Mapping the glycation sites in the neoglycoconjugate from hexasaccharide antigen of Vibrio cholerae,serotype Ogawa and the recombinant tetanus toxin C‐fragment carrier

We report herein the glycation sites in a vaccine candidate for cholera formed by conjugation of the synthetic hexasaccharide fragment of the O‐specific polysaccharide of Vibrio cholerae, serotype Ogawa, to the recombinant tetanus toxin C‐fragment (rTT–Hc) carrier. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry analysis of the vaccine revealed that it is composed of a mixture of neoglycoconjugates with carbohydrate : protein ratios of 1.9 : 1, 3.0 : 1, 4.0 : 1, 4.9 : 1, 5.9 : 1, 6.9 : 1, 7.9 : 1 and 9.1 : 1. Liquid chromatography tandem mass spectrometry (LC‐MS/MS) analysis of the tryptic and GluC V8 digests allowed identification of 12 glycation sites in the carbohydrate–protein neoglycoconjugate vaccine. The glycation sites are located exclusively on lysine (Lys) residues and are listed as follows: Lys 22, Lys 61, Lys 145, Lys 239, Lys 278, Lys 318, Lys 331, Lys 353, Lys 378, Lys 389, Lys 396 and Lys 437. Based on the 3‐D representation of the rTT–Hc protein, all the glycation sites correspond to lysines located at the outer surface of the protein. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of the glycation sites of Bacillus anthracis neoglycoconjugate vaccine by MALDI-TOF/TOF-CID-MS/MS and LC-ESI-QqTOF-tandem mass spectrometry

We present herein an efficient mass spectrometric method for the localization of the glycation sites of a model neoglycoconjugate vaccine formed by a construct of the tetrasaccharide side chain of the Bacillus anthracis exosporium and the protein carrier bovine serum albumin. The glycoconjugate was digested with both trypsin and GluC V8 endoproteinases, and the digests were then analyzed by MALDI-TOF/TOF-CID-MS/MS and nano-LC-ESI-QqTOF-CID-MS/MS. The sequences of the unknown peptides analyzed by MALDI-TOF/TOF-CID-MS/MS, following digestion with the GluC V8 endoproteinase, allowed us to recognize three glycopeptides whose glycation occupancies were, respectively, on Lys 235, Lys 420, and Lys 498. Similarly, the same analysis was performed on the tryptic digests, which permitted us to recognize two glycation sites on Lys 100 and Lys 374. In addition, we have also used LC-ESI-QqTOF-CID-MS/MS analysis for the identification of the tryptic digests. However, this analysis identified a higher number of glycopeptides than would be expected from a glycoconjugate composed of a carbohydrate-protein ratio of 5.4:1, which would have resulted in glycation occupancies of 18 specific sites. This discrepancy was due to the large number of glycoforms formed during the synthetic carbohydrate-spacer-carrier protein conjugation. Likewise, the LC-ESI-QqTOF-MS/MS analysis of the GluC V8 digest also identified 17 different glycation sites on the synthetic glycoconjugate.     

Exploring the frontiers of synthetic eumelanin polymers by high‐resolution matrix‐assisted laser/desorption ionization mass spectrometry

New trends in material science and nanotechnologies have spurred growing interest in eumelanins black insoluble biopolymers derived by tyrosinase‐catalysed oxidation of tyrosine via 5,6‐dihydroxyindole (DHI) and its 2‐carboxylic acid (DHICA). Efficient antioxidant and photoprotective actions, associated with peculiar optoelectronic properties, are recognised as prominent functions of eumelanin macromolecules within the human and mammalian pigmentary system, making them unique candidates for the realisation of innovative bio‐inspired functional soft materials, with structure‐based physical–chemical properties. An unprecedented breakthrough into the mechanism of synthetic eumelanin buildup has derived from a detailed investigation of the oxidative polymerization of DHI and its N‐methyl derivative (NMDHI) by linear and reflectron matrix‐assisted laser/desorption ionization mass spectrometry. Regular collections of oligomers of increasing masses, spanning the entire m/z ranges up to 5000 Da (>30‐mer) and 8000 Da (> 50‐mer) for the two building blocks, respectively, were disclosed. It is the first time that the in vitro polymerisation of dihydroxyindoles to form synthetic eumelanins is explored up to its high mass limits, giving at the same time information on the polymerisation mode, whether it follows a stepwise pattern (being this the conclusion in our case) or a staking sequencing of small‐sized entities. It also highlighted the influence of the N‐methyl substituent on the polymerization process; this opens the way to the production of N‐functionalized, synthetic eumelanin‐inspired soft materials, for possible future technological applications. Copyright © 2012 John Wiley & Sons, Ltd.     

