Accurate Identification of Closely Related Mycobacterium tuberculosis Complex Species by High Resolution Tandem Mass Spectrometry.

Rapid and accurate differentiation of Mycobacterium tuberculosis complex (MTBC) species from other mycobacterium is essential for appropriate therapeutic management, timely intervention for infection control and initiation of appropriate health care measures. However, routine clinical characterization methods for Mycobacterium tuberculosis (Mtb) species remain both, time consuming and labor intensive. In the present study, an innovative liquid Chromatography-Mass Spectrometry method for the identification of clinically most relevant Mycobacterium tuberculosis complex species is tested using a model set of mycobacterium strains. The methodology is based on protein profiling of Mycobacterium tuberculosis complex isolates, which are used as markers of differentiation. To test the resolving power, speed, and accuracy of the method, four ATCC type strains and 37 recent clinical isolates of closely related species were analyzed using this new approach. Using different deconvolution algorithms, we detected hundreds of individual protein masses, with a subpopulation of these functioning as species-specific markers. This assay identified 216, 260, 222, and 201 proteoforms for M. tuberculosis ATCC 27294™, M. microti ATCC 19422™, M. africanum ATCC 25420™, and M. bovis ATCC 19210™ respectively. All clinical strains were identified to the correct species with a mean of 95% accuracy. Our study successfully demonstrates applicability of this novel mass spectrometric approach to identify clinically relevant Mycobacterium tuberculosis complex species that are very closely related and difficult to differentiate with currently existing methods. Here, we present the first proof-of-principle study employing a fast mass spectrometry-based method to identify the clinically most prevalent species within the Mycobacterium tuberculosis species complex.

Polyphenols bind to low density lipoprotein at biologically relevant concentrations that are protective for heart disease.

There is ample evidence in the epidemiological literature that polyphenols, the major non-vitamin antioxidants in plant foods and beverages, have a beneficial effect on heart disease. Until recently other mechanisms which polyphenols exhibit such as cell signaling and regulating nitric oxide bioavailability have been investigated. The oxidation theory of atherosclerosis implicates LDL oxidation as the beginning step in this process. Nine polyphenols from eight different classes and several of their O-methylether, O-glucuronide and O-sulfate metabolites have been shown in this study to bind to the lipoproteins and protect them from oxidation at lysosomal/inflammatory pH (5.2), and physiological pH (7.4). Polyphenols bind to the apoprotein at pH 7.4 with Kb > 106 M-1 and the number of molecules of polyphenols bound per LDL particle under saturation conditions varied from 0.4 for ferulic acid to 13.1 for quercetin. Competition studies between serum albumin and LDL show that substantial lipoprotein binding occurs even in the presence of a great molar excess of albumin, the major blood protein. These in vitro results are borne out by published human supplementation studies showing that polyphenol metabolites from red wine, olive oil and coffee are found in LDL even after an overnight fast. A single human supplementation with various fruit juices, coffee and tea also produced an ex vivo protection against lipoprotein oxidation under postprandial conditions. This in vivo binding is heart-protective based on published olive oil consumption studies. Relevant to heart disease, we hypothesize that the binding of polyphenols and metabolites to LDL functions as a transport mechanism to carry these antioxidants to the arterial intima, and into endothelial cells and macrophages. Extracellular and intracellular polyphenols and their metabolites are heart-protective by many mechanisms and can also function as potent "intraparticle" and intracellular antioxidants due to their localized concentrations that can reach as high as the micromolar level. Low plasma concentrations make polyphenols and their metabolites poor plasma antioxidants but their concentration in particles such as lipoproteins and cells is high enough for polyphenols to provide cardiovascular protection by direct antioxidant effects and by other mechanisms such as cell signaling.

Analysis of Methylation, Acetylation, and Other Modifications in Bacterial Ribosomal Proteins.

A wide variety of posttranslational modifications of expressed proteins are known to occur in living organisms (Krishna R, Wold F. Post-translational modification of proteins. In: Meister A (ed) Advances in enzymology and related areas of molecular biology. Wiley, New York, 1993, pp 265-296). Although their presence in an organism cannot be predicted from the genome, these modifications can play critical roles in protein structure and function. The identification of posttranslational modifications is critical to our understanding of the functions of proteins involved in important biological pathways and mass spectrometry offers a fast, accurate method for observing them. A combined top-down/bottom-up approach can be used for identification and localization of posttranslational modifications of ribosomal proteins. This chapter describes procedures for analyzing Escherichia coli ribosomal proteins and their modifications by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. It also covers the analysis of gram-negative Caulobacter crescentus and gram-positive Bacillus subtilis ribosomal proteins by electrospray quadrupole time-of-flight (ESI-QTOF) mass spectrometry. Confirmation of the occurrence and localization of PTMs is obtained through mass spectrometric analysis of tryptic peptides.

