Direct and efficient monitoring of glycosyltransferase reactions on gold colloidal nanoparticles by using mass spectrometry

A simple and efficient assay for glycosyltransferase activity on gold colloidal nanoparticles (GCNPs) by using laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) is demonstrated by the enzymatic synthesis of the Lewis X trisaccharide on GCNPs containing GlcNAc residues. GCNPs containing multivalent sugars were well dispersed in aqueous solution and proved to be excellent acceptor substrates for the glycosyltransferase reaction. Direct LDI-TOF MS analysis of these GCNPs provided the ion peaks of the sugar derivatives, chemisorbed through S--Au linkages onto the GCNPs, even in the presence of contaminants such as proteins and salts. Thus, it enabled the rapid and direct detection of the enzymatic reaction on the GCNPs by subjecting a small amount (0.15 muL) of the reaction mixture to MS analysis without purification. Subsequent MS/MS analyses (LDI-LIFT-TOF/TOF method) of the product-carrying GCNPs enabled the structures of the sugar derivatives that had been constructed on the GCNPs by enzymatic glycosylation to be determined. A quantitative inhibition assay for glycosyltransferase by using LDI-TOF MS analysis on the GCNPs was demonstrated by using uridine 5'-diphosphate (UDP) as the inhibitor. This simple assay was then applied to the detection of the enzymatic activity of a crude cell extract of Escherichia coli, which produces Neisseria meningitidis beta-1,4-galactosyltransferase (beta-1,4-GalT). In this case, the GCNPs were roughly purified by means of ultrafiltration to remove the buffer and detergents before MS analysis. That the GCNPs are dissolved in solution in the reaction medium but are solid in the purification process is greatly advantageous for the simple and efficient detection of enzymatic activity in crude biological samples. Thus, GCNPs containing a variety of biomolecules may become a versatile and efficient tool for the rapid and direct monitoring of metabolism (metabolomics) in living cells when combined with LDI-TOF MS analysis.     

Determination of self-exchange rate of alkanethiolates in self-assembled monolayers on gold using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In this paper, we describe a new method for determining the exchange rates of alkanethiolates in self-assembled monolayers (SAMs) on gold using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze the compositions of the alkanethiolate in SAMs rapidly and directly. In particular, to investigate the self-exchange of alkanethiols, we prepared a deuterated alkanethiol that has the same molecular properties as the non-deuterated alkanethiol but a different molecular weight. SAMs consisting of deuterated alkanethiolates were immersed in a solution of the non-deuterated alkanethiol, and the influences of the immersion time, temperature, concentration, and solvent on the self-exchange rates were investigated. Furthermore, we assessed the exchange rates among alkanethiols with different carbon chain lengths and different size of ethylene glycol units. In addition, we performed molecular dynamics simulations using a model SAM system in order to understand the molecular mechanism of the exchange process.     

质谱在肽和蛋白质序列分析中的应用

了解肽和蛋白质的序列对理解其功能具有重要意义，测定其序列也是当前生命 科学研究中的重要内容之一，质谱作为高灵敏度的测定分子结构的仪器，其高灵敏 度、广泛的适用性及快速性等特性使它具有很大潜力发展成为辅助传统测序方法的 新方法，并得到了广泛的关注。从离子活化方法（包括碰撞诱导解离CID、源后裂 解PSD、源内裂解ISD等）、衍生化作用以及氨基酸残基消除方式（高能活化产生亚 稳离子、化学降解、酶降解）等多个角度介绍了利用质谱分析多肽和蛋白质序列的 方法，并对其发展前景作出展望。     

Fast quantification of endogenous carbohydrates in plasma using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry

Endogenous carbohydrates in biosamples are frequently highlighted as the most differential metabolites in many metabolomics studies. A simple, fast, simultaneous quantitative method for 16 endogenous carbohydrates in plasma has been developed using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry. In order to quantify 16 endogenous carbohydrates in plasma, various conditions, including columns, chromatographic conditions, mass spectrometry conditions, and plasma preparation methods, were investigated. Different conditions in this quantified analysis were performed and optimized. The reproducibility, precision, recovery, matrix effect, and stability of the method were verified. The results indicated that a methanol/acetonitrile (50:50, v/v) mixture could effectively and reproducibly precipitate rat plasma proteins. Cold organic solvents coupled with vortex for 1 min and incubated at –20°C for 20 min were the most optimal conditions for protein precipitation and extraction. The results, according to the linearity, recovery, precision, matrix effect, and stability, showed that the method was satisfactory in the quantification of endogenous carbohydrates in rat plasma. The quantified analysis of endogenous carbohydrates in rat plasma performed excellently in terms of sensitivity, high throughput, and simple sample preparation, which met the requirement of quantification in specific expanded metabolomic studies after the global metabolic profiling research.     

