Separation and characterization of multicomponent peptide mixtures by liquid chromatography-electrospray ionization mass spectrometry. Application to crude products of the synthesis of leuprolide

Leuprolide is a synthetic structural analogue of luteinizing hormone-releasing hormone used for the treatment of a large number of diseases related with the regulation of sexual hormones. Solid-phase peptide synthesis is used to obtain leuprolide peptidic hormone, but this synthetic procedure results in complex mixtures that need separation and characterization. Here, liquid chromatography coupled with mass spectrometry using electrospray ionization, (LC–ES-MS), was used for the separation and characterization of multicomponent peptide mixtures of crudes of synthesis of leuprolide. To optimize the LC separation process, the method of linear solvation energy relationships was applied and the powerful coupling LC–ES-MS permitted rapid and reliable characterization of the reaction product.     

Novel phosphoryl derivatization method for peptide sequencing by electrospray ionization mass spectrometry

A one-step phosphoryl derivatization method has been used in a peptide sequencing procedure for electrospray ionization tandem mass spectrometry (ESI-MS/MS). The sodiated derivatized peptides exhibit very simple dissociation patterns, in which two kinds of fragment ions, [b(n) + OH + Na]+ and [a(n) + Na]+, are formed. Since the amino acid residues are lost sequentially from the C-terminus, peptide sequences can be identified easily. The fragmentation efficiency of peptides increased as a result of the phosphorylation, and also provided peaks of useful intensity at lower m/z. A peptide with lysine at the C-terminus was derivatized and analyzed by ESI-MS/MS. Similar mass spectra, from which the sequence could be read out, were obtained. This is a novel derivatization method yielding neutral derivatives that should be suitable for peptide sequencing by LC/ESI-MS/MS.     

A fast and reliable method for the quantitative determination of benzimidazoles and metabolites in milk by LC-MS/MS with on-line sample treatment

A method to detect the presence of common wheat in durum wheat flour samples was developed and tested. Flour samples, or ground wheat samples, were digested by pepsin and chymotrypsin, and the peptide mixture obtained was analyzed by LC/ESI-MS and LC/ESI-MS/MS, which led to the identification of two marker peptides. One peptide was coded only in the DD genome, and thus present only in common wheat; the second was present in all wheat samples (both common and durum), so it was used as marker of the total wheat content. The ratio of the chromatographic areas of these two peptides, as determined by LC/ESI-MS, was related to the proportion of common wheat in the sample using a calibration curve that was constructed with standards of known composition. The proportions of common wheat in samples obtained by mixing different common and durum wheat varieties were accurately determined by this method. Finally, the method was applied in a survey of several durum wheat flour brands present on the Italian market. The results of the survey revealed that contamination of durum wheat flour with common wheat is commonplace.     

Quantitation of human glutathione S-transferases in complex matrices by liquid chromatography/tandem mass spectrometry with signature peptides

Direct quantitation of glutathione S-transferase (GST) isoforms [alpha (GST-A) and micro (GST-M)] in human liver cytosol was achieved by liquid chromatography/tandem mass spectrometry (LC/ESI-MS/MS) analysis of signature peptides of GST-A and GST-M and their corresponding stable isotopic peptide internal standards via multiple reaction monitoring (MRM). The selection of signature peptides was performed via trypsin digestion of commercially available cDNA-expressed GST-A1 and GST-M1, followed by LC/ESI-MS/MS with an ion trap mass spectrometer and sequencing with the TurboSEQUEST application. Quantitative analysis of the selected signature peptides in the multi-reaction monitoring (MRM) mode was performed using a triple-quadruple mass spectrometer. A series of human cytosol samples was quantitatively analyzed for levels of GST-A and GST-M. The total level of GST-A and GST-M obtained from this LC/ESI-MS/MS method was well correlated with the total level of GST determined by the 1-chloro-2,4-dinitrobenzene (CDNB) method.     

Investigation of synthetic peptide hormones by liquid chromatography coupled to pneumatically assisted electrospray ionization mass spectrometry: analysis of a synthesis crude of peptide triptorelin

Triptorelin, a synthetic peptide hormone used in the treatment of prostate cancer by means of reduction in the action of male hormone testosterone, is studied here. The synthetic procedure commonly results in unwanted side products that require extensive purification and characterization of the synthesis mixture. The chromatographic separation of triptorelin from the crude mixture was developed by applying the linear solvation energy relationship (LSER) methodology previously developed, to optimize the composition of the mobile phase in order to avoid lengthy empirical optimization procedures. Electrospray ionization mass spectrometry coupled to liquid chromatography (LC/ES-MS) was used to obtain reliable information on the inevitable side products. The knowledge of the identity of these impurities allows fast optimization of the synthetic procedure and also the therapeutic use of triptorelin peptide hormone.     

