mass range for analysis of proteins and nucleotides. ESI combined with Fourier-transform (FT) MS (9, 10) appears to have unusually superior attributes for proteins up to 67 kDa (11), with 105 resolving power and < 10-15 mol sample requirements (12)(13)(14)(15) (22) show that most protein product ions are formed by amide bond cleavage to yield multiply charged ions containing the N terminus (B-ions) and/or C terminus (Y"-ions), with these dissociating further at higher energies to form internal ions (23). A pair of B or of Y" fragment ions whose masses differ by that of an amino acid thus indicates its presence in the protein and its mass separation from a terminus; a series of such fragments provides a partial amino acid sequence in the protein.Within the past few years several research groups have demonstrated how MS can be used for identifica...

The plasminogen activation cascade system, directed by urokinase and the urokinase receptor, plays a key role in extracellular proteolysis during tissue remodeling. To identify molecular interaction partners of these trigger proteins on the cell, we combined covalent protein cross-linking with mass spectrometry based methods for peptide mapping and primary structure analysis of electrophoretically isolated protein conjugates. A specific tri-molecular complex was observed upon addition of pro-urokinase to human U937 cells. This complex included the urokinase receptor, pro-urokinase, and an unknown, high molecular weight urokinase receptorassociated protein. The tryptic peptide mixture derived from a cross-linked complex of pro-urokinase and the latter protein was analyzed by nanoelectrospray tandem mass spectrometric sequencing. This analysis identified the novel protein as the human homologue of a murine membrane-bound lectin with hitherto unknown function. The human cDNA was cloned and sequenced. The protein, designated uPARAP, is a member of the macrophage mannose receptor protein family and contains a putative collagen-binding (fibronectin type II) domain in addition to 8 C-type carbohydrate recognition domains. It proved capable of binding strongly to a single type of collagen, collagen V. This collagen binding reaction at the exact site of plasminogen activation on the cell may lead to adhesive functions as well as a contribution to cellular degradation of collagen matrices.

In proteomics it is essential to be able to detect proteins separated by gel electrophoresis at high sensitivity. Silver staining is currently the most popular method. Here we present silver staining protocols that are optimized for staining sensitivity, peptide recovery and compatibility with digestion and mass spectrometry.