Synthesis of two closely spaced cysteine barbiturates containing peptides by copper-catalyzed oxidation of contryphan disulfide

In this report, we are documenting the synthesis of peptide barbiturate through copper-catalyzed oxidation of peptide disulfide. Single disulfide-containing contryphans are used as models to access possibility of anchoring of barbituric acid (BRB) onto the peptide disulfide. Current method permits anchoring of two molecules of BRB onto the polypeptide and yield of peptide barbiturates varies from 59 to 84%. Formation of cysteine sulfenic acid (Cys-SOH) during oxidation of disulfide was confirmed using chemical probe of Cys-SOH dimedone. Mass spectrometric studies have confirmed the presence of cysteine barbiturate in anchored peptides. Based on the nature of reactive oxygen species involved in oxidation of peptide disulfide, possible mechanisms were proposed for anchoring of BRB onto the peptide disulfide through Cys-SOH. This is the first report on anchoring of two molecules of BRB onto the closely spaced cysteine residues of single polypeptide.     

Identification of isomeric 5‐hydroxytryptophan‐ and oxindolylalanine‐containing peptides by mass spectrometry

Cells continuously produce reactive oxidative species that can modify all cellular components. In proteins, for example, cysteine, methionine, tryptophan (Trp), and tyrosine residues are particularly prone to oxidation. Here, we report two new approaches to distinguish two isomeric oxidation products of Trp residues, i.e. 5‐hydroxytryptophan (5‐HTP) and oxindolylalanine (Oia) residues, in peptides. First, 2‐nitrobenzenesulfenyl chloride, known to derivatize Trp residues in position 2 of the indole ring, was used to label 5‐HTP residues. The mass shift of 152.98 m/z units allowed identifying 5‐HTP‐ besides Trp‐containing peptides by mass spectrometry, whereas Oia residues were not labeled. Second, fragmentation of the Oia‐ and 5‐HTP‐derived immonium ions at m/z 175.08 produced ions characteristic for each residue that allowed their identification even in the presence of y₁ ions at m/z 175.12 derived from peptides with C‐terminal arginine residues. The pseudo MS³ spectra acquired on a quadrupole time‐of‐flight hybrid mass spectrometer displayed two signals at m/z 130.05 and m/z 132.05 characteristic for Oia‐containing peptides and a group of six signals (m/z 103.04, 120.04, 130.04, 133.03, 146.04, and 148.04) for 5‐HTP‐cointaining peptides. In both cases, the relative signal intensities appeared to be independent of the sequence providing a specific fingerprint of each oxidative modification. Copyright © 2012 John Wiley & Sons, Ltd.     

氨基酸-磷酸非共价复合物离子的电喷雾电离质谱研究

分子间的非共价相互作用对于理解分子复合物的结构和性质有着非常重要的意义。而电喷雾电离质谱(ESI-MS)是团簇化学研究的一种重要工具,广泛用于重要生物分子复合物的形成和性质研究,包括氨基酸、多肽和核糖等。该文将一定化学剂量比的磷酸分别与L型精氨酸和L型色氨酸混合,通过电喷雾电离的方法,利用傅立叶变换离子回旋共振质谱仪对精氨酸-磷酸以及色氨酸-磷酸的非共价复合物离子进行了研究。结果显示,精氨酸和色氨酸均可与磷酸分子产生种类丰富、价态为+1和+2的复合物离子。而对不同体系的生成离子分布进行分析,则显示出不同体系间的差别。     

Unusual course of the reaction of N-(tosylmethyl)thiourea and N-(azidomethyl)thiourea with sodium enolate of dimedone

The end product of the reaction of N-(tosylmethyl)thiourea or N-(azidomethyl)thiourea with sodium enolate of dimedone is bis(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-enyl)methane (methylenebisdimedone) instead of the expected 8a-hydroxy-7,7-dimethyl-2-thioxoperhydroquinazolin-5-one.M. V. Lomonosov State Academy of Fine Chemical Technology, Moscow 117571, Russia; Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 1, pp. 70–72, January, 2000.     