Separation and identification of peptide mixtures in a synthesis crude of carbetocin by liquid chromatography/electrospray ionization mass spectrometry

In order to separate and characterize the target peptide and the side-product peptide compounds of a synthesis crude of the peptide hormone carbetocin, liquid chromatography coupled to high-flow electrospray ionization mass spectrometry (LC/eS-MS) has been used. Carbetocin is an important drug with recognized therapeutical application for stimulation of uterine contractions to facilitate parturition. Stepwise solid phase peptide synthesis (SPPS) commonly results in unwanted side products associated with incomplete peptide chains. Consequently, this procedure requires extensive purification and characterization of the final synthesis crude. The linear solvation energy relationship (LSER) method has been applied to optimize the proportion of organic modifier of the mobile phase used in the established LC method. On the other hand, ES-MS has allowed rapid and reliable identification of the target peptide and the other impurities present in the carbetocin synthesis products.     

Structural analysis of naphthoquinone protein adducts with liquid chromatography/tandem mass spectrometry and the scoring algorithm for spectral analysis (SALSA)

The relative reactivities of various naphthoquinone isomers (1,4-, 1,2- and 2-methyl-1,4-naphthoquinone) to two test proteins, apomyoglobin and human hemoglobin, were evaluated via liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The structural characterization of the resulting adducts was also obtained by LC/ESI-MS analysis of the intact proteins. The reactive sites of apomyoglobin and human hemoglobin with 1,4-naphthoquinone and 1,2-naphthoquinone were also identified through characterization of adducted tryptic peptides by use of high-pressure liquid chromatography/electrospray ionization with tandem mass spectrometry (HPLC/ESI-MS/MS), TurboSEQUEST, and the scoring algorithm for spectral analysis (SALSA). Four adducted peptides, which were formed by nucleophilic addition of a lysine amino acid residue to 1,4-naphthoquinone, were also identified, as was an adducted peptide from incubation of 1,2-naphthoquinone with apomyoglobin. In the case of incubation of human hemoglobin with the two naphthoquinones, two adducted peptides were identified from the N-terminal valine modification of the alpha and beta chains of human hemoglobin. The adducted protein formation may imply that naphthalene produces its in vivo toxicity through 1,2- and 1,4-naphthoquinone metabolites reacting with biomolecular proteins.     

Selective separations of peptides with sequence deletions, single amino acid polymorphisms, and/or epimeric centers using macrocyclic glycopeptide liquid chromatography stationary phases

Separating closely related peptides (those differing by one or two amino acids or the chirality of a single amino acid) can be challenging using reversed-phase liquid chromatography (LC), ion-exchange LC, or using ion-pairing agents. Also, the mobile phases that give the best separations in these modes may not be electrospray ionization mass spectrometry (ESI-MS) compatible. Forty-two peptides from 11 peptide families were separated on three macrocyclic glycopeptide stationary phases in reverse-phase mode using ESI-MS-compatible mobile phases. The peptide classes studied were angiotensin, bradykinin, alpha-bag cell factor, beta,gamma-bag cell factor, beta-casomorphin, dynorphin, enkephalin, leucokinin, lutinizing hormone releasing hormone, neurotinsin, substance P, and vasopressin. High selectivity was observed for single amino acid substitutions (achiral and chiral) regardless of the position of the substitution in the sequence. Mobile phase optimization, its effect on peptide elution behavior, and chromatographic efficiency is also discussed. Using LC-ESI-MS, a 2 ng limit of detection was obtained, two orders of magnitude lower than the UV detection limit.     

