Direct MALDI‐MS analysis of the disulfide bonds in peptide using thiosalicylic acid as a reactive matrix

The ability of a thiol‐containing molecule, thiosalicylic acid (TSA), to function as a reactive matrix for matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry analysis of peptides has been investigated. Although TSA has reducing characteristics, the use of TSA did not cause a reduction‐induced MALDI in‐source decay, probably because of the weak interactions between the thiol group in TSA and the carboxyl oxygen in the peptide. In contrast, when peptides containing disulfide bonds were analyzed by MALDI with TSA as the matrix, the disulfide bond was partially cleaved owing to the reaction with TSA, producing TSA‐adducted peptides. The reaction between the disulfide bond and TSA was suggested to be occurred in solution. The comparison of the MALDI mass spectra obtained using conventional matrix and TSA allows us to count the number of disulfide bonds in the peptides. Copyright © 2017 John Wiley & Sons, Ltd.     

Fragmentation of intra-peptide and inter-peptide disulfide bonds of proteolytic peptides by nanoESI collision-induced dissociation

Characterisation and identification of disulfide bridges is an important aspect of structural elucidation of proteins. Covalent cysteine-cysteine contacts within the protein give rise to stabilisation of the native tertiary structure of the molecules. Bottom-up identification and sequencing of proteins by mass spectrometry most frequently involves reductive cleavage and alkylation of disulfide links followed by enzymatic digestion. However, when using this approach, information on cysteine-cysteine contacts within the protein is lost. Mass spectrometric characterisation of peptides containing intra-chain disulfides is a challenging analytical task, because peptide bonds within the disulfide loop are believed to be resistant to fragmentation. In this contribution we show recent results on the fragmentation of intra and inter-peptide disulfide bonds of proteolytic peptides by nano electrospray ionisation collision-induced dissociation (nanoESI CID). Disulfide bridge-containing peptides obtained from proteolytic digests were submitted to low-energy nanoESI CID using a quadrupole time-of-flight (Q-TOF) instrument as a mass analyser. Fragmentation of the gaseous peptide ions gave rise to a set of b and y-type fragment ions which enabled derivation of the sequence of the amino acids located outside the disulfide loop. Surprisingly, careful examination of the fragment-ion spectra of peptide ions comprising an intramolecular disulfide bridge revealed the presence of low-abundance fragment ions formed by the cleavage of peptide bonds within the disulfide loop. These fragmentations are preceded by proton-induced asymmetric cleavage of the disulfide bridge giving rise to a modified cysteine containing a disulfohydryl substituent and a dehydroalanine residue on the C-S cleavage site.     

Fragmentation of peptides with intra-chain disulfide bonds in triple quadrupole mass spectrometry and its quantitative application to biological samples

A growing number of peptides are being used today in bioanalytical laboratories. Because of this, there is an increasing interest in the development of highly sensitive, specific and robust liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays for the quantitative analysis of peptides in biological samples. Among the mass spectrometers previously used for peptide quantification, triple quadrupole mass spectrometers are generally not considered the instrument of choice. With this instrumentation, collision cascades or multiple fragmentations tend to generate multiple peaks that have weak intensities. This leads to a loss in detection sensitivity. However, in cases where immonium product ions were formed in abundance, it was found that peptide quantification succeeded. A common feature of these peptides is their intra-loop structure. To elucidate the usefulness of this feature in fragmentation, several peptide analytes with intra-chain disulfide bonds were investigated in this study, including a newly synthesized analog having a single amino acid substitution. The results presented here indicate that abrupt bond cleavage from the intra-loop structure of peptides could be one of the premises for intense immonium ion generation. In contrast, any preferential cleavage of peptide bonds (e.g., proline effect) that gives rise to a linearized sequence would break the intactness of the loop and prevent it from completely dissociating. In addition, the utilization of immonium product ions in LC/MS/MS was demonstrated for the determination of peptides with intra-chain disulfide bonds in biological fluids.     