Quantification of HPLC-separated peptides and proteins by spectrofluorimetric detection of native fluorescence and mass spectrometry.

Due to relatively low reproducibility of the ionization and differences when using buffers as mobile phases, the quantitative analysis by electrospray ionization mass spectrometry (ESI-MS) can be often challenging. In the present study, the native fluorescence of phenylalanine, tyrosine, and tryptophan was investigated as an improvement tool for the analytical quantification of peptides and proteins by HPLC-ESI-MS. Natively fluorescent amino acids as well as peptides, proteins, and protein digests were successfully separated by HPLC, and quantified with a spectrofluorimetric detector and ESI-MS. The two detectors were connected in series and enabled the sequential measurements of the fluorescence intensities as well as the measurements of the ion signals and mass spectral characterization of separated polypeptides. Fluorescence detector provided better linearity and repeatability of quantification than mass spectrometer, and similar limits of detection for most of biomolecules analyzed. The fluorescence signal was linear over 3-4 orders of magnitude with limits of detection in picomole or high femtomole range, depending on nature and number of natively fluorescent amino acid residues present in the analyzed polypeptides. Hence, native fluorescence of phenylalanine, tyrosine, and tryptophan can be used as a label-free methodology to facilitate quantification of peptides and proteins by LC-ESI-MS.

Analysis of folate binding protein N-linked glycans by mass spectrometry.

The folate binding protein (FBP), also known as the folate receptor (FR), is a glycoprotein which binds the vitamin folic acid and its analogues. FBP contains multiple N-glycosilation sites, is selectively expressed in tissues and body fluids, and mediates targeted therapies in cancer and inflammatory diseases. Much remains to be understood about the structure, composition, and the tissue specificities of N-glycans bound to FBP. Here, we performed structural characterization of N-linked glycans originating from bovine and human milk FBPs. The N-linked glycans were enzymatically released from FBPs, purified, and permethylated. Native and permethylated glycans were further analyzed by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry (MS), while tandem MS (MS/MS) was used for their structural characterization. The assignment of putative glycan structures from MS and MS/MS data was achieved using Functional Glycomics glycan database and SimGlycan software, respectively. It was found that FBP from human milk contains putative structures that have composition consistent with high-mannose (Hex(5-6)HexNAc(2)) as well as hybrid and complex N-linked glycans (NeuAc(0-1)Fuc(0-3)Hex(3-6)HexNAc(3-5)). The FBP from bovine milk contains putative structures corresponding to high-mannose (Hex(4-9)HexNAc(2)) as well as hybrid and complex N-linked glycans (Hex(3-6)HexNAc(3-6)), but these glycans mostly do not contain fucose and sialic acid. Glycomic characterization of FBP provides valuable insight into the structure of this pharmacologically important glycoprotein and may have utility in tissue-selective drug targeting and as a biomarker.

Multimodal spectral imaging of cells using a transmission diffraction grating on a light microscope.

A multimodal methodology for spectral imaging of cells is presented. The spectral imaging setup uses a transmission diffraction grating on a light microscope to concurrently record spectral images of cells and cellular organelles by fluorescence, darkfield, brightfield, and differential interference contrast (DIC) spectral microscopy. Initially, the setup was applied for fluorescence spectral imaging of yeast and mammalian cells labeled with multiple fluorophores. Fluorescence signals originating from fluorescently labeled biomolecules in cells were collected through triple or single filter cubes, separated by the grating, and imaged using a charge-coupled device (CCD) camera. Cellular components such as nuclei, cytoskeleton, and mitochondria were spatially separated by the fluorescence spectra of the fluorophores present in them, providing detailed multi-colored spectral images of cells. Additionally, the grating-based spectral microscope enabled measurement of scattering and absorption spectra of unlabeled cells and stained tissue sections using darkfield and brightfield or DIC spectral microscopy, respectively. The presented spectral imaging methodology provides a readily affordable approach for multimodal spectral characterization of biological cells and other specimens.