Direct identification of trypanosomatids by matrix‐assisted laser desorption ionization–time of flight mass spectrometry (DIT MALDI‐TOF MS)

Accurate and rapid determination of trypanosomatids is essential in epidemiological surveillance and therapeutic studies. Matrix‐assisted laser desorption ionization/time of flight mass spectrometry (MALDI‐TOF MS) has been shown to be a useful and powerful technique to identify bacteria, fungi, metazoa and human intact cells with applications in clinical settings. Here, we developed and optimized a MALDI‐TOF MS method to profile trypanosomatids. trypanosomatid cells were deposited on a MALDI target plate followed by addition of matrix solution. The plate was then subjected to MALDI‐TOF MS measurement to create reference mass spectra library and unknown samples were identified by pattern matching using the BioTyper software tool. Several m/z peaks reproducibly and uniquely identified trypanosomatids species showing the potentials of direct identification of trypanosomatids by MALDI‐TOF MS. Moreover, this method discriminated different life stages of Trypanosoma cruzi, epimastigote and bloodstream trypomastigote and Trypanosoma brucei, procyclic and bloodstream. T. cruzi Discrete Typing Units (DTUs) were also discriminated in three clades. However, it was not possible to achieve enough resolution and software‐assisted identification at the strain level. Overall, this study shows the importance of MALDI‐TOF MS for the direct identification of trypanosomatids and opens new avenues for mass spectrometry‐based detection of parasites in biofluids. Copyright © 2016 John Wiley & Sons, Ltd.     

Separation with zwitterionic hydrophilic interaction liquid chromatography improves protein identification by matrix‐assisted laser desorption/ionization‐based proteomic analysis

Comprehensive proteomic analyses necessitate efficient separation of peptide mixtures for the subsequent identification of proteins by mass spectrometry (MS). However, digestion of proteins extracted from cells and tissues often yields complex peptide mixtures that confound direct comprehensive MS analysis. This study investigated a zwitterionic hydrophilic interaction liquid chromatography (ZIC‐HILIC) technique for the peptide separation step, which was verified by subsequent MS analysis. Human serum albumin (HSA) was the model protein used for this analysis. HSA was digested with trypsin and resolved by ZIC‐HILIC or conventional strong cation exchange (SCX) prior to MS analysis for peptide identification. Separation with ZIC‐HILIC significantly improved the identification of HSA peptides over SCX chromatography. Detailed analyses of the identified peptides revealed that the ZIC‐HILIC has better peptide fractionation ability. We further demonstrated that ZIC‐HILIC is useful for quantitatively surveying cell surface markers specifically expressed in undifferentiated embryonic stem cells. These results suggested the value of ZIC‐HILIC as a novel and efficient separation method for comprehensive and quantitative proteomic analyses. Copyright © 2009 John Wiley & Sons, Ltd.     

Matrix‐assisted laser desorption ionization time of flight mass spectrometry analysis of living cationic polymerization of vinyl ethers. I. Optimization of measurement conditions for poly(isobutyl vinyl ether)

Matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF‐MS) was utilized for the analysis of polymers obtained by the living cationic polymerization of isobutyl vinyl ether (IBVE) with the HCl‐VE adduct/SnCl₄/n‐Bu₄NCl initiating system in CH₂Cl₂ at −78 °C. Under optimized analysis conditions, well‐resolved spectra were obtained for samples with number‐average molecular weights of ≤10⁴ with the use of 1,8‐dihydroxy‐9(10H)‐anthracenone (dithranol) as a matrix and sodium trifluoroacetate as an added salt. The MS spectra showed only one series of peaks separated exactly by the mass of the IBVE. The observed mass of each peak was in good agreement with the theoretical one, which possesses one initiator fragment at the α end and one methoxy group originated from quenching with methanol at the ω end. Thus, detailed end group analysis is possible for poly(VE). © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 4023–4031, 2000     

Glycation pattern of peptides condensed with maltose,lactose and glucose determined by ultraviolet matrix‐assisted laser desorption/ionization tandem mass spectrometry

