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1.
Contemporary hydrogen deuterium exchange mass spectrometry.
Oganesyan, I, Lento, C, Wilson, DJ
Methods (San Diego, Calif.). 2018;:27-42
Abstract
Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) emerged as a tool for biochemistry and structural biology around 25 years ago. It has since become a key approach for studying protein dynamics, protein-ligand interactions, membrane proteins and intrinsically disordered proteins (IDPs). In HDX labeling, proteins are exposed to deuterated solvent (usually D2O) for a variable 'labeling time', resulting in isotope exchange of unprotected labile protons on the amide backbone and amino acid side chains. By comparing the levels of deuterium uptake in different regions of a protein, information on conformational and dynamic changes in the system can be acquired. When coupled with MS, HDX is suitable for probing allosteric effects in catalysis and ligand binding, epitope mapping, validation of biosimilars, drug candidate screening and mapping membrane-protein interactions among many other bioanalytical applications. This review introduces HDX-MS via a brief description of HDX-MS development, followed by an overview of HDX theory and ultimately an outline of methods and procedures involved in performing HDX-MS experiments.
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2.
Covalent labeling-mass spectrometry with non-specific reagents for studying protein structure and interactions.
Limpikirati, P, Liu, T, Vachet, RW
Methods (San Diego, Calif.). 2018;:79-93
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Abstract
Using mass spectrometry (MS) to obtain information about a higher order structure of protein requires that a protein's structural properties are encoded into the mass of that protein. Covalent labeling (CL) with reagents that can irreversibly modify solvent accessible amino acid side chains is an effective way to encode structural information into the mass of a protein, as this information can be read-out in a straightforward manner using standard MS-based proteomics techniques. The differential reactivity of proteins under two or more conditions can be used to distinguish protein topologies, conformations, and/or binding sites. CL-MS methods have been effectively used for the structural analysis of proteins and protein complexes, particularly for systems that are difficult to study by other more traditional biochemical techniques. This review provides an overview of the non-specific CL approaches that have been combined with MS with a particular emphasis on the reagents that are commonly used, including hydroxyl radicals, carbenes, and diethylpyrocarbonate. We describe the reagent and protein factors that affect the reactivity of amino acid side chains. We also include details about experimental design and workflow, data analysis, recent applications, and some future prospects of CL-MS methods.
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3.
Mass spectrometry for protein sialoglycosylation.
Zhang, Q, Li, Z, Wang, Y, Zheng, Q, Li, J
Mass spectrometry reviews. 2018;(5):652-680
Abstract
Sialic acids are a family of structurally unique and negatively charged nine-carbon sugars, normally found at the terminal positions of glycan chains on glycoproteins and glycolipids. The glycosylation of proteins is a universal post-translational modification in eukaryotic species and regulates essential biological functions, in which the most common sialic acid is N-acetyl-neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onic acid) (Neu5NAc). Because of the properties of sialic acids under general mass spectrometry (MS) conditions, such as instability, ionization discrimination, and mixed adducts, the use of MS in the analysis of protein sialoglycosylation is still challenging. The present review is focused on the application of MS related methodologies to the study of both N- and O-linked sialoglycans. We reviewed MS-based strategies for characterizing sialylation by analyzing intact glycoproteins, proteolytic digested glycopeptides, and released glycans. The review concludes with future perspectives in the field.
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4.
Research Techniques Made Simple: Mass Spectrometry for Analysis of Proteins in Dermatological Research.
Hammers, CM, Tang, HY, Chen, J, Emtenani, S, Zheng, Q, Stanley, JR
The Journal of investigative dermatology. 2018;(6):1236-1242
Abstract
Identifying previously unknown proteins or detecting the presence of known proteins in research samples is critical to many experiments conducted in life sciences, including dermatology. Sensitive protein detection can help elucidate new intervention targets and mechanisms of disease, such as in autoimmune blistering skin diseases, atopic eczema, or other conditions. Historically, peptides from highly purified single proteins were sequenced, with many limitations, by stepwise degradation from the N-terminus to the C-terminus with subsequent identification by UV absorbance spectroscopy of the released amino acids (i.e., Edman degradation). Recently, however, the availability of comprehensive protein databases from different species (derived from high-throughput next-generation sequencing of those organisms' genomes) and sophisticated bioinformatics analysis tools have facilitated the development and use of mass spectrometry for identification and global analysis of proteins, summarized as mass spectrometry-based proteomics. Mass spectrometry is an analytical technique measuring the mass (m)-to-charge (z) ratio of ionized biological molecules such as peptides. Proteins can be identified by correlating peptide-derived experimental mass spectrometry spectra with theoretical spectra predicted from protein databases. Here we briefly describe how this technique works, how it can be used for identification of proteins, and how this knowledge can be applied in elucidating human biology and disease.