On-line preconcentration and quantitative analysis of peptide hormone of brain and intestine using on-column transient isotachophoresis coupled with capillary electrophoresis/electrospray ionization mass spectrometry

A new approach was described to achieve very sensitive analysis of peptide hormone of brain and intestine by capillary electrophoresis coupled with a transient isotachophoresis (tITP) preconcentration method. The system used was electrospray ionization mass spectrometry (ESI-MS) as detector and equipped with a sheath flow configuration. The effects of sample matrix, pH and concentration of leading electrolyte (LE), sample injection time, and ESI-MS instrumental parameters on the efficiency of the sample stacking were investigated in detail. Under the optimized conditions, lower than micromole (0.01 microM) concentrations of the peptides were easily detected. Compared to traditional hydrodynamic injection methods, about 40-230-fold increase in detection sensitivity was obtained by this technique. A distinguishing feature of the described approach is that the background electrolyte can serve as terminating electrolyte (TE), which simplifies the process of the experiments. The method was further evaluated by the analysis of low concentration active peptide mixtures spiked in hypothalamus tissue of the rat.     

Determination of peptide hormones of brain and intestine by CE with ESI-MS detection

A new method for the determination of the peptide hormones of brain and intestine based on CE coupling with a DAD and ESI-MS was established. Several electrophoretic and ESI-MS parameters were investigated in detail, such as electrolyte nature and concentration, organic solvent and sheath liquid compositions, nebulization gas pressure and the ESI capillary voltage. Optimized conditions were achieved with 25 mM formic acid-ammonium formate (pH 2.9) as the optimal electrolyte, 2 mM formic acid in 80% methanol in water as the sheath liquid, and 20 kV applied voltage. Under the optimized conditions, four protonated peptides were separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath flow ESI interface. LODs for the four peptides (neurotensin hexapeptide, neurotensin, cholecystokinin tetrapeptide, and pentagastrin) were in the range of 0.10-0.60 micromol/L at an S/N of 3. The RSDs (n = 8) of the method were 0.70-1.5% for migration times and 1.6-6.1% for peak areas. This method is simple, rapid, and selective compared with RIA and ELISA techniques, and has been applied to the analysis of rat hypothalamus tissue.     

Analysis of ceramides in cosmetics by reversed-phase liquid chromatography/electrospray ionization mass spectrometry with collision-induced dissociation

A rapid, sensitive and selective method involving reversed-phase liquid chromatography (LC) with electrospray ionization (ESI) mass spectrometry (MS) was employed for determination of commercial ceramides in cosmetics for quality control of the product formulation. Using this LC/ESI-MS technique, simultaneous separation and characterization of ceramides and an impurity substance were possible. Informative fragmentation patterns were obtained by employing LC/ESI-MS in both positive and negative ionization modes to identify the structures of both sphingoid base and N-acyl chains of ceramides, and also of an impurity. The combination of positive and negative mass spectra can be used for unambiguous confirmation of ceramides and for characterization of unknown species. In-source collision-induced fragmentation resulted in characteristic product anions for the ceramides containing a phytosphingosine moiety at m/z 267, 255 and 225, and for those with a sphingosine moiety at m/z 263 and 237, regardless of the length of the fatty acyl chains. The detection limit was about 0.5 pmol in selected-ion monitoring mode. Quantification using internal standards showed good linearity and a relative standard deviation of 4%. These ceramides were more sensitively detected in positive than in negative ion mode.     

Study of non-covalent complexation between catechin derivatives and peptides by electrospray ionization mass spectrometry

The recent development of electrospray ionization mass spectrometry (ESI-MS) has allowed its use to study molecular interactions driven by non-covalent forces. ESI-MS has been used to detect non-covalent complexes between proteins and metals, ligands and peptides and interactions involving DNA, RNA, oligonucleotides and drugs. Surprisingly, the study of the interaction between polyphenolic molecules and peptides/proteins is still an area where ESI-MS has not benefited. With regard to the important influence of these interactions in the biological and food domains, ESI-MS was applied to the detection and the characterization of soluble polyphenol-peptide complexes formed in model solution. The ability to observe and monitor the weak interactions involved in such macromolecular complexation phenomena was demonstrated for monomeric and dimeric flavonoid molecules (catechin-derived compounds) largely encountered in plants and plant derived products. Intact non-covalent polyphenol-peptide complexes were observed by ESI-MS using different experimental conditions. Utilizing mild ESI interface conditions allowed the detection of 1 : 1 polyphenol-peptide complexes in all tested solutions and 2 : 1 complexes for the dimers and galloylated polyphenols (flavanols). These results show that there is a preferential interaction between polymerized and/or galloylated polyphenols and peptide compared with that between monomeric polyphenols and peptides. Thus, ESI-MS shows potential for the study of small polyphenolic molecule-peptide interactions and determination of stoichiometry.     