Molecular design of glycoprotein mimetics: glycoblotting by engineered proteins with an oxylamino-functionalized amino acid residue

The general and efficient method for the site-directed glycosylation of proteins is a key step in order to understand the biological importance of the carbohydrate chains of proteins and to control functional roles of the engineered glycoproteins in terms of the development of improved glycoprotein therapeutics. We have developed a novel method for site-directed glycosylation of proteins based on chemoselective blotting of common reducing sugars by genetically encoded proteins. The oxylamino-functionalized L-homoserine residues, 2-amino-4-O-(N-methylaminooxy) butanoic acid and 2-amino-4-aminooxy butanoic acid, were efficiently incorporated into proteins by using the four-base codon/anticodon pair strategy in Escherichia coli in vitro translation. Direct and chemoselective coupling between unmodified simple sugars and N-methylaminooxy group displayed on the engineered streptavidin allowed for the combinatorial synthesis of novel glycoprotein mimetics.     

Per-O-methylation reaction for structural analysis of carbohydrates by mass spectrometry

Per-O-methylation of carbohydrates is an important sample preparation step in structural analysis of complex carbohydrates, which has generated considerable interest as shown by thousands of citations in the last 10 years. This article provides a critical overview of the per-O-methylation methods applied for structural analysis of carbohydrates by mass spectrometry. The understanding of the O-methylation mechanism can help the researchers to apply the adequate O-methylation method and can generate new ideas in the effort of improving this reaction. The per-O-methylation of carbohydrates is relied upon stepwise reactions. The parameters that affect the reaction are discussed for the most important methods and are critically commented for each reaction step. The limits of each method are emphasized. The improvements of the per-O-methylation reaction are described in detail with their advantages and disadvantages and some illustrative examples are given. The methods that give complete O-methylation in non-hazardous conditions with high yields within minutes at room temperature with a very low amount of side-products are especially highlighted.     

Probing of arginine residues in peptides and proteins using selective tagging and electrospray ionization mass spectrometry

A general labelling method is presented which allows the determination of the number of guanidine groups (related to arginine and homoarginine in peptides and proteins) by means of mass spectrometry. It implies a guanidine-selective derivatization step with 2,3-butanedione and an arylboronic acid under aqueous, alkaline conditions (pH 8-10). The reaction mixture is then directly analysed by electrospray ionization mass spectrometry without further sample pretreatment. Other amino acids are not affected by this reaction although it is demonstrated that lysine side-chains may be unambiguously identified when they are converted to homoarginine prior to derivatization. Guanidine functionalities, as e.g. in the amino acid arginine, are easily identified by the characteristic mass shift between underivatized and derivatized analyte. The tagging procedure is straightforward and selective for guanidine groups. The influence of several experimental parameters, especially the pH of the solution and the choice of reagents, is examined and the method is applied to various arginine-containing peptides and to lysozyme as a representative protein. Possible applications of this technique and its limitations are discussed.     

Fast and eco-friendly method using atmospheric solids analysis probe mass spectrometry to characterize orange varieties