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5.
Mass Spectrometry-Based Identification of Extracellular Domains of Cell Surface N-Glycoproteins: Defining the Accessible Surfaceome for Immunophenotyping Stem Cells and Their Derivatives.
Fujinaka, CM, Waas, M, Gundry, RL
Methods in molecular biology (Clifton, N.J.). 2018;:57-78
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Abstract
Human stem cells and their progeny are valuable for a variety of research applications and have the potential to revolutionize approaches to regenerative medicine. However, we currently have limited tools to permit live isolation of homogeneous populations of cells apt for mechanistic studies or cellular therapies. While these challenges can be overcome through the use of immunophenotyping based on accessible cell surface markers, the success of this process depends on the availability of reliable antibodies and well-characterized markers, which are lacking for most stem cell lineages. This chapter outlines an iterative process for the development of new cell surface marker barcodes for identifying and selecting stem cell derived progeny of specific cell types, subtypes, and maturation stages, where antibody-independent identification of cell surface proteins is achieved using a modern chemoproteomic approach to specifically identify N-glycoproteins localized to the cell surface. By taking advantage of a large repository of available cell surfaceome data, proteins that are unlikely to confer cell type specificity can be rapidly eliminated from consideration. Subsequently, targeted quantitation by mass spectrometry can be used to refine candidates of interest, and a bioinformatic visualization tool is key to mapping experimental data to candidate protein sequences for the purpose of epitope selection during the antibody development phase. Overall, the process of developing cell surface barcodes for immunophenotyping is iterative and can include multiple rounds of discovery, refinement, and validation depending on the phenotypic resolution required.
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Assembling the Community-Scale Discoverable Human Proteome.
Wang, M, Wang, J, Carver, J, Pullman, BS, Cha, SW, Bandeira, N
Cell systems. 2018;(4):412-421.e5
Abstract
The increasing throughput and sharing of proteomics mass spectrometry data have now yielded over one-third of a million public mass spectrometry runs. However, these discoveries are not continuously aggregated in an open and error-controlled manner, which limits their utility. To facilitate the reusability of these data, we built the MassIVE Knowledge Base (MassIVE-KB), a community-wide, continuously updating knowledge base that aggregates proteomics mass spectrometry discoveries into an open reusable format with full provenance information for community scrutiny. Reusing >31 TB of public human data stored in a mass spectrometry interactive virtual environment (MassIVE), the MassIVE-KB contains >2.1 million precursors from 19,610 proteins (48% larger than before; 97% of the total) and doubles proteome coverage to 6 million amino acids (54% of the proteome) with strict library-scale false discovery controls, thereby providing evidence for 430 proteins for which sufficient protein-level evidence was previously missing. Furthermore, MassIVE-KB can inform experimental design, helps identify and quantify new data, and provides tools for community construction of specialized spectral libraries.
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Pitfalls in the detection of citrullination and carbamylation.
Verheul, MK, van Veelen, PA, van Delft, MAM, de Ru, A, Janssen, GMC, Rispens, T, Toes, REM, Trouw, LA
Autoimmunity reviews. 2018;(2):136-141
Abstract
Carbamylation and citrullination are both post-translational modifications against which (auto)antibodies can be detected in sera of rheumatoid arthritis (RA) patients. Carbamylation is the chemical modification of a lysine into a homocitrulline, whereas citrullination is an enzymatic conversion of an arginine into a citrulline. It is difficult to distinguish between the two resulting amino acids due to similarities in structure. However, differentiation between citrulline and homocitrulline is important to understand the antigens that induce antibody production and to determine which modified antigens are present in target tissues. We have observed in literature that conclusions are frequently drawn regarding the citrullination or carbamylation of proteins based on reagents that are not able to distinguish between these two modifications. Therefore, we have analyzed a wide spectrum of methods and describe here which method we consider most optimal to distinguish between citrulline and homocitrulline. We have produced several carbamylated and citrullinated proteins and investigated the specificity of (commercial) antibodies by both ELISA and western blot. Furthermore, detection methods based on chemical modifications, such as the anti-modified citrulline-"Senshu" method and also mass spectrometry were investigated for their capacity to distinguish between carbamylation and citrullination. We observed that some antibodies are able to distinguish between carbamylation and citrullination, but an overlap in reactivity is often present in the commercially available anti-citrulline antibodies. Finally, we conclude that the use of mass spectrometry is currently essential to differentiate between citrullinated and carbamylated proteins present in complex biological samples.