Synthesis and healing properties of poly(arylether sulfone)–poly(alkylthioether) multiblock copolymers containing disulfide bonds

Thermoplastic elastomers composed of soft and hard segments are important elastic and processable synthetic polymers. The microphase‐separated soft domains show low glass transition temperature and possess sufficient chain mobility at room temperature. In this study, we report the synthesis and healing properties of multiblock copolymers containing disulfide bonds as dynamic covalent bonds. The multiblock copolymers composed of poly(arylether sulfone) and poly(alkylthioether) segments were synthesized by oxidative coupling polymerization of the corresponding thiol‐terminated oligomers. Atomic force microscopy phase images, differential scanning calorimetry, and dynamic mechanical analysis curves indicated the microphase‐separated morphology of the multiblock copolymer. Self‐healing properties of the polymer were evaluated by changes in the elongation at break of the cut/adhered samples. The elongation recovery increased with UV irradiation time, and the multiblock copolymer showed a 93% recovery after UV irradiation for 5 h. The healing efficiency induced by UV irradiation, determined by subtracting the recovery without UV irradiation, was calculated to be 51%. According to the UV spectra and solubility changes after UV irradiation, the main healing factor in this study was the crosslinking reactions caused by thiyl radicals generated from UV irradiation instead of disulfide exchange reactions. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 3545–3553     

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.     

样品制备方法对高效液相色谱-串联质谱分析人乳内源肽的影响

人乳内源肽是乳蛋白在乳腺中被降解形成的具有生理功能的肽,是人乳的重要组成部分,研究人乳内源肽对于婴儿健康具有重要的意义.高效液相色谱-串联质谱(LC-MS/MS)联用技术的应用,促使人乳内源肽的研究取得了突破性的进展.人乳中内源肽含量低、干扰组分多,样品制备方法是影响分析结果的关键步骤.为了研究样品制备方法对分析结果的...     

Studying disulfide bond rearrangement by MALDI-RTOF PSD and MALDI-TOF/RTOF high-energy CID (20 keV) experiments of peptides derived from ammodytoxins

Ammodytoxins (Atxs) are presynaptically neurotoxic phospholipases present in Vipera ammodytes ammodytes snake venom. Atxs show a high sequence homology and contain 14 cysteines which form seven biologically relevant disulfide bridges-connecting non-neighboring cysteines. Formic acid cleavage was performed to confirm protein sequences by MALDI RTOF MS and resulted in 95.6% sequence coverage exhibiting only few formylations. Cysteine-containing peptides showed adjacent signals 2 and/or 4 Da lower (according to the number of cysteines present in the peptide) than the theoretical molecular weight indicating disulfide bridge rearrangement. Post-source decay (PSD) and high-energy collision-induced dissociation (CID) at 20 keV experiments showed fragmentation pattern unique for the reduced, thiol group containing and the oxidized, disulfide bridge harboring peptides. Besides typical low-energy fragment ions observed during PSD experiments (a-, b-, y-type ions), additional high-energy fragment ions (c-, x-, w-, d-type and internal fragments) of significant intensity were generated during fragmentation at 20 keV. In the case of charge directing N- and C-termini, x- and w-type ions were also observed during PSD. Good and up to complete sequence coverage was achieved for all studied peptides from Atxs in the case of high-energy CID, whereas PSD lacked information particularly for larger peptides.     

Hydrogen‐deuterium exchange of α‐carbon protons and fragmentation pathways in N‐methylated glycine and alanine‐containing peptides derivatized by quaternary ammonium salts

Recently, we developed a selective and efficient method of hydrogen‐deuterium exchange (HDX) at the α‐carbon (α‐C) of sarcosine residue (N‐methylglycine) in model peptides [B?chor et al. J. Mass Spectrom. 2014, 49, 43]. Here, we report the influence of quaternary ammonium (QA) group on HDX at the α‐C of sarcosine and N‐methylalanine in peptides. The obtained results suggest a significant acceleration of the HDX in sarcosine residue caused by the presence of QA. The effect depends on the distance between the sarcosine residue and QA moiety. The deuterons, introduced at α‐C, are resistant to the back‐exchange in acidic aqueous solution. The collision induced dissociation of the deuterium‐labeled analogs of QA‐tagged oligosarcosine peptides without mobile hydrogen revealed the mobilization of the hydrogens localized at α‐C of sarcosine residue. Copyright © 2014 John Wiley & Sons, Ltd.     

