Solid-state glycation of beta-lactoglobulin monitored by electrospray ionisation mass spectrometry and gel electrophoresis techniques

Glycation of beta-lactoglobulin (beta-Lg) with either lactose or galactose in a solid-state medium was monitored using gel electrophoresis techniques and liquid chromatography coupled to electrospray ionisation mass spectrometry (LC/ESI-MS). The kinetics of glycation monitored by SDS polyacrylamide gel electrophoresis showed a molecular weight increase over time of the beta-Lg bands for both sugars, but no significant amounts of aggregated proteins were observed. The isoelectric point of the protein, observed by isoelectric focusing gel electrophoresis, was dramatically affected by galactosylation. LC/MS measurements of beta-Lg variants A and B, over the whole glycation reaction time, showed a larger extent of glycation with galactose (from 4 up to 22 adducts) as compared with lactose (from 0 up to 14 adducts), and confirmed that early Maillard reaction products were the main species observed. Based on the relative abundances obtained from the deconvoluted mass spectra after a 8 h 15 min incubation time at 60 degrees C, the mean values of lactose and galactose molecules bound to the protein species were calculated to be 10.4 and 17.9, and 10.5 and 18.6, for variants A and B, respectively. Furthermore, the charge state distribution data obtained by ESI-MS was studied using different methanol percentages, and indicated that adduct formation with lactose, but more significantly galactose, tends to improve the stability properties of the native protein towards denaturation.     

Mass spectrometry‐based proteomics of oxidative stress: Identification of 4‐hydroxy‐2‐nonenal (HNE) adducts of amino acids using lysozyme and bovine serum albumin as model proteins

Modification of proteins by 4‐hydroxy‐2‐nonenal (HNE), a reactive by‐product of ω6 polyunsaturated fatty acid oxidation, on specific amino acid residues is considered a biomarker for oxidative stress, as occurs in many metabolic, hereditary, and age‐related diseases. HNE modification of amino acids can occur either via Michael addition or by formation of Schiff‐base adducts. These modifications typically occur on cysteine (Cys), histidine (His), and/or lysine (Lys) residues, resulting in an increase of 156 Da (Michael addition) or 138 Da (Schiff‐base adducts), respectively, in the mass of the residue. Here, we employed biochemical and mass spectrometry (MS) approaches to determine the MS “signatures” of HNE‐modified amino acids, using lysozyme and BSA as model proteins. Using direct infusion of unmodified and HNE‐modified lysozyme into an electrospray quadrupole time‐of‐flight mass spectrometer, we were able to detect up to seven HNE modifications per molecule of lysozyme. Using nanoLC‐MS/MS, we found that, in addition to N‐terminal amino acids, Cys, His, and Lys residues, HNE modification of arginine (Arg), threonine (Thr), tryptophan (Trp), and histidine (His) residues can also occur. These sensitive and specific methods can be applied to the study of oxidative stress to evaluate HNE modification of proteins in complex mixtures from cells and tissues under diseased versus normal conditions.     

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.     

Characterizing closely spaced, complex disulfide bond patterns in peptides and proteins by liquid chromatography/electrospray ionization tandem mass spectrometry

Identifying the Cys residues involved in disulfide linkages of peptides and proteins that contain complex disulfide bond patterns is a significant analytical challenge. This is especially true when the Cys residues involved in the disulfide bonds are closely spaced in the primary sequence. Peptides and proteins that contain free Cys residues located near disulfide bonds present the additional problem of disulfide shuffling via the thiol-disulfide exchange reaction. In this paper, we report a convenient method to identify complex disulfide patterns in peptides and proteins using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with partial reduction by tris(2-carboxyethyl)phosphine (TCEP). The method was validated using well-characterized peptides and proteins including endothelin, insulin, alpha-conotoxin SI and immunoglobulin G (IgG2a, mouse). Peptide or protein digests were treated with TCEP in the presence of an alkylation reagent, maleimide-biotin (M-biotin) or N-ethylmaleimide (NEM), followed by complete reduction with dithiothreitol and alkylation by iodoacetamide (IAM). Subsequently, peptides that contained alkylated Cys were analyzed by capillary LC/ESI-MS/MS to determine which Cys residues were modified with M-biotin/NEM or IAM. The presence of the alkylating reagent (M-biotin or NEM) during TCEP reduction was found to minimize the occurrence of the thiol-disulfide exchange reaction. A critical feature of the method is the stepwise reduction of the disulfide bonds and the orderly, sequential use of specific alkylating reagents.     

