Matrix assisted laser desorption ionization-time-of-flight mass spectrometry identification of mycobacterium bovis in bovinae

In this study, Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry was used to identify Mycobacterium bovis from cattle and buffalo tissue isolates from the North and South regions of Brazil, grown in solid medium and previously identified by Polymerase Chain Reaction (PCR) based on Region of Difference 4 (RD4), sequencing and spoligotyping. For this purpose, the protein extraction protocol and the mass spectra reference database were optimized for the identification of 80 clinical isolates of mycobacteria. As a result of this optimization, it was possible to identify and differentiate M. bovis from other members of the Mycobacterium tuberculosis complex with 100% specificity, 90.91% sensitivity and 91.25% reliability. MALDI-TOF MS methodology described herein provides successful identification of M. bovis within bovine/bubaline clinical samples, demonstrating its usefulness for bovine tuberculosis diagnosis in the future.Instituto de BiotecnologíaFil: Bacanelli, Gisele. Federal University of Mato Grosso do Sul. Biotechnology and Biodiversity of the Central Western Region Postgraduate Program; BrasilFil: Olarte, Larissa C. Federal University of Mato Grosso do Sul. Biochemistry and Molecular Biology Multicentric Postgraduate Program; BrasilFil: Silva, Marcio Roberto. Empresa Brasileira de Pesquisa Agropecuária (Embrapa). Gado de Leite; BrasilFil: Rodrigues, Rudielle A. Federal University of Mato Grosso do Sul. Faculty of Veterinary Medicine. Veterinary Sciences Postgraduate Program; BrasilFil: Carneiro, Paulo A. M. Michigan State University. Center for Comparative Epidemiology; Estados UnidosFil: Kannene, John B. Michigan State University. Center for Comparative Epidemiology; Estados UnidosFil: Pasquatti, Taynara N. Dom Bosco Catholic University; BrasilFil: Takatani, Haruo. Agricultural Defense Agency of Amazonas; BrasilFil: Zumarraga, Martin Jose. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Etges, Rodrigo N. Secretary of Agriculture, Livestock and Irrigation; BrasilFil: Araujo, Flabio Ribeiro de. Empresa Brasileira de Pesquisa Agropecuária (Embrapa). Gado de Corte; BrasilFil: Verbisck, Newton V. Empresa Brasileira de Pesquisa Agropecuária (Embrapa). Gado de Corte; Brasi

Structure analysis of biologically important prokaryotic glycopolymers

Of the many post-translational modifications organisms can undertake, glycosylation is the most prevalent and the most diverse. The research in this thesis focuses on the structural characterisation of glycosylation in two classes of glycopolymer (lipopolysaccharide (LPS) and glycoprotein) in two domains of life (bacteria and archaea). The common theme linking these subprojects is the development and application of high sensitivity analytical techniques, primarily mass spectrometry (MS), for studying prokaryotic glycosylation. Many prokaryotes produce glycan arrangements with extraordinary variety in composition and structure. A further challenge is posed by additional functionalities such as lipids whose characterisation is not always straightforward. Glycosylation in prokaryotes has a variety of different biological functions, including their important roles in the mediation of interactions between pathogens and hosts. Thus enhanced knowledge of bacterial glycosylation may be of therapeutic value, whilst a better understanding of archaeal protein glycosylation will provide further targets for industrial applications, as well as insight into this post- translational modification across evolution and protein processing under extreme conditions. The first sub-project focused on the S-layer glycoprotein of the halophilic archeaon Haloferax volcanii, which has been reported to be modified by both glycans and lipids. Glycoproteomic and associated MS technologies were employed to characterise the N- and O-linked glycosylation and to explore putative lipid modifications. Approximately 90% of the S-layer was mapped and N-glycans were identified at all the mapped consensus sites, decorated with a pentasaccharide consisting of two hexoses, two hexuronic acids and a methylated hexuronic acid. The O-glycans are homogeneously identified as a disaccharide consisting of galactose and glucose. Unexpectedly it was found that membrane-derived lipids were present in the S- layer samples despite extensive purification, calling into question the predicted presence of covalently linked lipid. The H. volcanii N-glycosylation is mediated by the products of the agl gene cluster and the functional characterisation of members of the agl gene cluster was investigated by MS analysis of agl-mutant strains of the S-layer. Burkholderia pseudomallei is the causative agent of melioidosis, a serious and often fatal disease in humans which is endemic in South-East Asia and other equatorial regions. Its LPS is vital for serum resistance and the O-antigen repeat structures are of interest as vaccine targets. B. pseudomallei is reported to produce several polysaccharides, amongst which the already characterised ‘typical’ O-antigen of K96243 represents 97% of the strains. The serologically distinct ‘atypical’ strain 576 produces a different LPS, whose characterisation is the subject of this research project. MS strategies coupled with various hydrolytic and chemical derivatisation methodologies were employed to define the composition and potential sequences of the O-antigen repeat unit. These MS strategies were complemented by a novel NMR technique involving embedding of the LPS into micelles. Taken together the MS and NMR data have revealed a highly unusual O-antigen structure for atypical LPS which is remarkably different from the typical O-antigen. The development of structural analysis tools in MS and NMR applicable to the illustrated types of glycosylation in these prokaryotes will give a more consistent approach to sugar characterisation and their modifications thus providing more informative results for pathogenicity and immunological studies as well as pathway comparisons.Open Acces