An improved LC‐MS/MS method for simultaneous determination of 1,5‐dicaffeoylquinic acid and its active metabolites in human plasma and its application to a pharmacokinetic study in patients

In this study, a sensitive, selective and reproducible liquid chromatography–tandem mass spectrometry method for the simultaneous determination of 1,5‐dicaffeoylquinic acid (1,5‐DCQA) and its active metabolites, 1‐caffeoyl‐5‐feruoylquinic acid and 1,5‐O‐diferuoylquinic acid, in human plasma, using puerarin as internal standard, was developed and validated. Analytes were extracted from plasma samples by liquid–liquid extraction with ethyl acetate, separated on a C₁₈ reversed‐phase column with water containing 5 mM ammonium acetate and acetonitrile as the mobile phase and detected by electrospray ionization mass spectrometry in negative selected reaction monitoring mode. The accuracy and precision of the method were acceptable and linearity was good over the range 1–200 ng/mL for each analyte. In addition, the selectivity, extraction recovery and matrix effect were satisfactory too. The validated LC‐MS/MS method was successfully applied to phase II clinical pharmacokinetic study of 1,5‐DCQA in patients. Copyright © 2010 John Wiley & Sons, Ltd.     

基于液相色谱-串联质谱法的肉类特征肽段鉴别及掺假测定

建立了液相色谱-串联质谱技术鉴别肉类特征肽段及定量检测羊肉中常见外源肉掺假的方法。样品经蛋白质提取、胰蛋白酶水解和固相萃取小柱净化后,利用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱（UPLC-Q/Exactive-HRMS）和Proteinpilot软件,实现蛋白质和多肽的鉴定;再通过基本局部比对搜索工具（BLAST）与Uniprot数据库对比分析,筛选出羊肉、鸭肉、猪肉和鸡肉的20个物种特征性多肽标志物;最后利用高效液相色谱-三重四极杆质谱（UPLC-QqQ-MS）系统对羊肉、鸭肉、猪肉和鸡肉的特征性多肽进行了验证和多反应监测（MRM）定量研究。将鸭肉、猪肉和鸡肉分别按照质量分数为1%、5%、10%、20%、50%的比例掺加到羊肉中,得到鸭肉最低掺假检出限为0.25%、猪肉最低掺假检出限为0.17%、鸡肉最低掺假检出限为0.10%。     

Rapid and sensitive identification of ricin in environmental samples based on lactamyl agarose beads using LC‐MS/MS (MRM)

Ricin, a plant‐derived toxin extracted from the seeds of Ricinus communis (castor bean plant), is one of the most toxic proteins known. Ricin's high toxicity, widespread availability, and ease of its extraction make it a potential agent for bioterrorist attacks. Most ricin detection methods are based on immunoassays. These methods may suffer from low efficiency in matrices containing interfering substances, or from false positive results due to antibody cross reactivity, with highly homologous proteins. In this study, we have developed a simple, rapid, sensitive, and selective mass spectrometry assay, for the identification of ricin in complex environmental samples. This assay involves three main stages: (a) Ricin affinity capture by commercial lactamyl‐agarose (LA) beads. (b) Tryptic digestion. (c) LC‐MS/MS (MRM) analysis of tryptic fragments. The assay was validated using 60 diverse environmental samples such as soil, asphalt, and vegetation, taken from various geographic regions. The assay's selectivity was established in the presence of high concentrations of competing lectin interferences. Based on our findings, we have defined strict criteria for unambiguous identification of ricin. Our novel method, which combines affinity capture beads followed by MRM‐based analysis, enabled the identification of 1 ppb ricin spiked into complex environmental matrices. This methodology has the potential to be extended for the identification of ricin in body fluids from individuals exposed (deliberately or accidentally) to the toxin, contaminated food or for the detection of the entire family of RIP‐II toxins, by applying multiplex format.     