Evaluation of chromatographic versus electrophoretic behaviour of a series of therapeutical peptide hormones

In this work, models describing the effect of pH on chromatographic and electrophoretic behaviour for a series of polyprotic therapeutic peptide hormones were compared. taking into account the species in solution and the activity coefficients. The usefulness of the proposed equations is twofold, they permit the determination of the acidity constants in water and in the hydroorganic mobile phases used in liquid chromatography (LC) and capillary electrophoresis (CE) and can also be used for the selection of the optimum pH for the separation of mixtures of the modelled compounds. The proposed relationships allow an important reduction of the experimental data needed for the development of new separation methods. The accuracy of the proposed equations is verified by modelling the chromatographic and electrophoretic behaviour of a series of polyprotic therapeutic peptide hormones. By calculating the values of predicted resolutions, selection of the optimum pH to perform LC or CE separations of their mixtures becomes a rapid and simple process.     

Liquid chromatography-electrospray mass spectrometry of multicomponent peptide mixtures. Characterization of a mixture from the synthesis of the hormone goserelin

In order to separate and characterize the target peptide and the side-product peptide compounds of a synthesis mixture of the peptide hormone goserelin, liquid chromatography coupled to high-flow electrospray ionization mass spectrometry (LC-ES-MS) has been used. Goserelin is an important drug with recognized therapeutical application for palliative treatment of prostatic and breast carcinomas. Stepwise solid-phase peptide synthesis commonly results in unwanted side-products associated with incomplete peptide chains. Consequently, this procedure requires extensive purification and characterization of the final synthesis mixture. The method of linear solvation energy relationships has been applied to optimize the proportion of organic modifier of the mobile phase used in the established LC method. On the other hand, ES-MS has allowed rapid and reliable identification of the target peptide and the other impurities present in the goserelin synthesis products.     

Simultaneous detection of type A and type B trichothecenes in cereals by liquid chromatography-electrospray ionization mass spectrometry using NaCl as cationization agent

A LC/MS method for the simultaneous determination of both type A and type B trichothecenes by using an electrospray ionization (ESI) interface in the positive ionization mode with a single quadrupole analyzer is described. In order to enhance the ionization of both groups of trichothecenes, the sodium ion was used as cationization agent by adding sodium chloride to the eluent. All LC/MS parameters were optimized. The newly developed LC/ESI-MS method was applied to the analysis of a wheat reference material and cereal-based foods and feeds.     

Separation of potentially therapeutic peptide hormones by liquid chromatography. Optimisation of the composition and pH of the mobile phase

The aim of this work is to optimise the proportion of the organic modifier and the pH of the mobile phase, in order to separate a series of peptide hormones with therapeutic interest in the molecular mass range from 500 to 6000. The composition of the mobile phase was optimised by establishing relationships between retention parameters and either the scale of solvent polarity, or the Kamlet–Taft multiparameter solvent scale of the eluent, using linear solvation energy relationships. Likewise, linear correlations between the chromatographic retention and Reichardt’s E^N_T parameter were obtained. These relationships allowed an important reduction of the experimental retention data needed for developing a given separation. In addition, a model describing the effect of the correctly measured pH of the mobile phase on retention in LC was established and tested for the series of selected peptides using an octadecylsilica column. The proposed equations permit the prediction of the optimum pH and also permit the determination of the acidity constants of the peptides in the hydro-organic mixtures using a minimum number of measurements.     

Determination of peanut allergens in cereal-chocolate-based snacks: metal-tag inductively coupled plasma mass spectrometry immunoassay versus liquid chromatography/electrospray ionization tandem mass spectrometry

A comparison of two methods for the identification and determination of peanut allergens based on europium (Eu)-tagged inductively coupled plasma mass spectrometry (ICP-MS) immunoassay and on liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) with a triple quadrupole mass analyzer was carried out on a complex food matrix like a chocolate rice crispy-based snack. The LC/MS/MS method was based on the determination of four different peptide biomarkers selective for the Ara h2 and Ara h3/4 peanut proteins. The performance of this method was compared with that of a non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) method with ICP-MS detection of the metal used to tag the antibody for the quantitative peanut protein analysis in food. The limit of detection (LOD) and quantitation of the ICP-MS immunoassay were 2.2 and 5 microg peanuts g(-1) matrix, respectively, the recovery ranged from 86 +/- 18% to 110 +/- 4% and linearity was proved in the 5-50 microg g(-1) range. The LC/MS/MS method allowed us to obtain LODs of 1 and 5 microg protein g(-1) matrix for Ara h3/4 and Ara h2, respectively, thus obtaining significantly higher values with respect to the ELISA ICP-MS method, taking into account the different expression for concentrations. Linearity was established in the 10-200 microg g(-1) range of peanut proteins in the food matrix investigated and good precision (RSD <10%) was demonstrated. Both the two approaches, used for screening or confirmative purposes, showed the power of mass spectrometry when used as a very selective detector in difficult matrices even if some limitations still exist, i.e. matrix suppression in the LC/ESI-MS/MS procedure and the change of the Ag/Ab binding with matrix in the ICP-MS method.     

