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N-glycosylation plays an important role in the structure and function of membrane and secreted proteins. The spike protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is heavily glycosylated and the major target for developing vaccines, therapeutic drugs and diagnostic tests. The first major SARS-CoV-2 variant carries a D614G substitution in the spike (S-D614G) that has been associated with altered conformation, enhanced ACE2 binding, and increased infectivity and transmission. In this report, we used mass spectrometry techniques to characterize and compare the N-glycosylation of the wild type (S-614D) or variant (S-614G) SARS-CoV-2 spike glycoproteins prepared under identical conditions. The data showed that half of the N-glycosylation sequons changed their distribution of glycans in the S-614G variant. The S-614G variant showed a decrease in the relative abundance of complex-type glycans (up to 45%) and an increase in oligomannose glycans (up to 33%) on all altered sequons. These changes led to a reduction in the overall complexity of the total N-glycosylation profile. All the glycosylation sites with altered patterns were in the spike head while the glycosylation of three sites in the stalk remained unchanged between S-614G and S-614D proteins., (© 2021. The Author(s).)

It is essential to have a simple, quick and sensitive method for the detection and quantification of botulinum neurotoxins, the most toxic substances and the causative agents of botulism. Type C botulinum neurotoxin (BoNT/C) represents one of the seven members of distinctive BoNT serotypes (A to G) that cause botulism in animals and avians. Here we report the development of optimized peptide substrates for improving the detection of BoNT/C and /CD mosaic toxins using an Endopep-MS assay, a mass spectrometry-based method that is able to rapidly and sensitively detect and differentiate all types of BoNTs by extracting the toxin with specific antibodies and detecting the unique cleavage products of peptide substrates. Based on the sequence of a short SNAP-25 peptide, we conducted optimization through a comprehensive process including length determination, terminal modification, single and multiple amino acid residue substitution, and incorporation of unnatural amino acid residues. Our data demonstrate that an optimal peptide provides a more than 200-fold improvement over the substrate currently used in the Endopep-MS assay for the detection of BoNT/C1 and /CD mosaic. Using the new substrate in a four-hour cleavage reaction, the limit of detection for the BoNT/C1 complex spiked in buffer, serum and milk samples was determined to be 0.5, 0.5 and 1 mouseLD 50 /mL, respectively, representing a similar or higher sensitivity than that obtained by traditional mouse bioassay. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxins (BoNTs) cause the disease called botulism, which can be lethal. BoNTs are proteins secreted by some species of clostridia and are known to cause paralysis by interfering with nerve impulse transmission. Although the human lethal dose of BoNT is not accurately known, it is estimated to be between 0.1 μg to 70 μg, so it is important to enable detection of small amounts of these toxins. Our laboratory previously reported on the development of Endopep-MS, a mass-spectrometric-based endopeptidase method to detect, differentiate, and quantify BoNT immunoaffinity purified from complex matrices. In this work, we describe the application of Endopep-MS for the analysis of thirteen blinded samples supplied as part of the EQuATox proficiency test. This method successfully identified the presence or absence of BoNT in all thirteen samples and was able to successfully differentiate the serotype of BoNT present in the samples, which included matrices such as buffer, milk, meat extract, and serum. Furthermore, the method yielded quantitative results which had z-scores in the range of -3 to +3 for quantification of BoNT/A containing samples. These results indicate that Endopep-MS is an excellent technique for detection, differentiation, and quantification of BoNT in complex matrices. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most poisonous substances known to humankind. It is essential to have a simple, quick, and sensitive method for the detection and quantification of botulinum toxin in various media, including complex biological matrices. Our laboratory has developed a mass spectrometry-based Endopep–MS assay that is able to rapidly detect and differentiate all types of BoNTs by extracting the toxin with specific antibodies and detecting the unique cleavage products of peptide substrates. Botulinum neurotoxin type E (BoNT/E) is a member of a family of seven distinctive BoNT serotypes (A–G) and is the causative agent of botulism in both humans and animals. To improve the sensitivity of the Endopep–MS assay, we report here the development of novel peptide substrates for the detection of BoNT/E activity through systematic and comprehensive approaches. Our data demonstrate that several optimal peptides could accomplish 500-fold improvement in sensitivity compared with the current substrate for the detection of both not-trypsin-activated and trypsin-activated BoNT/E toxin complexes. A limit of detection of 0.1 mouse LD 50 /ml was achieved using the novel peptide substrate in the assay to detect not-trypsin-activated BoNT/E complex spiked in serum, stool, and food samples. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxins (BoNTs) are highly toxic proteases produced by anaerobic bacteria. Traditionally, a mouse bioassay (MBA) has been used for detection of BoNTs, but for a long time, laboratories have worked with alternative methods for their detection. One of the most promising in vitro methods is a combination of an enzymatic and mass spectrometric assay called Endopep-MS. However, no comprehensive validation of the method has been presented. The main purpose of this work was to perform a validation for the qualitative analysis of BoNT-A, B, C, C/D, D, D/C, and F in serum. The limit of detection (LOD), selectivity, precision, stability in matrix and solution, and correlation with the MBA were evaluated. The LOD was equal to or even better than that of the MBA for BoNT-A, B, D/C, E, and F. Furthermore, Endopep-MS was for the first time successfully used to differentiate between BoNT-C and D and their mosaics C/D and D/C by different combinations of antibodies and target peptides. In addition, sequential antibody capture was presented as a new way to multiplex the method when only a small sample volume is available. In the comparison with the MBA, all the samples analyzed were positive for BoNT-C/D with both methods. These results indicate that the Endopep-MS method is a valid alternative to the MBA as the gold standard for BoNT detection based on its sensitivity, selectivity, and speed and that it does not require experimental animals. [ABSTRACT FROM AUTHOR]