Comprehensive Profiling for Histone H4of Human Liver Cells Using High Resolution LTQ-Orbitrap Mass Spectrometry

The post translational modifications of histone variants are playing an important role in the structure of chro‐ matin, the regulation of gene activities and the diagnosis of diseases, and conducting in‐depth researches and discovering new sites depend on new and rational analytical methods to some extent. In this work, the combinatorial method of high resolution LTQ‐Orbitrap mass spectrometry and multiple enzymes was employed to identify the post translational modifications (PTMs) of histone H4 of human liver cells. The novel methylation site, argnine 67 (R 67), was observed besides some sites reported previously such as lysine 31 (K 31), lysine 44 (K 44), argnine 55 (R 55) and lysine 59 (K 59) in the global domain. Meanwhile, various combinations of acetylation of lysine 5 (K 5), lysine 8 (K 8), lysine 12 (K 12), lysine 16 (K 16) and methylation of lysine 20 (K 20) in the NH₂‐terminal tails were also identified after the LC‐MS/MS analysis of trypsin, Arg‐C, Glu‐C and chymotrypsin digests.     

Study of peptides containing modified lysine residues by tandem mass spectrometry: precursor ion scanning of hexanal-modified peptides

Upon hexanal-modification in the presence of NaCNBH(3), the oxidized B chain of insulin becomes mono- and further dialkylated on both the N-terminal and Lys(29) residues. A pseudo-MS(3) study was performed with a triple-quadrupole mass spectrometer on the different modified lysine-containing species to gain further insights into the characteristic fragmentation pattern. These fragmentations, in good agreement with true MS(3) measurements obtained using an ion trap mass spectrometer, highlighted characteristic monoalkylated lysine (immonium-NH(3)) and protonated modified caprolactam ions at m/z 168 and 213, respectively. In contrast, no fragment ion derived from a modified lysine residue (immonium or caprolactam) was observed when dialkylation occurs on Lys(29). However, a fragment ion corresponding to a protonated dihexylamine was observed at m/z 186. This loss, characteristic of dialkylated lysine fragmentation, was also observed upon dialkylation of N(alpha)-acetyllysine with either hexanal or pentanal. On the other hand, acetylation and malondialdehyde-modification of the N(alpha)-acetyllysine side chain led mainly to the corresponding modified (immonium-NH(3)) fragment ions at m/z 126 and 138, respectively. Finally, it was demonstrated that precursor ion scanning for both m/z 168 and 213 ions led to specific and sensitive identification of peptides containing hexanal-modified lysine residues within an unfractionated tryptic digest of hexanal-modified apomyoglobin. Thus, Lys(42), Lys(45), Lys(62), Lys(63), Lys(77), Lys(87), Lys(96), Lys(98), Lys(145) and Lys(147) were found to be modified upon reaction with hexanal.     

Rapid screening and identification of cytochalasins by electrospray tandem mass spectrometry

The cytochalasin class of fungal metabolites was analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) with the aim of developing a methodology for their rapid identification in microbial extracts. ESI-MS analyses of reference cytochalasins were performed and several product ions were produced in MS/MS experiments on parent ions that are structurally characteristic. A precursor ion search was performed to detect cytochalasins in an ethyl acetate extract of fungal strain RK97-F21. Three cytochalasins were detected and one of the components was identified as epoxycytochalasin H by comparing the tandem mass spectra of the product ions with those of reference compounds. This finding was further validated by LC/MS and LC/MS/MS experiments.     