Protein glycation is the non‐enzymatic condensation of sugars with proteins. Although commonly occurring in both the therapeutic and food/beverage industries, protein glycation has not been the focus of many proteomic investigations. This study aims to establish a reliable mass spectrometric method for screening large tandem mass spectrometric (MSMS) datasets for protein glycation with glucose, lactose and maltose. Control experiments using a standard peptide containing a single glycation site led to the discovery of characteristic neutral loss fragmentation patterns in MSMS analysis for glucose, lactose and maltose condensed with peptides. Valid in both tandem time‐of‐flight (TOFTOF) and quadrupole ion trap time‐of‐flight matrix‐assisted laser desorption/ionization (QIT TOF MALDI) mass spectrometers, these neutral loss signatures were then applied to elucidation of modified peptides from a complex human serum albumin (HSA) digest glycated with each of the proposed sugars. Screening of these large datasets was made possible by specifically designed software solutions that enable the input of detailed user‐defined post‐translational modifications that are not included in the universally available databases such as Unimod. Copyright © 2010 John Wiley & Sons, Ltd.     

Study of the cyanoacrylate fuming mechanism by matrix‐assisted laser desorption/ionization mass spectrometry

Despite cyanoacrylate fuming being widely used in the forensic science field, its mechanism is not well understood. In this study, matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry is used to study latent fingerprints that have been cyanoacrylate fumed in an attempt to gain insight into the fuming mechanism. In the negative mode mass spectrometry data, four compounds related to the polymerization of cyanoacrylate are identified and their structures are determined from accurate mass and MS/MS. A mechanism is proposed for the formation of these compounds that are regarded as intermediates in the polymerization reaction. In addition, based on the fuming of standard endogenous compounds, we suggest that fatty acids and amino acids are the major catalytic nucleophiles that initiate the polymerization reactions.     

Application of porous metal enrichment probe sampling to single cell analysis using matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF‐MS)

There is an increasing need for analyzing metabolism in a single cell, which is important to understand the nature of cellular heterogeneity, disease, growth and specialization, etc. However, single cell analysis is often challenging for the traces of samples. In the present study, porous metal enrichment probe sampling combined with matrix‐assisted laser desorption ionization time of flight mass spectrometry ( MALDI‐TOF‐MS) has been applied for in situ analysis of live onion epidemic cell. Porous probe, treated by corroding copper wire with HCl, was directly inserted into a single cell to get cell solution. A self‐made linear actuator was enough to control the penetration of probe into the target cell accurately. Then samples on the tip of probe were eluted and detected by a commercial MALDI‐TOF‐MS directly. The formation of porous microstructure on the probe surface increased the adsorptive capacity of cell solution. The sensitivity of porous probe sampling was 6 times higher than uncorroded probes generally. This method provides a sensitive and convenient way for the sampling and detection of single cell solution. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of the metabolites of gigantol in rat urine by ultra‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Gigantol is a typical bibenzyl compound isolated from Dendrobii Caulis that has been widely used as a medicinal herb in China for the treatment of diabetic cataract, cancer and arteriosclerosis obliterans and as a tonic for stomach nourishment, saliva secretion promotion and fever reduction. However, few studies have been carried out on its in vivo metabolism. In the present study, a rapid and sensitive method based on ultra‐performance liquid chromatography/electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (UPLC‐Q/TOF‐MS) in positive ion mode was developed and applied to identify the metabolites of gigantol in rat urine after a single oral dose (100 mg/kg). Chromatographic separation was performed on an Acquity UPLC HSS T3 column (100 × 2.1 mm i. d., 1.8 µm) using acetonitrile and 0.1% aqueous formic acid as mobile phases. A total of 11 metabolites were detected and identified as all phase II metabolites. The structures of the metabolites were identified based on the characteristics of their MS, MS² data and chromatographic retention times. The results showed that glucuronidation is the principal metabolic pathway of gigantol in rats. The newly identified metabolites are useful to understand the mechanism of elimination of gigantol and, in turn, its effectiveness and toxicity. As far as we know, this is the first attempt to investigate the metabolic fate of gigantol in vivo. Copyright © 2014 John Wiley & Sons, Ltd.     

