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Leveraging orthogonal mass spectrometry based strategies for comprehensive sequencing and characterization of ribosomal antimicrobial peptide natural products.
Moyer, TB, Parsley, NC, Sadecki, PW, Schug, WJ, Hicks, LM
Natural product reports. 2021;(3):489-509
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Abstract
Covering: Up to July 2020Ribosomal antimicrobial peptide (AMP) natural products, also known as ribosomally synthesized and post-translationally modified peptides (RiPPs) or host defense peptides, demonstrate potent bioactivities and impressive complexity that complicate molecular and biological characterization. Tandem mass spectrometry (MS) has rapidly accelerated bioactive peptide sequencing efforts, yet standard workflows insufficiently address intrinsic AMP diversity. Herein, orthogonal approaches to accelerate comprehensive and accurate molecular characterization without the need for prior isolation are reviewed. Chemical derivatization, proteolysis (enzymatic and chemical cleavage), multistage MS fragmentation, and separation (liquid chromatography and ion mobility) strategies can provide complementary amino acid composition and post-translational modification data to constrain sequence solutions. Examination of two complex case studies, gomesin and styelin D, highlights the practical implementation of the proposed approaches. Finally, we emphasize the importance of heterogeneous AMP peptidoforms that confer varying biological function, an area that warrants significant further development.
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Critical role of mass spectrometry proteomics in tear biomarker discovery for multifactorial ocular diseases (Review).
Ma, JYW, Sze, YH, Bian, JF, Lam, TC
International journal of molecular medicine. 2021;(5)
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Abstract
The tear film is a layer of body fluid that maintains the homeostasis of the ocular surface. The superior accessibility of tears and the presence of a high concentration of functional proteins make tears a potential medium for the discovery of non‑invasive biomarkers in ocular diseases. Recent advances in mass spectrometry (MS) have enabled determination of an in‑depth proteome profile, improved sensitivity, faster acquisition speed, proven variety of acquisition methods, and identification of disease biomarkers previously lacking in the field of ophthalmology. The use of MS allows efficient discovery of tear proteins, generation of reproducible results, and, more importantly, determines changes of protein quantity and post‑translation modifications in microliter samples. The present review compared techniques for tear collection, sample preparation, and acquisition applied for the discovery of tear protein markers in normal subjects and multifactorial conditions, including dry eye syndrome, diabetic retinopathy, thyroid eye disease and primary open‑angle glaucoma, which require an early diagnosis for treatment. It also summarized the contribution of MS to early discovery by means of disease‑related protein markers in tear fluid and the potential for transformation of the tear MS‑based proteome to antibody‑based assay for future clinical application.
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Using X-ray Footprinting and Mass Spectrometry to Study the Structure and Function of Membrane Proteins.
Gupta, S
Protein and peptide letters. 2019;(1):44-54
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Abstract
BACKGROUND Membrane proteins are crucial for cellular sensory cascades and metabolite transport, and hence are key pharmacological targets. Structural studies by traditional highresolution techniques are limited by the requirements for high purity and stability when handled in high concentration and nonnative buffers. Hence, there is a growing requirement for the use of alternate methods in a complementary but orthogonal approach to study the dynamic and functional aspects of membrane proteins in physiologically relevant conditions. In recent years, significant progress has been made in the field of X-ray radiolytic labeling in combination with mass spectroscopy, commonly known as X-ray Footprinting and Mass Spectrometry (XFMS), which provide residue-specific information on the solvent accessibility of proteins. In combination with both lowresolution biophysical methods and high-resolution structural data, XFMS is capable of providing valuable insights into structure and dynamics of membrane proteins, which have been difficult to obtain by standalone high-resolution structural techniques. The XFMS method has also demonstrated a unique capability for identification of structural waters and their dynamics in protein cavities at both a high degree of spatial and temporal resolution, and thus capable of identifying conformational hot-spots in transmembrane proteins. CONCLUSION We provide a perspective on the place of XFMS amongst other structural biology methods and showcase some of the latest developments in its usage for studying conformational changes in membrane proteins.
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Fast Photochemical Oxidation of Proteins Coupled with Mass Spectrometry.
Shi, L, Gross, ML
Protein and peptide letters. 2019;(1):27-34
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Abstract
BACKGROUND Determination of the composition and some structural features of macromolecules can be achieved by using structural proteomics approaches coupled with mass spectrometry (MS). One approach is hydroxyl radical protein footprinting whereby amino-acid side chains are modified with reactive reagents to modify irreversibly a protein side chain. The outcomes, when deciphered with mass-spectrometry-based proteomics, can increase our knowledge of structure, assembly, and conformational dynamics of macromolecules in solution. Generating the hydroxyl radicals by laser irradiation, Hambly and Gross developed the approach of Fast Photochemical Oxidation of Proteins (FPOP), which labels proteins on the sub millisecond time scale and provides, with MS analysis, deeper understanding of protein structure and protein-ligand and protein- protein interactions. This review highlights the fundamentals of FPOP and provides descriptions of hydroxyl-radical and other radical and carbene generation, of the hydroxyl labeling of proteins, and of determination of protein modification sites. We also summarize some recent applications of FPOP coupled with MS in protein footprinting. CONCLUSION We survey results that show the capability of FPOP for qualitatively measuring protein solvent accessibility on the residue level. To make these approaches more valuable, we describe recent method developments that increase FPOP's quantitative capacity and increase the spatial protein sequence coverage. To improve FPOP further, several new labeling reagents including carbenes and other radicals have been developed. These growing improvements will allow oxidative- footprinting methods coupled with MS to play an increasingly significant role in determining the structure and dynamics of macromolecules and their assemblies.