Fragmentation behavior of glycated peptides derived from D-glucose, D-fructose and D-ribose in tandem mass spectrometry

Nonenzymatic glycosylation (or glycation) is a common nonenzymatic side-chain specific sequence-independent posttranslational modification formed by the reaction of reducing carbohydrates with free amino groups. Thus, proteins can react with aldoses or ketoses to yield Amadori or Heynes compounds, respectively. Here, the fragmentation behavior of D-glucose and D-ribose-derived Amadori peptides as well as D-fructose-derived Heynes peptides were studied by collision-induced fragmentation (CID) after electrospray (ESI) or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). All three sugar moieties displayed characteristic fragmentation patterns accompanying the parent and the fragment ions, which could be explained by consecutive losses of water and formaldehyde. Glucose-derived Amadori parent and fragment ions displayed losses of 18, 36, 54, 72, and 84 u at a characteristic intensity distribution compared with losses of 18, 36, 54, 72, 84, and 96 u for D-fructose-derived ions and losses of 18, 36, and 54 u for ribose-derived ions. Furthermore, each sugar moiety produced indicative lysine-derived immonium ions that were successfully used in a precursor ion scan analysis to identify Amadori peptides in a tryptic digest of bovine serum albumin (BSA) glycated with D-glucose. BSA was modified on lysine residues at positions 36, 160, 235, 256, 401, and 548.     

Separation of Amadori peptides from their unmodified analogs by ion-pairing RP-HPLC with heptafluorobutyric acid as ion-pair reagent

Glycation is a common class of nonenzymatic posttranslational modifications relevant for several diseases and cell aging in general, such as D-glucose-derived modifications at the ɛ-amino groups of lysine residues in blood proteins, especially albumin, immunoglobulin, and hemoglobin, for diabetic patients. These Amadori compounds are identified on the peptide level after enzymatic digestion and chromatographic separation by mass spectrometry. Their syntheses usually rely on a global glycation approach. Both areas require the reliable separation of glycated peptides from their unmodified congeners present in different ratios, which is typically not achieved by standard eluent systems in ion-pairing RP-HPLC (IP-RPLC). Here, we compare aqueous acetonitrile and methanol gradients containing either trifluoroacetic acid (TFA) or heptafluorobutyric acid (HFBA) as ion-pairing agents to separate such peptide pairs. TFA-containing eluents resulted in rather low resolutions, and the glycated and unglycated peptides often coeluted. HFBA increased the retention times of the unmodified peptide more than for the glycated peptide thereby improving the separation of all eight studied peptide pairs, even achieving baseline separations for some sequences. Thus the use of HFBA as ion-pair reagent provides a universally applicable eluent system in IP-RPLC to separate glycated peptides from their unmodified counterparts, even at the preparative scale required for synthetic peptides.     

Open-tubular capillary electrochromatography-mass spectrometry with sheathless nanoflow electrospray ionization for analysis of amino acids and peptides

A novel, rugged sheathless capillary electrochromatography-electrospray ionization (CEC-ESI) device, in which an open-tubular separation capillary and an electrospray tip are integrated with a Nafion tubing junction, is coupled to mass spectrometry (MS) for the analysis of amino acids and peptides. A stable electrospray was generated at nanoflow rates by applying a positive electrical potential at the Nafion membrane junction. To sustain the stable spray, an electroosmotic flow (EOF) to the spray was supported by coating the fused silica capillary with Lupamin, a high-molecular-weight linear positively charged polyvinylamine (PVAm) polymer, which also minimizes analyte adsorption. Electrochromatographic separation of amino acids and peptides was further enhanced by the chromatographic selectivity of Lupamin stationary phase for these molecules. The device was very reliable and reproducible for CEC-ESI-MS analyses of amino acids and peptides for over a hundred injections. The separation and detection behaviors of amino acids and peptides under different conditions including pH, concentration, and composition of mobile phases on Lupamin-coated and uncoated capillaries have been investigated. The relationship between nano electrospray stability and EOF is discussed.     

Differentiation of three pairs of Boc-beta,gamma- and gamma,beta-hybrid peptides by electrospray ionization tandem mass spectrometry