MALDI-ToF mass spectrometry biomarker profiling via multivariate data analysis application in the biopharmaceutical bioprocessing industry

PhD ThesisMatrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF MS) is a technique by which protein profiles can be rapidly produced from biological samples. Proteomic profiling and biomarker identification using MALDI-ToF MS have been utilised widely in microbiology for bacteria identification and in clinical proteomics for disease-related biomarker discovery. To date, the benefits of MALDI-ToF MS have not been realised in the area of mammalian cell culture during bioprocessing. This thesis explores the approach of ‘intact-cell’ MALDI-ToF MS (ICM-MS) combined with projection to latent structures – discriminant analysis (PLS-DA), to discriminate between mammalian cell lines during bioprocessing. Specifically, the industrial collaborator, Lonza Biologics is interested in adopting this approach to discriminate between IgG monoclonal antibody producing Chinese hamster ovaries (CHO) cell lines based on their productivities and identify protein biomarkers which are associated with the cell line productivities. After classifying cell lines into two categories (high/low producers; Hs/Ls), it is hypothesised that Hs and Ls CHO cells exhibit different metabolic profiles and hence differences in phenotypic expression patterns will be observed. The protein expression patterns correlate to the productivities of the cell lines, and introduce between-class variability. The chemometric method of PLS-DA can use this variability to classify the cell lines as Hs or Ls. A number of differentially expressed proteins were matched and identified as biomarkers after a SwissProt/TrEMBL protein database search. The identified proteins revealed that proteins involved in biological processes such as protein biosynthesis, protein folding, glycolysis and cytoskeleton architecture were upregulated in Hs. This study demonstrates that ICM-MS combined with PLS-DA and a protein database search can be a rapid and valuable tool for biomarker discovery in the bioprocessing industry. It may help in providing clues to potential cell genetic engineering targets as well as a tool in process development in the bioprocessing industry. With the completion of the sequencing of the CHO genome, this study provides a foundation for rapid biomarker profiling of CHO cell lines in culture during recombinant protein manufacturing.Lonza Biologics

MALDI Imaging Mass Spectrometry (MALDI-IMS)—Application of Spatial Proteomics for Ovarian Cancer Classification and Diagnosis

MALDI imaging mass spectrometry (MALDI-IMS) allows acquisition of mass data for metabolites, lipids, peptides and proteins directly from tissue sections. IMS is typically performed either as a multiple spot profiling experiment to generate tissue specific mass profiles, or a high resolution imaging experiment where relative spatial abundance for potentially hundreds of analytes across virtually any tissue section can be measured. Crucially, imaging can be achieved without prior knowledge of tissue composition and without the use of antibodies. In effect MALDI-IMS allows generation of molecular data which complement and expand upon the information provided by histology including immuno-histochemistry, making its application valuable to both cancer biomarker research and diagnostics. The current state of MALDI-IMS, key biological applications to ovarian cancer research and practical considerations for analysis of peptides and proteins on ovarian tissue are presented in this review

Deciphering the interplay of molecular alterations underpinning renal cell carcinoma by label-free mass spectrometry and clinical proteomics: A systems medicine approach for precision diagnosis

Renal neoplasia is the 14th most common tumor type diagnosed worldwide. With a vast heterogeneity, renal neoplasia encompasses different subtypes. 90% of the neoplasms arise from the epithelial layer of the nephron and vary from benign renal masses (renal oncocytoma, RO) to more indolent or aggressive cancers (renal cell carcinomas, RCC). As RCC subtypes, clear cell (ccRCC) subtype is the most predominant subtype, followed by papillary (pRCC) and chromophobe (chRCC). Despite the different outcomes, some overlapped histological and morphological features difficult their differentiation and diagnosis. Therefore, new approaches for a clear and accurate diagnosis are still needed. To achieve this goal, renal tissue biopsies diagnosed with ccRCC (n = 7), pRCC (n = 5), chRCC (n = 5), RO (n = 5) and normal adjacent tissue (NAT, n= 5) were enrolled in this study. As a very resourceful tool for proteome analysis and biomarker discovery, mass spectrometry (MS)-based methods were used to interrogate the proteome of each tumor in order to undisclosed differences trough which to develop faster and accurate diagnostics. The results achieved with this doctoral thesis include i) the accomplishment of an effective ultrasonic workflow to recover the proteome of optimal cutting temperature (OCT)-embedded tissues, ii) a novel analytical approach based on MALDI-MS profiling to distinguish chRCC from RO, iii) a 109-protein panel to discriminate between chRCC and RO and NAT, iv) a top 24-protein panel to diagnose ccRCC, pRCC, chRCC and RO based on absolute concentration values, v) the translation and validation of three promising biomarkers by immunohistochemical analysis, and vi) an approach for phosphopeptide enrichment. This work brings new insights into the different mechanisms underlying formation of these tumors as well as it provides valuable information to improve clinical diagnosis by opening new avenues for immunohistochemistry and mass spectrometry-based approaches