Orange fruit is one of the most popular types of fruit in the world, and its juice is the main product of its processing. This study aimed to evaluate a simple, fast, and eco-friendly methodology, atmospheric solids analysis probe mass spectrometry (ASAP-MS), to assess the chemical profile of four oranges varieties (Valencia, Folha Murcha, Pera, and Iapar). The oranges' varieties were evaluated for the physicochemical composition (extraction yield, pH, total titratable acidity, total soluble solids [TSS], °Brix), ratio (TSS/TA), and bioactive compounds (ASAP-MS analysis). The characterization of oranges resulted in great values of oranges yield according to the varieties (44.00–48.10% [w/w], adequate and characteristic acidity [0.73–1.35%], soluble solids content (10.24–13.80°Brix), pH (3.30–3.96), and ratio (7.59–19.90) level for this fruit. This powerful method showed that all analysis procedures were simple, fast, and easy because there is no need to prepare the sample and the analysis time lasted 2 min. Besides, results obtained exhibited a vast array of chemical groups. Principal component analysis (PCA) defined and distinguished the varieties of the orange. Therefore, ASAP-MS and PCA showed that they are very attractive candidates for routine analysis to monitor the varieties of the orange with its pronounced advantages, besides being contributing to the environment because it does not use any quantities of organic solvents. This methodology was applied for the first time to this type of sample.     

High-throughput glycosylation analysis of intact monoclonal antibodies by mass spectrometry coupled with capillary electrophoresis and liquid chromatography

The analysis of monoclonal antibodies glycosylation is a crucial quality control attribute of biopharmaceutical drugs. High throughput screening approaches for antibody glycoform analysis are required in various stages of process optimization. Here, we present high throughput screening suitable mass spectrometry-based workflows for the analysis of intact antibody glycosylation out of cell supernatants. Capillary electrophoresis and liquid chromatography were coupled with quadrupole time-of-flight mass spectrometry or Orbitrap mass spectrometry. Both separation methods offer fast separation (10–15 min) and the capability to prevent the separated cell supernatant matrix to enter the mass spectrometry by post-separation valving. Both mass spectrometry instruments provide comparable results and both are sufficient to determine the glycosylation pattern of the five major glycoforms of the measured antibodies. However, the Orbitrap yields higher sensitivity of 25 μg/mL (CE-nanoCEasy-Orbitrap mass spectrometry) and 5 μg/mL (liquid chromatography-Orbitrap mass spectrometry). Data processing was optimized for a faster processing and easier detection of low abundant glycoforms based on averaged charge-deconvoluted mass spectra. This approach combines a non-target glycoform analysis while yielding the same glycosylation pattern as the traditional approach based on extracted ion traces. The presented methods enable the high throughput screening of the glycosylation pattern of antibodies down to low μg/mL-range out of cell supernatant without any sample preparation.     

Classification of secondary ion mass spectrometry (SIMS) micrographs to characterize chemical phases

This work demonstrates the potential of multivariate image analysis methods in the extraction of useful, problem dependent information from SIMS images. Specific algorithms have been developed to classify SIMS micrographs manually as well as automatically. A feature selection has been achieved by means of principal component analysis with a subsequent image classification.As an application example for these improved digital image processing tools chemical phases within a soldered industrial metal sample have been identified. This is of highly practical value as it was assumed that during the soldering process inhomogeneities occur along the joint site which cause a cracking of the brazed material under mechanical strain conditions.     

Using accurate mass electrospray ionization-time-of-flight mass spectrometry with in-source collision-induced dissociation to sequence peptide mixtures

Although data-dependent LC-MS-MS with database searching has become au courant for identifying proteins, the technique is constrained by duty-cycle inefficiency and the inability of most tandem mass analyzers to accurately measure peptide product ion masses. In this work, a novel approach is presented for simultaneous peptide fragmentation and accurate mass measurement using in-source collision-induced dissociation (CID) on electrospray ionization (ESI)-time-of-flight (TOF) MS. By employing internal mass reference compounds, mass measurement accuracy within +/-5 ppm for tryptic peptide precursors and +/-10 ppm for most sequence-specific product ions was consistently achieved. Analysis of a complex solution containing several digested protein standards did not adversely affect instrument performance.     