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Analytical barriers in clinical B-type natriuretic peptide measurement and the promising analytical methods based on mass spectrometry technology.
Xiao, P, Li, H, Li, X, Song, D
Clinical chemistry and laboratory medicine. 2019;(7):954-966
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Abstract
B-type natriuretic peptide (BNP) is a circulating biomarker that is mainly applied in heart failure (HF) diagnosis and to monitor disease progression. Because some identical amino acid sequences occur in the precursor and metabolites of BNP, undesirable cross-reactions are common in immunoassays. This review first summarizes current analytical methods, such as immunoassay- and mass spectrometry (MS)-based approaches, including the accuracy of measurement and the inconsistency of the results. Second, the review presents some promising approaches to resolve the current barriers in clinical BNP measurement, such as how to decrease cross-reactions and increase the measurement consistency. Specific approaches include research on novel BNP assays with higher-specificity chemical antibodies, the development of International System of Units (SI)-traceable reference materials, and the development of structure characterization methods based on state-of-the-art ambient and ion mobility MS technologies. The factors that could affect MS analysis are also discussed, such as biological sample cleanup and peptide ionization efficiency. The purpose of this review is to explore and identify the main problems in BNP clinical measurement and to present three types of approaches to resolve these problems, namely, materials, methods and instruments. Although novel approaches are proposed here, in practice, it is worth noting that the BNP-related peptides including unprocessed proBNP were all measured in clinical BNP assays. Therefore, approaches that aimed to measure a specific BNP or proBNP might be an effective way for the standardization of a particular BNP form measurement, instead of the standardization of "total" immunoreactive BNP assays in clinical at present.
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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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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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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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Advances in the Biology of Seed and Vegetative Storage Proteins Based on Two-Dimensional Electrophoresis Coupled to Mass Spectrometry.
Mouzo, D, Bernal, J, López-Pedrouso, M, Franco, D, Zapata, C
Molecules (Basel, Switzerland). 2018;(10)
Abstract
Seed storage proteins play a fundamental role in plant reproduction and human nutrition. They accumulate during seed development as reserve material for germination and seedling growth and are a major source of dietary protein for human consumption. Storage proteins encompass multiple isoforms encoded by multi-gene families that undergo abundant glycosylations and phosphorylations. Two-dimensional electrophoresis (2-DE) is a proteomic tool especially suitable for the characterization of storage proteins because of their peculiar characteristics. In particular, storage proteins are soluble multimeric proteins highly represented in the seed proteome that contain polypeptides of molecular mass between 10 and 130 kDa. In addition, high-resolution profiles can be achieved by applying targeted 2-DE protocols. 2-DE coupled with mass spectrometry (MS) has traditionally been the methodology of choice in numerous studies on the biology of storage proteins in a wide diversity of plants. 2-DE-based reference maps have decisively contributed to the current state of our knowledge about storage proteins in multiple key aspects, including identification of isoforms and quantification of their relative abundance, identification of phosphorylated isoforms and assessment of their phosphorylation status, and dynamic changes of isoforms during seed development and germination both qualitatively and quantitatively. These advances have translated into relevant information about meaningful traits in seed breeding such as protein quality, longevity, gluten and allergen content, stress response and antifungal, antibacterial, and insect susceptibility. This review addresses progress on the biology of storage proteins and application areas in seed breeding using 2-DE-based maps.
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Ultrahigh resolution metabolomics for S-containing metabolites.
Nakabayashi, R, Saito, K
Current opinion in biotechnology. 2017;:8-16
Abstract
The advent of the genome-editing era greatly increases the opportunities for synthetic biology research that aims to enhance production of potentially useful bioactive metabolites in heterologous hosts. A wide variety of sulfur (S)-containing metabolites (S-metabolites) are known to possess bioactivities and health-promoting properties, but finding them and their chemical assignment using mass spectrometry-based metabolomics has been difficult. In this review, we highlight recent advances on the targeted metabolomic analysis of S-metabolites (S-omics) in plants using ultrahigh resolution mass spectrometry. The use of exact mass and signal intensity differences between 32S-containing monoisotopic ions and counterpart 34S isotopic ions exploits an entirely new method to characterize S-metabolites. Finally, we discuss the availability of S-omics for synthetic biology.