Abstract: Clostridium botulinum neurotoxins (BoNTs) cause the life-threatening disease botulism through the inhibition of neurotransmitter release by cleaving essential SNARE proteins. There are seven serologically distinctive types of BoNTs and many subtypes within a serotype have been identified. BoNT/A5 is a recently discovered subtype of type A botulinum neurotoxin which possesses a very high degree of sequence similarity and identity to the well-studied A1 subtype. In the present study, we examined the endopeptidase activity of these two BoNT/A subtypes and our results revealed significant differences in substrate binding and cleavage efficiency between subtype A5 and A1. Distinctive hydrolysis efficiency was observed between the two toxins during cleavage of the native substrate SNAP-25 versus a shortened peptide mimic. N-terminal truncation studies demonstrated that a key region of the SNAP-25, including the amino acid residues at 151 through 154 located in the remote binding region of the substrate, contributed to the differential catalytic properties between A1 and A5. Elevated binding affinity of the peptide substrate resulted from including these important residues and enhanced BoNT/A5's hydrolysis efficiency. In addition, mutations of these amino acid residues affect the proteolytic performance of the two toxins in different ways. This study provides a better understanding of the biological activity of these toxins, their performance characteristics in the Endopep-MS assay to detect BoNT in clinical samples and foods, and is useful for the development of peptide substrates. [Copyright &y& Elsevier]

Abstract: Botulinum neurotoxins (BoNTs) are a family of seven toxin serotypes that are the most toxic substances known to humans. Intoxication with BoNT causes flaccid paralysis and can lead to death if untreated with serotype-specific antibodies. Supportive care, including ventilation, may be necessary. Rapid and sensitive detection of BoNT is necessary for timely clinical confirmation of clinical botulism. Previously, our laboratory developed a fast and sensitive mass spectrometry (MS) method termed the Endopep–MS assay. The BoNT serotypes are rapidly detected and differentiated by extracting the toxin with serotype-specific antibodies and detecting the unique and serotype-specific cleavage products of peptide substrates that mimic the sequence of the BoNT native targets. To further improve the sensitivity of the Endopep–MS assay, we report here the optimization of the substrate peptide for the detection of BoNT/A. Modifications on the terminal groups of the original peptide substrate with acetylation and amidation significantly improved the detection of BoNT/A cleavage products. The replacement of some internal amino acid residues with single or multiple substitutions led to further improvement. An optimized peptide increased assay sensitivity 5-fold with toxin spiked into buffer solution or different biological matrices. [Copyright &y& Elsevier]

Histone methylation represents one of the most critical epigenetic events in DNA function regulation in eukaryotic organisms. Classic molecular biology and genetics tools provide significant knowledge about mechanisms and physiological roles of histone methyltransferases and demethylases in various cellular processes. In addition to this stream line, development and application of chemistry and chemistry-related techniques are increasingly involved in biological study, and offer information otherwise difficult to obtain by standard molecular biology methods. Herein, we review recent achievements and progress in developing and applying chemical and biochemical approaches in the study of histone methylation, including chromatin immunoprecipitation, chemical ligation, mass spectrometry, biochemical methylation and demethylation assays, and inhibitor development. These technological advances allow histone methylation to be studied from genome-wide level to molecular and atomic levels. With ChIP technology, information can be obtained about precise mapping of histone methylation patterns at specific promoters, genes, or other genomic regions. MS is particularly useful in detecting and analyzing methylation marks in histone and nonhistone protein substrates. Chemical approaches that permit site-specific incorporation of methyl groups into histone proteins greatly facilitate the investigation of biological impacts of methylation at individual modification sites. Discovery and design of selective organic inhibitors of histone methyltransferases and demethylases provide chemical probes to interrogate methylation-mediated cellular pathways. Overall, these chemistry-related technological advances have greatly improved our understanding of the biological functions of histone methylation in normal physiology and diseased states, and also are of great potential to translate basic epigenetics research into diagnostic and therapeutic applications in the clinic. © 2010 Wiley Periodicals, Inc. Med Res Rev., 32, No. 4, 815-867, 2012 [ABSTRACT FROM AUTHOR]