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High Resolution Mass Spectrometric Analysis of Secoiridoids and Metabolites as Biomarkers of Acute Olive Oil Intake-An Approach to Study Interindividual Variability in Humans.
Silva, S, Garcia-Aloy, M, Figueira, ME, Combet, E, Mullen, W, Bronze, MR
Molecular nutrition & food research. 2018;(2)
Abstract
SCOPE Phenolic compounds are minor components of extra virgin olive oil (EVOO). Secoiridoids are the major components contributing to the phenolic content of EVOO. Information is lacking regarding their potential as biomarkers for EVOO intake. METHODS AND RESULTS Healthy volunteers (n = 9) ingested 50 mL of EVOO in a single dose containing 322 mg kg-1 total phenolic content (caffeic acid equivalents) and 6 mg 20 g-1 hydroxytyrosol and its derivatives. Plasma is collected before (0 h) and at 0.5, 1, 2, 4, and 6 h after ingestion. Urine samples are collected prior to ingestion (0 h) and at 0-4, 4-8, 8-15, and 15-24 h. Samples are analyzed by UPLC coupled with an Exactive Orbitrap MS. Partial least squares discriminant analysis with orthogonal signal correction is applied to screen for metabolites that allow sample discrimination. Plasma biomarkers and urine biomarkers are selected although individual variability is observed among volunteers. Results are in accordance with in vitro experiments performed (in vitro digestion and hepatic microsomal activity assays). CONCLUSIONS Plasma (elenolic acid + H2 ; p-HPEA-EA + H2 + glucuronide) and urinary (3,4-DHPEA-EA, 3,4-DHPEA-EA + H2 +glucuronide, methyl 3,4-DHPEA-EA + H2 +glucuronide) secoiridoid compounds are selected as biomarkers to monitor EVOO intake showing good predictive ability according to multivariate analysis.
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High resolution-ion mobility mass spectrometry as an additional powerful tool for structural characterization of mycotoxin metabolites.
Righetti, L, Fenclova, M, Dellafiora, L, Hajslova, J, Stranska-Zachariasova, M, Dall'Asta, C
Food chemistry. 2018;:768-774
Abstract
This work was designed as a proof of concept, to demonstrate the successful use of the comparison between theoretical and experimental collision cross section (CCS) values to support the identification of isomeric forms. To this purpose, thirteen mycotoxins were considered and analyzed using drift time ion mobility mass spectrometry. A good linear correlation (r2 = 0.962) between theoretical and experimental CCS was found. The average ΔCCS was 3.2%, fully consistent with the acceptability threshold value commonly set at 5%. The agreement between theoretical and experimental CCS obtained for mycotoxin glucuronides suggested the potential of the CCS matching in supporting the annotation procedure.
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10.
Study of the metabolomics characteristics of patients with metabolic syndrome based on liquid chromatography quadrupole time-of-flight mass spectrometry.
Wu, N, Wang, W, Yi, M, Cheng, S, Wang, D
Annales d'endocrinologie. 2018;(1):37-44
Abstract
BACKGROUND Metabolic syndrome (MS) is a disease with complex pathophysiology and pathogenesis involving multiple systems of the human body. This study aimed to identify serum metabolites that are relevant to MS. MATERIAL AND METHODS This study involved 40 patients with MS and 28 healthy adults, and the following data were statistically analyzed: basic clinical data, blood lipids, fasting blood glucose, blood pressure, waist circumference, and visceral fat coefficient. Serum samples from both groups were collected and analyzed by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF/MS); multivariate and univariate statistical methods were used to identify potential MS biomarkers and MS-related metabolic pathways. In addition, leucine and valine levels in serum from MS patients and normal subjects were measured using enzyme-linked immunosorbent assays (ELISAs). RESULTS In this study, 23 potential biomarkers were identified in the plasma of MS patients. These biomarkers were mainly related to metabolism; the tricarboxylic acid cycle; galactose metabolism; arachidonic acid metabolism; valine, leucine, and isoleucine degradation; and valine, leucine, and isoleucine biosynthesis. ELISAs were utilized to verify serum leucine and valine levels, and the results supported the experimental metabolomics results. CONCLUSIONS In total, 23 MS-related metabolites were identified in the serum; these differential metabolites were mainly associated with lipid metabolism, amino acid metabolism, glucose metabolism, purine metabolism, and other related metabolic pathways. This study shows that LC/MS-based metabolomics methods can be used to investigate the pathological changes in MS patients and identify biomarkers for the early diagnosis of MS.