A new series of Boc-N-beta(3), gamma(4)-/gamma(4), beta(3)-isomeric hybrid peptides (containing repeats of beta(3)-Caa and gamma(4)-Caa's, Caa = C-linked carbo beta(3)-/gamma(4)-amino acids derived from D-xylose) have been differentiated by both positive and negative ion electrospray ionization (ESI) ion-trap and high resolution quadrupole time-of-flight/tandem mass spectrometry (Q-TOF MS/MS). MS(n) of protonated isomeric peptides and [M + H - Boc + H](+) produce characteristic fragmentation involving the peptide backbone, the Boc-group, and the side chain. The positional isomers are differentiated from one another by the presence of y(n) (+), b(n) (+), and other fragment ions of different m/z values. It is observed that the peptides with beta-Caa at the N-terminus produce extensive fragmentation, whereas gamma-Caa gave rise to much less fragmentation. Peptides with gamma-Caa at the N-terminus lose NH(3), whereas this process is absent for the carbopeptides with beta-Caa at the N-terminus. Two pairs of dipeptide diastereomers are clearly differentiated by the collision-induced dissociation (CID) of their protonated molecules. The loss of 2-methylprop-1-ene is more pronounced for Boc-NH-(R)-beta-Caa-(R)-gamma-Caa-OCH(3) (6) and Boc-NH-(R)-gamma-Caa-(R)-beta-Caa-OCH(3) (12), whereas it is insignificant or totally absent for its protonated diastereomeric pair Boc-NH-(S)-beta-Caa-(S)-gamma-Caa-OCH(3) (1) and Boc-NH-(S)-gamma-Caa-(S)-beta-Caa-OCH(3) (7). Further, ESI negative ion tandem mass spectrometry has also been found to be useful for differentiating these isomeric peptide acids. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Initiation of free‐radical polymerization by peroxypivalates studied by electrospray ionization mass spectrometry

Initiation by tert‐butyl peroxypivalate (TBPP), tert‐amyl peroxypivalate (TAPP), 1,1,3,3‐tetramethylbutyl peroxypivalate (TMBPP), or 1,1,2,2‐tetramethylpropyl peroxypivalate (TMPPP) of radical polymerization of methyl methacrylate in toluene solution at 90 °C was studied via polymer end‐group analysis using electrospray ionization mass spectrometry (ESI‐MS). Conclusive peak assignments allowed for measuring the type and concentration of the fragments that actually initiate macromolecular growth after thermal decomposition of these peroxypivalates. It was found that the pivaloyloxy radical moiety undergoes instantaneous decarboxylation to yield an initiating tert‐butyl radical. The alkoxy radical moiety, on the other hand, may generate, via β‐scission reaction, different types of carbon‐centered radicals (together with a ketone) or may undergo a 1,5‐H‐shift reaction, by which reaction an oxygen‐centered radical is transformed into a carbon‐centered hydroxy radical. This hydrogen shift reaction was found in case of TMBPP. Surprisingly, no evidence for initiating alkoxy radicals could be found, not even in case of initiation by TBPP, where the intermediate tert‐butoxy radical undergoes a rapid chain‐transfer reaction with the solvent toluene. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 4266–4275, 2004     

电喷雾离子源中样品离子化能量转移理论的初探

电喷雾离子源（electrospray ionization,ESI）不仅可以用于小分子的检测,也能够用于蛋白质、多肽等大分子的研究。本文通过对离子化过程的系统分析,提出了基于能量最低原理的离子化过程能量转移理论。样品分子在由液相转移到气相形成离子化气体的过程中受到静电力、分子间的范德华力等多种力的作用。样品的离子化是多种力共同作用的结果,在不同的离子化阶段,不同形式的力的作用也不尽相同。电荷在样品表面蒸发和多电荷离子的形成之间存在竞争。对不同结构的分子,分子形态、构象改变导致的两相间转移Gibbs自由能变化不同,可能导致离子蒸发、大分子形成多电荷离子、产生链弹射等行为。离子化能量转移理论不仅能够对已有的3种理论加以简化统一,也可以说明溶剂、电解质离子等在离子化过程中的作用,为优化不同结构与形态样品的质谱检测、了解离子化的真实过程提供了一种可能的依据。     

Mass spectrometry in coupling with affinity capture-release and isotope-coded affinity tags for quantitative protein analysis

Affinity capture-release electrospray ionization mass spectrometry (ACESIMS) and isotope-coded affinity tags (ICAT) are two recently introduced techniques for the quantitation of protein activity and content with applications to clinical enzymology and functional proteomics, respectively. One common feature of these methods is that they use biotinylated tags that function as molecular handles for highly selective and reversible affinity capture of conjugates from complex biological mixtures such as cell homogenates and sub-cellular organelles. ACESIMS uses synthetic substrate conjugates specifically to target cellular enzymes that, when deficient, are the cause of genetic diseases. Multiplex determination of enzyme activities is used for the diagnosis of lysosomal storage diseases. The ICAT method relies on selective conjugation of cysteine thiol groups in proteins, followed by enzymatic digestion and quantitative analysis of peptide conjugates by mass spectrometry. Another common feature of the ACESIMS and ICAT approaches is that both use conjugates labeled with stable heavy isotopes as internal standards for quantitation. Selected applications of the ACESIMS and ICAT techniques are presented that include molecular-level diagnosis of genetic diseases in children and quantitative determination of protein expression in cells.     