Future perspective of nanoparticle interaction‐assisted laser desorption/ionization mass spectrometry for rapid,simple, direct and sensitive detection of microorganisms

The introduction of nanoparticles into mass spectrometric research greatly influenced the applicability of this technique into various omics. Surface‐modified or functionalized nanoparticles (NPs) have recently extended the use of mass spectrometry into microorganism research. We survey the application of unmodified NPs, for microorganism research, on the basis of our expertise in this area within the recent years in this decade. The use of unmodified NPs in mass spectrometry, especially with respect to microorganisms, is an untreaded research area, which we have ventured to probe and have been fruitful. On the basis of our experience, we provide an insight into the principle behind the use of unmodified NPs and provide guidelines to be followed to obtain significant results. We also brief the current scenario of nanoparticle interaction‐assisted laser desorption/ionization mass spectrometry (NPILDI‐MS) for rapid, simple, direct and sensitive detection of microorganisms on the basis of our past and present reports, quoting examples of successful application of this technique. Finally, we address the future of the NPILDI‐MS technique and the tools needed to reach those visions. Copyright © 2012 John Wiley & Sons, Ltd.     

Laser‐induced decomposition of 2,2‐dimethoxy‐2‐phenylacetophenone and benzoin in methyl methacrylate homopolymerization studied via matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Pulsed laser polymerization (PLP) experiments were performed on the bulk polymerization of methyl methacrylate (MMA) at ?34 °C. The aim of this study was to investigate the polymer end groups formed during the photoinitiation process of MMA monomer using 2,2‐dimethoxy‐2‐phenylacetophenone (DMPA) and benzoin as initiators via matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. Analysis of the MALDI‐TOF spectra indicated that the two radical fragments generated upon pulsed laser irradiation show markedly different reactivity toward MMA: whereas the benzoyl fragment—common to both DMPA and benzoin—clearly participates in the initiation process, the acetal and benzyl alcohol fragments cannot be identified as end groups in the polymer. The complexity of the MALDI‐TOF spectrum strongly increased with increasing laser intensity, this effect being more pronounced in the case of benzoin. This indicates that a cleaner initiation process is at work when DMPA is used as the photoinitiator. In addition, the MALDI‐TOF spectra were analyzed to extract the propagation‐rate coefficient, k_p, of MMA at ?34 °C. The obtained value of k_p = 43.8 L mol^?1 s^?1 agrees well with corresponding numbers obtained via size exclusion chromatography (k_p = 40.5 L mol^?1 s^?1). © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 675–681, 2002; DOI 10.1002/pola.10150     

Characterization and de novo sequencing of snow crab tropomyosin enzymatic peptides by both electrospary ionization and matrix‐assisted laser desorption ionization QqToF tandem mass spectrometry

The protein tropomyosin (TM) is a known major allergen present in shellfish causing frequent food allergies. TM is also an occupational allergen generated in the working environment of snow crab (Chionoecetes opilio) processing plants. The TM protein was purified from both claw and leg meats of snow crab and analyzed by electrospray ionization and matrix‐assisted laser desorption/ionization (MALDI) using hybrid quadruple time‐of‐flight tandem mass spectrometry (QqToF‐MS). The native polypeptide molecular weight of TM was determined to be 32 733 Da. The protein was further characterized using the ‘bottom‐up’ MS approach. A peptide mass fingerprinting was obtained by two different enzymatic digestions and de novo sequencing of the most abundant peptides performed. Any post‐translational modifications were identified by searching their calculated and predicted molecular weights in precursor ion spectra. The immunological reactivity of snow crab extract was evaluated using specific antibodies and allergenic reactivity assessed with serum of allergic patients. Subsequently, a signature peptide for TM was identified and evaluated in terms of identity and homology using the basic local alignment search tool (BLAST). The identification of a signature peptide for the allergen TM using MALDI‐QqToF‐MS will be critical for the sensitive and specific quantification of this highly allergenic protein in the work place. Copyright © 2010 John Wiley & Sons, Ltd.     

A recommended and verified procedure for in situ tryptic digestion of formalin‐fixed paraffin‐embedded tissues for analysis by matrix‐assisted laser desorption/ionization imaging mass spectrometry

Matrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a molecular imaging technology uniquely capable of untargeted measurement of proteins, lipids, and metabolites while retaining spatial information about their location in situ. This powerful combination of capabilities has the potential to bring a wealth of knowledge to the field of molecular histology. Translation of this innovative research tool into clinical laboratories requires the development of reliable sample preparation protocols for the analysis of proteins from formalin‐fixed paraffin‐embedded (FFPE) tissues, the standard preservation process in clinical pathology. Although ideal for stained tissue analysis by microscopy, the FFPE process cross‐links, disrupts, or can remove proteins from the tissue, making analysis of the protein content challenging. To date, reported approaches differ widely in process and efficacy. This tutorial presents a strategy derived from systematic testing and optimization of key parameters, for reproducible in situ tryptic digestion of proteins in FFPE tissue and subsequent MALDI IMS analysis. The approach describes a generalized method for FFPE tissues originating from virtually any source.     