Abstract: Botulinum neurotoxins (BoNTs) are the most toxic substances known to humankind. Rapid and sensitive detection of BoNTs is necessary for timely clinical confirmation of the disease state in botulism. BoNTs cleave proteins and peptide mimics at specific sites. A mass spectrometry (MS)-based method, Endopep–MS, can detect these cleavages and has detection limits of 0.05–0.5 mouse LD50 (U) in serum, depending on the BoNT serotypes. In this method, the products generated from cleavage of peptide substrates using antibody affinity-purified toxins are detected by MS. Nonspecific bound endogenous proteases or peptidases in stool can coextract with the toxin, cleaving the peptide substrates and reducing the sensitivity of the method. Here we report a method to reduce nonspecific substrate cleavage by reducing stool protease coextraction in the Endopep–MS assay. [Copyright &y& Elsevier]

Cervical cancer is among the most common cancers in women worldwide. Discovery of biomarkers for the early detection of cervical cancer would improve current screening practices and reduce the burden of disease. In this study, we report characterization of the human cervical mucous proteome as the first step towards protein biomarker discovery. The protein composition was characterized using one- and two-dimensional gel electrophoresis, and liquid chromatography coupled with mass spectrometry. We chose to use this combination of traditional biochemical techniques and proteomics to allow a more comprehensive analysis. A total of 107 unique proteins were identified, with plasma proteins being most abundant. These proteins represented the major functional categories of metabolism, immune response, and cellular transport. Removal of high molecular weight abundant proteins by immunoaffinity purification did not significantly increase the number of protein spots resolved. We also analyzed phosphorylated and glycosylated proteins by fluorescent post-staining procedures. The profiling of cervical mucous proteins and their post-translational modifications can be used to further our understanding of the cervical mucous proteome. [ABSTRACT FROM AUTHOR]

N-terminal derivatization of peptides with Edman's reagent, phenyl isothiocyanate (PITC), promotes gas-phase Edman cleavage that yields abundant complementary b1 and Yn-1 ion pairs by tandem mass spectrometry (MS/ MS). The fonnation of b1 ions can be utilized as a mass tag to enhance the interpretation of MS/MS spectra and increase the confidence of peptide identification during mass spectrometry analysis. Derivatization of tryptic peptides with another isothiocyanate analogue, 4-sulfophenyl isothiocyanate, also produces signature ions resulting from Edman cleavage and facilitates peptide sequencing on linear or branched peptides. The limitation of these derivatizations, however, is reduced MS signal intensities of modified peptides, due presumably to the tags themselves. Here we have demonstrated that several other isothiocyanate analogues bearing basic moieties can derivatize peptides and significantly improve the MS sensitivity of tagged analytes, while promoting Edman fragmentation and maintaining other sequence fragments as well. [ABSTRACT FROM AUTHOR]

Protein ubiquitination plays an important role in the degradation and other functional regulation of cellular proteins in organisms ranging from yeasts to mammals. Trypsin digestion of ubiquitin conjugated proteins produces diglycine branched peptides in which the C-terminal Gly-Gly fragment of ubiquitin is attached to the ϵ-amino group of a modified lysine residue within the peptide. This provides a platform for mapping ubiquitination sites using mass spectrometry. Here we report the development of a novel strategy for determining posttraslational protein ubiquitination based on the N-terminal sulfonation of diglycine branched peptides. In contrast to conventional tandem MS spectra of native tryptic peptides, MALDI MS/MS analysis of a sulfonated tryptic peptide containing a diglycine branch generates a unique spectrum composed of a signature portion and a sequence portion. The signature portion of the spectrum consists of several intense ions resulting from the elimination of the tags, the N-terminal residues at the peptide and the branch, and their combination. This unique ion distribution pattern can distinguish ubiquitination modificatons from others and can identify the first N-terminal residues of the peptides as well. The sequence portion consists of an exclusive series of y-type ions and y' ions (differing by the loss of one glycine residue from the sulfonated diglycine branch) that can directly reveal the amino acid sequence of the peptide and the precise location of the ubiquitination site. The technique is demonstrated for a series of synthetic peptides and is validated by a model protein, tetraubiquitin. Our results show that the MALDI MS/MS analysis of sulfonated tryptic peptides can provide a highly effective method for the determination of ubiquitination substrates, ubiquitination sites on protein targets, and modification sites on ubiquitins themselves. [ABSTRACT FROM AUTHOR]

Abstract: Proteomics have been used widely to study proteins in complex materials such as cells, body fluids, tissues, and organisms. Application of advance proteomic techniques for the characterization of disease-specific proteins may provide information for the detection of potential infectious agents. In this report, two proteomics techniques, a two-dimensional differential gel electrophoresis (2D-DIGE) and a one-dimensional gel electrophoresis and one-dimensional liquid chromatography coupled with mass spectrometry (GeLC-MS/MS), were applied for investigating viral proteins from cultured cells inoculated with a clinical sample. The 2D-DIGE method identified five viral proteins of vaccinia virus that are only present in infected cells, these results are in agreement with findings determined by genome based methods. The GeLC-MS/MS method identified eight vaccinia virus proteins out of 428 proteins detected in the sample. These results demonstrate that proteomic techniques can be used effectively for the detection of infectious agents. Given that the methods are capable of applying to proteins without a prior knowledge of the pathogen present, proteomics has a potential of being developed as a molecular tool for pathogen discovery, and disease diagnosis of emerging infectious diseases and for bioterrorism defense. [Copyright &y& Elsevier]