Structural studies of the allelic wheat glutenin subunits 1Bx7 and 1Bx20 by matrix-assisted laser desorption/ionization mass spectrometry and high-performance liquid chromatography/electrospray ionization mass spectrometry

Structural studies of the high molecular mass (HMM) glutenin subunits 1Bx7 (from cvs Hereward and Galatea) and 1Bx20 (from cv. Bidi17) of bread wheat were conducted using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS). For all three proteins, MALDI-TOFMS analysis showed that the isolated fractions contained a second component with a mass about 650 Da lower than the major component. The testing and correction of the gene-derived amino acid sequences of the three proteins were performed by direct MALDI-TOFMS analysis of their tryptic peptide mixture. Analysis of the digest was performed by recording several MALDI mass spectra of the mixture at low, medium and high mass ranges, optimizing the matrix and the acquisition parameters for each mass range. Complementary data were obtained by RP-HPLC/ESI-MS analysis of the tryptic digest. This resulted in coverage of about 98% of the sequences. In contrast to the gene-derived data, the results obtained demonstrate the insertion of the sequence QPGQGQ between Trp716 and Gln717 of subunit 1Bx7 (cv. Galatea) and a possible single amino acid substitution within the T20 peptide of subunit 1Bx20. Moreover, the mass spectrometric data demonstrated that the lower mass components present in all the fractions correspond to the major components but lack about six amino acid residues, which are probably lost from the protein C-terminus. Finally, the results obtained provide evidence for the lack of glycosylation or other post-translational modifications of these subunits.     

Tracing glycoprotein structures: electrospray ionization tandem mass spectrometric analysis of sugar-peptide adducts

Owing to the diversity of carbohydrate structures and their significance for the function of many biopolymers, structural analysis of various carbohydrate-related compounds is of great importance. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to establish the fragmentation behaviour of a range of sugar-peptide adducts as model compounds of widespread glycoprotein structures. The compounds used in this study were chosen to provide correlation of distinct fragment ions with specific structural differences, namely position and type of carbohydrate-peptide bond and structure of the sugar moiety. All compounds show N- and C-terminal sequence ions along with losses of up to three water molecules. Fructose-related Amadori compounds exhibit M + 78 modified N-terminal peptide fragment ions. Fragmentation of glucose-peptide esters is characterized by the sugar ring fragmentation. Additionally, under the ESI-MS conditions applied, the esters studied undergo intramolecular reaction giving cyclic sugar-peptide structures that can be traced by the presence of N-terminal peptide M + 42 adducts. Detailed analysis of cyclic fructose-related compound comprising structural features of both studied groups revealed a rich fragmentation pattern derived from amino acid residues and water molecules losses from [M - 2H(2)O + H](+) ion. Also, some interesting differences were found with respect to the nature of carbohydrate moieties.     

Novel Rearrangement of Small Peptides in Electrospray Ionization Tandem Mass Spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is a powerful method for sequencing peptides. A novel fragmentation pattern with the loss of a neutral fragment of 45 Da was observed with the dipeptides, tripeptides,tetrapeptides and pentapeptides containing phenylalanine or histidine residues. A novel rearrangement reaction with the extrusion of a formamide piece was studied and the rearrangement mechanism was proposed and confirmed by deuterium labeling experiments with ESI-MS^n and high-resolution mass spectrometry. These findings are potentially helpful in identifying the specific sequence pattern in the peptide sequencing.     

Structural characterization of intact antibodies by high‐resolution LTQ Orbitrap mass spectrometry

Recombinant monoclonal antibodies (MAbs) can be heterogeneous due to modifications that can occur during expression, purification or during storage. These large multichain proteins (～150 kDa) are structurally challenging for detailed characterization to identify the sites of modifications. We report the use of LTQ Orbitrap mass spectrometry to accurately measure the average masses of individual glycoforms by direct infusion of an intact antibody. To identify the site‐specific modification of methionines in the antibody caused by forced oxidation, we used a ‘middle‐down’ approach. The antibody was subjected to limited digestion using the endoproteinase Lys‐C and reduced to generate Fab heavy chain, single chain Fc and light chain fragments (～25 kDa each). These species were subjected to on‐line liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis using an LTQ Orbitrap, where these large precursors were dissociated by higher‐energy collisions in the C‐trap. High resolution and accuracy achieved for resulting fragments allowed us to show in a site‐specific manner that only the methionines in the Fc heavy chain were oxidized under the studied conditions. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of binding sites in carboplatin-bound cytochrome c using electrospray ionization mass spectrometry and tandem mass spectrometry