Ionene-dynamically coated capillary for analysis of urinary and recombinant human erythropoietin by capillary electrophoresis and online electrospray ionization mass spectrometry

In this article, a series of ionene polymers were synthesized and used to coat fused-silica capillaries for the separation of recombinant and urinary human erythropoietin (rhEPO and uEPO) standards by CE. The influence of the charge density of coatings on the separation of rhEPO and uEPO glycoforms was investigated. Then, we further studied the method for fast separation and detection of rhEPO and uEPO standards by CE-ESI-MS. The influence of several CE and MS operating parameters, such as the concentration of CE running buffer, applied external pressure, and the composition and flow rate of sheath liquid on CE-ESI-MS was studied. The results demonstrated that when the capillary was permanently coated with 6,6-ionene and the pH value of acetic acid-ammonium acetate running buffer was 4.80 and 5.50, respectively, a significantly reproducible separation was achieved for rhEPO and uEPO glycoforms. In the online CE-ESI-MS experiments, we not only achieved the online MS signal of uEPO, but also obtained baseline separation of three major rhEPO glycoforms successfully and reproducibly on the 6,6-ionene-coated capillaries. Furthermore, the standard mixture of rhEPO and uEPO was separated, and two incompletely resolved peaks that were identified to be rhEPO and uEPO by the unique MS "fingerprint" were obtained. Additionally, the molecular weight of rhEPO and uEPO were verified and compared to the results by MALDI-TOF-MS. It can be concluded that, in contrast to other indirect methods, the online CE-ESI-MS technique with the combination of the advantages of both CE and MS shows great potential for the separation and detection of rhEPO doping directly in competitive sports.     

基质辅助激光解吸电离质谱和电喷雾电离质谱在辣根过氧化物酶糖肽结构分析中的应用

糖链结构的质谱解析是今后糖蛋白分析中的重要研究内容,其中完整糖肽的分析,由于可以同时获得糖基化位点和对应糖链的结构信息,更具有重要意义和研究前景。本工作对质谱软电离技术在完整糖肽分析中的应用进行了研究,其中包括了基质辅助激光解吸电离(matrix-assisted laser desorption ionization, MALDI)和电喷雾电离(electrospray ionization, ESI)技术。通过平行使用两种串联质谱(tandem mass spectrometry, MS/MS)分析策略: MALDI-MS/MS和ESI-MS/MS对目标糖蛋白——辣根过氧化物酶进行分析,并讨论了其互补性。结果表明,MALDI和ESI技术各有优劣,结合串联质谱分析,可获得糖肽的糖链结构信息;两条路线互补使用,在揭示蛋白质糖基化修饰(位点和结构)的研究中十分必要。     

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.     

二肽衍生物的电喷雾质谱研究

基于HIV整合酶核心结构域,合成了以HIV整合酶为靶标的二肽衍生物,采用多级质谱技术（二级、三级）研究二肽衍生物在质谱条件下的化学键断裂途径,发现主要的断裂方式为：氨基与羰基间的NH-CO键的断裂以及N-(苯并噻唑-2-基)甲酰氨基与亚甲基间的CO-C间的断裂。     

自建电喷雾解吸电离源（DESI）在大分子分析中的应用及离子化机理探索

自建了简易的电喷雾解吸电离源(DESI),优化了DESI源喷口的位置和角度,并将其用于常见多肽和蛋白质的分析。多肽和小质量蛋白质(<20 kDa)可以容易地从表面解吸电离,生成清晰的质谱。而牛血清白蛋白(66.4 kDa)不能产生清晰的多电荷分布的质谱,说明当前DESI源的设计可能存在一个电离的分子量上限。通过比较不同的实验条件并对比ESI-MS,发现溶剂分子的挥发过程对电荷分布以及峰宽均有显著影响,可能是由于ESI更软引起。载样表面的性质对DESI-MS的信号强度有较大影响。金表面的自组装单分子膜(SAM)相对于纯金表面有较好的绝缘性,并有助于产生较强信号,说明来自表面的电子转移(电中和)是电喷雾解吸电离过程中的一个重要因素。该文的研究有助于对DESI-MS的实验条件和载样表面的选择,同时增进了对电喷雾解吸电离机理的了解。     