Identification of carbonylation sites in apomyoglobin after exposure to 4‐hydroxy‐2‐nonenal by solid‐phase enrichment and liquid chromatography–electrospray ionization tandem mass spectrometry

Identification of protein carbonylation because of covalent attachment of a lipid peroxidation end‐product was performed by combining proteolytic digestion followed by solid‐phase hydrazide enrichment and liquid chromatography (LC)–electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using both collision‐induced dissociation (CID) and electron capture dissociation (ECD). To evaluate this approach, we selected apomyoglobin and 4‐hydroxy‐2‐nonenal (4‐HNE) as a model protein and a representative end‐product of lipid peroxidation, respectively. Although the characteristic elimination of 4‐HNE (156 Da) in CID was found to serve as a signature tag for the modified peptides, generation of nearly complete fragment ion series because of efficient peptide backbone cleavage (in most cases over 75%) and the capability to retain the labile 4‐HNE moiety of the tryptic peptides significantly aided the elucidation of primary structural information and assignment of exact carbonylation sites in the protein, when ECD was employed. We have concluded that solid‐phase enrichment with both CID‐ and ECD‐MS/MS are advantageous during an in‐depth interrogation and unequivocal localization of 4‐HNE‐induced carbonylation of apomyoglobin that occurs via Michael addition to its histidine residues. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization and quantitative amino acids analysis of analgesic peptides in cinobufacini injection by size exclusion chromatography,matrix‐assisted laser desorption/ionization time of flight mass spectrometry and gas chromatography mass spectrometry

Cinobufacini injection that comes from the water extract of Bufo bufo gargarizans Cantor skin is widely used for cancer treatment in China. Peptide is one of its major types of constituents, however the biological effects and content of this injection are little reported. In present study, the analgesic effect of peptides was determined and evaluated by in‐vivo models. To characterize and quantitatively analyze these peptides, a reliable and efficient method combining size exclusion chromatography and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry with amino acid analysis was developed. The peptides presented as a series of analogs with similar molecular weights mostly ranging from 2 to 8 kDa. The amino acid analysis by gas chromatography mass spectrometry (GC‐MS) was developed to determine both free and combined amino acids (FAA and CAA) in cinobufacini injection. This method achieved good linearity (R², 0.9909–0.9999) and low limit of detection and quantification. FAA and CAA samples were efficiently analyzed by modified Phenomenex EZ: faast procedure. For the sample analysis, the method showed good repeatability (relative standard deviation, RSD ≤ 10%). For most FAA and CAA the mean recoveries were >80% with RSD <10%. The GC‐MS based method is useful for quality assurance of both FAA and CAA in cinobufacini injection. Copyright © 2014 John Wiley & Sons, Ltd.     

The in vivo absorbed constituents and metabolites of Danshen decoction in rats identified by HPLC with electrospray ionization tandem ion trap and time‐of‐flight mass spectrometry

Danshen, the dried root and rhizome of Salvia miltiorrhiza Bunge, is widely used for the treatment of cardiovascular and cerebrovascular diseases. This research focuses on the in vivo metabolism of Danshen decoction (DSD) in rats. After oral administration of DSD, the absorptive constituents and their metabolites in urine and plasma were analyzed by HPLC coupled with a photodiode array detector and electrospray ionization hybrid ion trap and time‐of‐flight mass spectrometry. Samples were separated on a C₁₈ column by gradient elution using 0.1% (v/v) aqueous formic acid and acetonitrile. As a result, 93 compounds from urine and 38 compounds from plasma were identified. Among them, lipo‐soluble diterpenoids (24 in urine and 15 in plasma) were reported for the first time as in vivo metabolites of DSD. According to the quantities and contents of the identified compounds, tanshinone IIA, cryptotanshinone and tanshinone I were deduced to be the major absorptive diterpenoids of DSD. Moreover, nine water‐soluble phenolics (caffeic acid, ferulic acid, danshensu, etc.) were proved to be the major absorptive constituents as reported. Most of the absorbed constituents underwent sulfation, glucuronidation, hydrogenation and hydroxylation in vivo. This investigation provided scientific evidence to obtain a more comprehensive metabolic profile of DSD. Copyright © 2014 John Wiley & Sons, Ltd.