Interaction of carboplatin with cytochrome c (Cyt. c) has been investigated by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS). ESI-MS studies revealed that the ring-opened adducts of carboplatin with Cyt. c were formed in the stoichiometric ratio of 1:1 and 2:1 at pH 5.0 and 37 degrees C and in the stoichiometric ratio of 1:1 only at pH 7.0 and 37 degrees C. It was also found that Cyt. c could be cleaved by carboplatin at pH 2.5 and 50 degrees C. The cleaved fragments of Cyt. c were determined by ESI-MS and MS/MS analysis to be Glu66 approximately Met80, Ac-Gly01 approximately Met65, Glu66 approximately Glu104, Ac-Gly01 approximately Met80 and Ile81 approximately Glu104. The carboplatin prefers to anchor to Met65 first, then to Met80. To further confirm the binding site of Met, AcMet-Gly was used as the model molecule to investigate its interaction with carboplatin and its hydrolysis reaction. On the basis of species detected during the reaction monitored by ESI-MS, a possible pathway of the cleavage reaction was proposed.     

Fragmentation behavior of Amadori‐peptides obtained by non‐enzymatic glycosylation of lysine residues with ADP‐ribose in tandem mass spectrometry

Mono‐ and poly‐adenosine diphosphate (ADP)‐ribosylation are common post‐translational modifications incorporated by sequence‐specific enzymes at, predominantly, arginine, asparagine, glutamic acid or aspartic acid residues, whereas non‐enzymatic ADP‐ribosylation (glycation) modifies lysine and cysteine residues. These glycated proteins and peptides (Amadori‐compounds) are commonly found in organisms, but have so far not been investigated to any great degree. In this study, we have analyzed their fragmentation characteristics using different mass spectrometry (MS) techniques. In matrix‐assisted laser desorption/ionization (MALDI)‐MS, the ADP‐ribosyl group was cleaved, almost completely, at the pyrophosphate bond by in‐source decay. In contrast, this cleavage was very weak in electrospray ionization (ESI)‐MS. The same fragmentation site also dominated the MALDI‐PSD (post‐source decay) and ESI‐CID (collision‐induced dissociation) mass spectra. The remaining phospho‐ribosyl group (formed by the loss of adenosine monophosphate) was stable, providing a direct and reliable identification of the modification site via the b‐ and y‐ion series. Cleavage of the ADP‐ribose pyrophosphate bond under CID conditions gives access to both neutral loss (347.10 u) and precursor‐ion scans (m/z 348.08), and thereby permits the identification of ADP‐ribosylated peptides in complex mixtures with high sensitivity and specificity. With electron transfer dissociation (ETD), the ADP‐ribosyl group was stable, providing ADP‐ribosylated c‐ and z‐ions, and thus allowing reliable sequence analyses. Copyright © 2010 John Wiley & Sons, Ltd.     

电喷雾质谱在寡糖序列分析中的应用

选取具有不同结构特征的N-糖链、硫酸软骨素寡糖、人乳寡糖以及海洋来源的壳寡糖、褐藻胶寡糖、卡拉胶寡糖和硫酸岩藻寡糖等,对电喷雾质谱在寡糖的主链序列、分支位点、硫酸基取代位置确定、单糖组成和聚合度分析等方面的应用技术及碎片离子的断裂规律进行了总结.根据相邻同类碎片离子之间的质荷比差值可初步判断寡糖的单糖组成类型;通过与色谱分离技术联用或衍生化方法可提高寡糖的分辨率和离子化效率,并测得寡糖的分子量及聚合度;借助串联质谱及对寡糖还原端的特异性标记,可获得寡糖的还原端残基和部分序列信息;根据寡糖产生的特征碎片离子及其丰度大小可判断残基的特定位置和类型.另外,寡糖的分支通常作为一个整体发生糖苷键断裂或产生D离子,据此可判断分支点的位置;根据硫酸寡糖产生的特异性跨环断裂碎片,可以确定硫酸基的连接位置.这些规律和方法的总结为未知寡糖的结构和序列的分析提供了启发和指导.     