Structural characterization of novel chemotactic and mastoparan peptides from the venom of the social wasp Agelaiapallipes pallipes by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

High-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) techniques were applied for the detection, purification, monitoring, and sequencing of two novel and biologically active peptides occurring at very low levels in the venom of the wasp Agelaia pallipes pallipes. These peptides were sequenced under LC/ESI-MS/MS conditions and designated as Agelaia-CP (I/L-L-G-T-I-L-G-L-L-K-G-I/L-NH2, MW 1207.8 Da) and Agelaia-MP (I/L-N-W-L-K-L-G-K-A-I-I-D-A-I/L-NH2, MW 1565.0 Da). The peptide Agelaia-CP showed no hemolytic activity, but it behaved as a mast cell degranulator and induced a potent chemotaxis in polymorphonucleated leukocyte (PMNL) cells, typical of a wasp chemotactic peptide. The peptide Agelaia-MP showed both powerful mast cell degranulation and hemolysis of washed rat red blood cells, and is thus assigned as a new member of the mastoparan family of peptides. Both peptides seem to be directly involved in the strong inflammatory reactions associated with wasp stings.     

蛋白质组质谱分析中基于串并联支持向量机的肽段色谱保留时间预测方法

基于质谱的大规模蛋白质鉴定中,在线液相色谱分离发挥了重要作用。色谱保留时间(retention time,RT)是肽段鉴定和定量的重要信息。由于整个色谱分析运行时间中,流动相中的有机相采用了非线性浓度曲线以及样品中肽段之间的相互影响等因素,基于肽段序列的RT预测还存在精度不高、模型推广性能差等问题。本文提出了一种基于串并联支持向量机(serial and parallel support vector machine,SP-SVM)的RT预测方法,能够表征洗脱过程中有机相浓度的非线性变化和肽段之间的相互影响,显著提高了肽段保留时间预测的精度。利用复杂样本数据集验证结果表明,预测RT和实验RT之间的决定系数达到了0.95,超过95%的鉴定肽段的RT预测误差范围小于总运行时间的20%,超过70%的鉴定肽段的RT预测误差范围小于总运行时间的10%。本文提出的模型的性能达到了目前已知的最好水平。     

Analysis of sulfated peptides from the skin secretion of the Pachymedusa dacnicolor frog using IMAC-Ga enrichment and high-resolution mass spectrometry

Immobilized metal ion affinity chromatography (IMAC) has been widely used for the enrichment of phosphopeptides, whereas no report exists describing the use of IMAC columns for the enrichment of sulfopeptides. In this study, we used IMAC-Ga microcolumns for the enrichment of sulfopeptides from a complex mixture of peptides, extracted from skin secretions of the Pachymedusa dacnicolor frog. The enriched fraction obtained by IMAC-Ga was analyzed by liquid chromatograpy/electrospray ionization mass spectrometry (LC/ESI-MS) in an Orbitrap XL and by matrix-assisted laser desorption/ionization time-of-flight time-of-flight (MALDI-TOF/TOF) in an ABI 4800 instrument. From this fraction, different sulfated and non-sulfated peptides belonging to the caerulin and bradykinin families were structurally characterized. Other interesting negatively charged groups, such as phosphate adducts of dermaseptins and pyridoxal phosphate attached to a protease inhibitor, were also characterized. Unexpectedly, some dermaseptin antimicrobial peptides were also enriched by IMAC-Ga and a Sauvatine-like peptide was also fully sequenced. Furthermore, neutral loss of sulfated peptides and their fragmentation patterns in the gas phase were also compared using collision-induced dissociation (CID) and high-energy collision dissociation (HCD). Our present study provides evidence that IMAC-Ga enrichment is a fast, useful and promising method for high-throughput analysis of sulfated-peptides, since high-resolution mass spectrometers can be used for this purpose.