Fast and selective extraction of chloramphenicol from soil by matrix solid‐phase dispersion using molecularly imprinted polymer as dispersant

The molecularly imprinted polymer (MIP) was synthesized and used as dispersant of matrix solid‐phase dispersion (MSPD) for the extraction of chloramphenicol (CAP) in soil samples. The satisfactory recovery of CAP was obtained by the optimized extraction conditions: 1:2 as the ratio of sample to MIPs; 5 min as the dispersion time; 30% aqueous methanol as washing solvent and methanol as elution solvent. The CAP extracted from soil was determined by LC‐MS/MS. The slight ion suppression phenomenon was observed for the CAP when the sample was cleaned up by MSPD with MIP as dispersant, when compared with C18 as MSPD dispersant, which caused significant ion suppression. LOD of CAP is 4.1 ng/g. RSDs of intra‐ and inter‐day tests ranging from 3.1 to 6.2% and from 3.9 to 8.3% are obtained. At all three fortified levels (20, 100 and 500 ng/g), recoveries of CAP are in the range of 86.9–92.6%. The effect of ageing time of spiked soil sample on the CAP recovery was examined. The CAP recovery decreased from 91.0 to 36.9% when the ageing time changed from 1 day to 4 wk.     

Behavior of peptides and computer-assisted optimization of peptides separations in a normal-phase thin-layer chromatography system with and without the addition of ionic liquid in the eluent

The addition of an ionic liquid into the mobile phase appeared to be useful in optimization of chromatographic separation of peptides. Different behavior of peptides in thin-layer chromatography (TLC) was observed after addition of 1-ethyl-3-methylimidazolium tetra fluoroborate to the eluent in comparison to the system without the ionic liquid. Nonlinear dependence of the retention coefficient, R(M), of peptides on the volume percentage of acetonitrile in the eluent was found in normal-phase TLC with and without immidazolium tetra fluoroborate in the mobile phase. In general, R(M) increased with increasing concentration of acetonitrile. In TLC systems without the ionic liquid, R(M) can be described well with a quadratic function. On the other hand, in a TLC system with an ionic liquid as the additive to the mobile phase, the retention behavior is better described with a third-degree polynomial function. The potential usefulness of ionic liquids for optimization of separation of peptides was demonstrated. Optimization of the separation conditions was supported by a commercially available computer program.     

磺化修饰结合离子交换色谱和生物质谱富集鉴定含组氨酸肽段

通过在肽段的N端引入磺酸基,从而使含组氨酸的肽段与其他肽段在pH<3.0的条件下产生电荷差异,建立了一种基于强阳离子交换色谱(SCX)结合生物质谱富集鉴定含组氨酸肽段的方法,并以含有组氨酸的标准蛋白质为模型,进行了方法学考察。结果表明,经N端磺酸化后,含组氨酸的肽段能有效地被阳离子交换色谱富集,且在肽的N端引入磺酸基促进了肽的裂解,使之产生简单而信息丰富的二级质谱图,从而得到完美的质谱鉴定结果。这说明磺化修饰结合强阳离子交换色谱用于含组氨酸肽段的富集鉴定是可行的,且具有在蛋白质组研究中应用的潜力。     

An LC–MS/MS method for simultaneous determination of 1,5‐dicaffeoylquinic acid and 1‐O‐acetylbritannilactone in rat plasma and its application to a pharmacokinetic study

A selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous quantitative determination of 1,5‐dicaffeoylquinic acid (1,5‐DCQA) and 1‐O‐ acetylbritannilactone (1‐O‐ ABL) in rat plasma. Chromatographic separation was performed on a Zorbax Eclipse XDB‐C₁₈ column using isocratic mobile phase consisting of methanol–water–formic acid (70:30:0.1, v /v/v) at a flow rate of 0.25 mL/min. The detection was achieved using a triple‐quadrupole tandem MS in selected reaction monitoring mode. The calibration curves of all analytes in plasma showed good linearity over the concentration ranges of 0.850–213 ng/mL for 1,5‐DCQA, and 0.520–130 ng/mL for 1‐O‐ ABL, respectively. The extraction recoveries were ≥78.5%, and the matrix effect ranged from 91.4 to 102.7% in all the plasma samples. The method was successfully applied for the pharmacokinetic study of the two active components in the collected plasma following oral administration of Inula britannica extract in rats.     