Liquid chromatography/mass spectrometry in anabolic steroid analysis--optimization and comparison of three ionization techniques: electrospray ionization,atmospheric pressure chemical ionization and atmospheric pressure photoionization

The applicability of liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the detection of the free anabolic steroid fraction in human urine was examined. Electrospray ionization (ESI), atmospheric pressure chemical ionization and atmospheric pressure photoionization methods were optimized regarding eluent composition, ion source parameters and fragmentation. The methods were compared with respect to specificity and detection limit. Although all methods proved suitable, LC/ESI-MS/MS with a methanol-water gradient including 5 mM ammonium acetate and 0.01% acetic acid was found best for the purpose. Multiple reaction monitoring allowed the determination of steroids in urine at low nanogram per milliliter levels. LC/MS/MS exhibited high sensitivity and specificity for the detection of free steroids and may be a suitable technique for screening for the abuse of anabolic steroids in sports.     

Comparison of Electrospray Ionization and Fast Atom Bombardment Mass Spectrometry for the Analysis of Saponins from Alfalfa,Clover, and Mungbeans

The determination of seven saponins in crude plant extracts by electrospray ionization mass spectrometry (ESI-MS) and fast atom bombardment mass spectrometry (FAB-MS) is described. Distinct protonated and natriated (Na-adduct) molecular ions in ESI-MS spectra readily provide molecular weight information, which can be further verified using clusters of molecular ions. Saponin mixtures can be analyzed by ESIMS on varying the potential difference between the capillary and skimmer in the ESI source to decompose impurities. ESI-MS uses less amount of sample than that required by FAB-MS. ESI-MS does not produce structural information, however. The FAB-MS spectra consist mainly of protonated and deprotonated molecular ions with limited structural information. (-)-FAB-MS is more suitable for analyzing saponin samples than the (+)-FAB-MS.     

Combination of liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry for the detection of 21 anabolic steroid residues in bovine urine

For the detection of anabolic steroid residues in bovine urine, a highly sensitive liquid chromatographic/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method was developed using both positive and negative ionization. For four compounds the ESI mode was not sensitive enough and gas chromatographic/mass spectrometric GC/MS detection was therefore still necessary as a complementary method. The sample clean-up consisted of solid-phase extraction (SPE) on a C(18) column followed by enzymatic hydrolysis and a second solid-phase extraction on a combination of a C(18) and a NH(2) column. After this last SPE clean-up, the eluate was split into two equal aliquots. One aliquot was further purified and after derivatization used for GC/MS analysis. The other aliquot was analyzed with LC/MS/MS in both ESI+ and ESI- modes. The method was validated according to the European Commission Decision 2002/657/EC. Decision limits (CCalpha) were between 0.16 and 1 ng ml(-1) for the compounds detected with the LC/MS/MS method. The developed method is used in routine analysis in our laboratory.     

Comparative LC-MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products

Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for N(epsilon)-fructoselysine (FL), N(epsilon)-carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34+/-3.81 nmol CML per micromol of free Lys (Lys(free)) and 81.5+/-87.8 nmol Pyr micromol(-1) Lys(free)(-1) vs. 3.72+/-1.29 nmol FL micromol(-1) Lys(free)(-1). In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47+/-0.08 nmol FL micromol(-1) of protein-bound Lys (Lys(p-b)), 0.04+/-0.03 nmol CML micromol(-1) Lys(p-b)(-1) and 0.06+/-0.02 nmol Pyr micromol(-1)Lys(p-b)(-1). It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products.