Study of the Transient Reactive Pd(IV) Intermediate in the Pd(OAc)2‐Catalyzed Oxidative Coupling Reaction System by Electrospray Ionization Tandem Mass Spectrometry

Pd‐catalyzed oxidative coupling reaction was of great importance in the aromatic C? H activation and the formation of new C? O and C? C bonds. Sanford has pioneered practical, directed C? H activation reactions employing Pd(OAc)₂ as catalyst since 2004. However, until now, the speculated reactive Pd(IV) transient intermediates in these reactions have not been isolated or directly detected from reaction solution. Electrospray ionization tandem mass spectrometry (ESI‐MS/MS) was used to intercept and characterize the reactive Pd(IV) transient intermediates in the solutions of Pd(OAc)₂‐catalyzed oxidative coupling reactions. In this study, the Pd(IV) transient intermediates were detected from the solution of Pd(OAc)₂‐catalyzed oxidative coupling reactions by ESI‐MS and the MS/MS of the intercepted Pd(IV) transient intermediate in reaction system was the same with the synthesized authentic Pd(IV) complex. Our ESI‐MS(/MS) studies confirmed the presence of Pd(IV) reaction transient intermediates. Most interestingly, the MS/MS of Pd(IV) transient intermediates showed the reductive elimination reactivity to Pd(II) complexes with new C? O bond formation into product in gas phase, which was consistent with the proposed reactivity of the Pd(IV) transient intermediates in solution.     

Internal energy distribution of peptides in electrospray ionization : ESI and collision-induced dissociation spectra calculation

The internal energy of ions and the timescale play fundamental roles in mass spectrometry. The main objective of this study is to estimate and compare the internal energy distributions of different ions (different nature, degree of freedom 'DOF' and fragmentations) produced in an electrospray source (ESI) of a triple-quadrupole instrument (Quattro I Micromass). These measurements were performed using both the Survival Yield method (as proposed by De Pauw) and the MassKinetics software (kinetic model introduced by Vékey). The internal energy calibration is the preliminary step for ESI and collision-induced dissociation (CID) spectra calculation. meta-Methyl-benzylpyridinium ion and four protonated peptides (YGGFL, LDIFSDF, LDIFSDFR and RLDIFSDF) were produced using an electrospray source. These ions were used as thermometer probe compounds. Cone voltages (V(c)) were linearly correlated with the mean internal energy values () carried by desolvated ions. These mean internal energy values seem to be slightly dependent on the size of the studied ion. ESI mass spectra and CID spectra were then simulated using the MassKinetics software to propose an empirical equation for the mean internal energy () versus cone voltage (V(c)) for different source temperatures (T): < E(int) > = [405 x 10(-6) - 480 x 10(-9) (DOF)] V(c)T + E(therm)(T). In this equation, the E(therm)(T) parameter is the mean internal energy due to the source temperature at 0 V(c).     

Electrospray mass spectrometry and molecular modeling study of formation and stability of silver complexes with diazaperylene and bisisoquinoline

The complex formation of the following diazaperylene ligands (L) 1,12‐diazaperylene 1 , 1,1′‐bisisoquinoline 2 , 2,11‐disubstituted 1,12‐diazaperylenes (alkyl = methyl, ethyl, isopropyl, 3 , 5 , 7 ), 3,3′‐disubstituted 1,1′‐bisisoquinoline (alkyl = methyl, ethyl, isopropyl, 4 , 6 , 8 and with R = phenyl, 11 and with pyridine 12 ), and the 5,8‐dimethoxy‐substituted diazaperylene 9 , 6,6′‐dimethoxy‐substituted bisisoquinoline 10 with AgBF4 was investigated. Collision‐induced dissociation measurements were used to evaluate the relative stabilities of the ligands themselves and for the [1:1]⁺ complexes as well as for the homoleptic and heteroleptic silver [1:2]⁺ complexes in the gas phase. This method is very useful in rapid screening of the stabilities of new complexes in the gas phase. The influence of the spatial arrangement of the ligands and the type of substituents employed for the complexation were examined. The effect of the preorganization of the diazaperylene on the threshold activation voltages and thus of the relative binding energies of the different complexes are discussed. Density functional theory calculations were used to calculate the optimized structures of the silver complexes and compared with the stabilities of the complexes in the gas phase for the first time.