Rapid determination of yunaconitine and related alkaloids in aconites and aconite‐containing drugs by ultra high‐performance liquid chromatography–tandem mass spectrometry

Yunaconitine (YAC) is a toxic aconite alkaloid that is considered to be a hidden aconite poison since it is frequently found in body fluids from aconite poisoning patients, but has not been well studied in commonly used herbal drugs. In this paper, a rapid and sensitive ultra high‐performance liquid chromatography–tandem mass spectrometry (UHPLC‐MS/MS) detection combined with microwave‐assisted extraction (MAE) was developed for high throughput simultaneous determination of YAC and six other toxic aconite alkaloids in 31 samples of crude, processed aconites and aconite‐containing drugs. The optimized method showed excellent linearity, precision, accuracy and recovery for all target compounds with short run time. YAC was detected in some samples with contents from 0.015 to 10.41 mg/g. This is the first report on the determination of YAC in Radix Aconiti, Radix Aconiti Kusnezoffii and aconite‐containing drugs. This newly developed method facilitates the rapid screening of YAC and related toxic aconite alkaloids and allows YAC to be used as a chemical marker for the quality control of aconites and aconite‐containing drugs. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

Characterization of polyethylenimine by electrospray ionization and matrix-assisted laser desorption/ionization

Branched polyethylenimines (PEIs) with lower average molecular weights (600, 1200 and 1800 Da) have been studied by Electrospray Ionization (ESI) and Matrix‐Assisted Laser Desorption/Ionization (MALDI) mass spectrometry. In both, ESI and MALDI mass spectra, the main distribution arises from protonated PEI oligomers with NH₂ end groups, [PEI + H]⁺, which are observed at m/z 43n + 18. A trace of sodium contamination in the PEI samples results in the presence of a series that appears at m/z 43n + 40 [PEI + Na]⁺. However, only the MALDI mass spectra show a [PEI + K]⁺ series at m/z 43n + 56, because of matrix contamination with potassium, and a series generated by condensation of the matrix with PEI at m/z 43n + 30. Collisionally activated dissociation tandem mass spectrometry (CAD (MS/MS)) of protonated PEI oligomers is shown to yield three fragment ion series b_n, and K_n. The experiments have demonstrated the capabilities of these mass spectrometry techniques, along with CAD MS/MS to detect and characterize such polar synthetic polymers. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

Fast,selective, and sensitive analysis of low-abundance peptides in human plasma by electromembrane extraction

A totally new concept based on electrokinetic migration was evaluated for the extraction of three biologically active peptides from human plasma. Angiotensin 2, leu-enkephalin, and endomorphin 1 migrated from a diluted human plasma sample (2 mL, positive electrode), through a supported liquid membrane (SLM) of 1-octanol, di-isobutylketon, and di-(2-ethylhexyl) phosphate (DEHP) (55:35:10, w/w/w), and into an acidified acceptor solution (25 μL 50 mM HCl, negative electrode) by the application of an electrical potential (20 V) across the SLM. After only five min of extraction, the acceptor solution was injected and analyzed directly by liquid chromatography. The three peptides were quantified by tandem mass spectrometry, with acceptable linearity ranging from 100.0 to 1000.0 pg mL⁻¹ (r² in the range 0.9736–0.9988), and repeatability (RSD) ranging between 15% and 24% (n = 5), using plasma spiked with the three peptides in 100 pg mL⁻¹ concentration. The estimated detection limits (S/N ratio of 3:1) for angiotensin 2, leu-enkephalin, and endomorphin 1, were 60, 24, and 24 pg mL⁻¹, respectively. With this novel approach based on electromembrane extraction (EME) coupled to LC–MS/MS, endogenous concentrations of the peptides were detected in non-spiked human plasma samples, with a total analysis time less than 50 min. These experimental findings were highly interesting, and showed the opportunities for EME with regard to future peptide extractions.