Glycosylation characterization of recombinant human erythropoietin produced in glycoengineered Pichia pastoris by mass spectrometry

Glycosylation plays a critical role in the in vivo efficacy of both endogenous and recombinant erythropoietin (EPO). Using mass spectrometry, we characterized the N‐/O‐linked glycosylation of recombinant human EPO (rhEPO) produced in glycoengineered Pichia pastoris and compared with the glycosylation of Chinese hamster ovary (CHO) cell‐derived rhEPO. While the three predicted N‐linked glycosylation sites (Asn24, Asn38 and Asn83) showed complete site occupancy, Pichia‐ and CHO‐derived rhEPO showed distinct differences in the glycan structures with the former containing sialylated bi‐antennary glycoforms and the latter containing a mixture of sialylated bi‐, tri‐ and tetra‐antennary structures. Additionally, the N‐linked glycans from Pichia‐produced rhEPO were similar across all three sites. A low level of O‐linked mannosylation was detected on Pichia‐produced rhEPO at position Ser126, which is also the O‐linked glycosylation site for endogenous human EPO and CHO‐derived rhEPO. In summary, the mass spectrometric analyses revealed that rhEPO derived from glycoengineered Pichia has a highly uniform bi‐antennary N‐linked glycan composition and preserves the orthogonal O‐linked glycosylation site present on endogenous human EPO and CHO‐derived rhEPO. Copyright © 2013 John Wiley & Sons, Ltd.     

Accurate mass measurements by Fourier transform mass spectrometry in the study of advanced glycation end products/peptides

The Maillard reaction occurring between sugars and amino groups is important in living systems. When amino groups belonging to protein chains are involved, the Maillard reaction has been invoked as responsible for protein cross-linking and the production of 'toxic' compounds. The reaction leads to the production of a heterogeneous group of substances, usually called advanced glycation end products (AGEs). Classical analytical approaches, such as spectroscopic (ultraviolet, fluorescence) and mass spectrometric (matrix-assisted laser desorption/ionization, liquid chromatography/electrospray ionization mass spectrometry) methods, have shown that the digestion mixture is highly complex. However, there are clear differences between the digestion mixtures of glycated and unglycated human serum albumin (HSA). In the former case, possible glycated peptides belonging to the AGE peptide class may be identified. Tandem mass spectrometric experiments on selected species seemed to be promising as regards structural information, but it was thought of interest to undertake the present investigation, based on liquid chromatography/electrospray ionization Fourier transform mass spectrometry, in order to obtain definitive results on their elemental composition. Using this approach, about 20 glycated peptides were detected and their possible structures were postulated by examining the known sequence of HSA.     

Simultaneous determination of 13 carbohydrates using high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection and mass spectrometry

A simple, accurate, and highly sensitive method was developed for the determination of 13 carbohydrates in polysaccharide of Spirulina platensis based on high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection and mass spectrometry. Samples were extracted with deionized water using ultrasonic‐assisted extraction, and the ultrasound‐assisted extraction conditions were optimized by Box–Behnken design. Then the extracted polysaccharide was hydrolyzed by adding 1 mol/L trifluoroacetic acid before determination by high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection and confirmed by high‐performance anion‐exchange chromatography coupled with mass spectrometry. The high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection method was performed on a CarboPac PA20 column by gradient elution using deionized water, 0.1 mol/L sodium hydroxide solution, and 0.4 mol/L sodium acetate solution. Excellent linearity was observed in the range of 0.05–10 mg/L. The average recoveries ranged from 80.7 to 121.7%. The limits of detection and limits of quantification for 13 carbohydrates were 0.02–0.10 and 0.2–1.2  μg/kg, respectively. The developed method has been successfully applied to ambient samples, and the results indicated that high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection and mass spectrometry could provide a rapid and accurate method for the simultaneous determination of carbohydrates.     

Applications of mass spectrometry to food proteins and peptides

The application of mass spectrometry (MS) to large biomolecules has been revolutionized in the past decade with the development of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) techniques. ESI and MALDI permit solvent evaporation and sublimation of large biomolecules into the gaseous phase, respectively. The coupling of ESI or MALDI to an appropriate mass spectrometer has allowed the determination of accurate molecular mass and the detection of chemical modification at high sensitivity (picomole to femtomole). The interface of mass spectrometry hardware with computers and new extended mass spectrometric methods has resulted in the use of MS for protein sequencing, post-translational modifications, protein conformations (native, denatured, folding intermediates), protein folding/unfolding, and protein-protein or protein-ligand interactions. In this review, applications of MS, particularly ESI-MS and MALDI time-of-flight MS, to food proteins and peptides are described.