Many useful energy transfer concepts originate from nature. For example, fluorescent proteins have evolved to perform energy transfer with extraordinary efficiency. While fluorescent proteins are commonly used as probes of protein localization in biological cells, their native functions in the host organisms are light absorption, energy transfer to other proteins, or light emission to the environment. 1 For example, green fluorescent protein (GFP) from the jellyfish Aequorea victoria absorbs energy transferred from blue luminescent protein (aequorin) and then emits green light. 1,2 Cyanobacteria and eukaryotic algae (red algae, glaucophytes, and criptomonads) contain three classes of fluorescent phycobiliproteins: phycoerythrins, allophycocyanins, and phycocyanins. 3 These are assembled into photosynthetic protein complexes called phycobilisomes. 3,4 The phycobiliproteins B-phycoerythrin (B-PE) and R-phycoerythrin (R-PE) each contain more than 30 phycoerythrobilin (PEB) and phycourobilin (PUB) chromophores that are assembled in a 240 kDa multimeric complex (Rβ) 6 γ. Hence, phycobilin chromophores, which are linear tetrapyrole compounds bound to polypeptide chains by thioether bonds, are responsible for the excellent spectroscopic properties of phycobiliproteins as well as energy transfer involving phycobiliproteins. 4 Phycobilisomes absorb sunlight and then transfer energy via F€ orster resonance energy transfer (FRET) to chlorophyll a. 4 Phycobiliproteins possess unique characteristics that make them suitable for fluorescence detection and energy transfer, which include extremely high absorption coefficients over a broad part

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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.

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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.

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.

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Advances in massively parallel sequencing, of complete bacterial genomes, have led to many novel findings in the field of genomics. However, these data often lack correlation with expressed protein profiles. It has been demonstrated that even very closely related genomes, such as in mycobacteria, express drastically different phenotypes. These phenotypes often have major roles in pathogenicity. Therefore, it is just as important to have a method for examining the proteome of a bacterium as well as its genome. These studies are further complicated in mycobacteria due to the cell wall and mycolic acid. A comprehensive method for the identification and characterization of the whole mycobacterium protein profile is needed. In the present study, a simple, sensitive, and specific liquid chromatography tandem mass spectrometry method was developed for the extraction, purification and profiling the mycobacterial proteome in various species. During development, sonication and bead-beating cell lysis protocol was tested using 15% Acetonitrile and 6 M guanidine-HCl (GuHCl) as extraction solvent. Sonication lysis in 6 M GuHCl with glass beads was the preferred method for cell lysis. This method was developed using reverse phase liquid chromatography and a Q Exactive ™ Plus Orbitrap™ mass spectrometer for peptide and protein identification. Bottom-up liquid chromatography-mass spectrometry LC–MS analysis resulted in identification of greater than 2500 proteins.