Copper-induced oligomerization of peptides: a model study

In this work, copper-binding of the tetraglycine peptide (Gly-Gly-Gly-Gly) was studied by electrospray ionization mass spectrometry. Experiments were performed under alkaline conditions, in the presence of ethanolamine (pH 10.95). We observed that the presence of copper(II) ions induces the aggregation of the peptide and the formation of copper-bound complexes with higher molecular mass is favored, such as the oligomer complexes [3M+2Cu-3H](+) and [4M+3Cu-5H](+). At 1:1 peptide-copper(II) ion ratio, the singly charged [3M+2Cu-3H](+) oligomer complex is the base peak in the mass spectrum. Metal ion-induced oligomer-ization of neurotoxic peptides is well known in the literature; however, there are very few examples in which such oligomerization was directly observed by mass spectrometry. Our results show that application of short peptides can be useful to study the -mechanism of metal ion binding and metal ion-induced oligomerization of peptides.     

Complexes of silver(I) with peptides and proteins as produced in electrospray mass spectrometry

Silver(I) forms aqueous phase complexes with both sulfur and nonsulfur containing peptides and proteins. These complexes were introduced into the gas phase via electrospray, and their structures probed by means of tandem mass spectrometry. Experiments with di-, tri-, and oligopeptides show that the abundance of silver(I)-containing ions increases relative to that of proton-containing ions as peptide length increases. This increase is much more dramatic for methionine-containing peptides. Collision-induced dissociation of silver-peptide complexes yields a multitude of product ions that are silver containing. However, even for methioninecontaining peptides, very few of these product ions contain the methionine residue. The solution-phase structure and the gas-phase structure of the silver/peptide complex are not identical. The methionine sulfur acts as the silver anchoring point in solution. Desolvation in the gas phase leads to a rearrangement of the silver/peptide complex such that the silver ion becomes chelated to the nitrogen and oxygen atom on the peptide backbone in addition to the methionine sulfur. This rearrangement decreases the importance of the silver/sulfur bond to the extent that it is frequently broken upon collision activation and leads to the formation of silver/peptide product ions that are nonsulfur bearing.     

Selective identification and quantitative analysis of methionine containing peptides by charge derivatization and tandem mass spectrometry

To enable the development of a tandem mass spectrometry (MS/MS) based methodology for selective protein identification and differential quantitative analysis, a novel derivatization strategy is proposed, based on the formation of a "fixed-charge" sulfonium ion on the side-chain of a methionine amino acid residue contained within a protein or peptide of interest. The gas-phase fragmentation behavior of these side chain fixed charge sulfonium ion containing peptides is observed to result in exclusive loss of the derivatized side chain and the formation of a single characteristic product ion, independently of charge state or amino acid composition. Thus, fixed charge containing peptide ions may be selectively identified from complex mixtures, for example, by selective neutral loss scan mode MS/MS methods. Further structural interrogation of identified peptide ions may be achieved by subjecting the characteristic MS/MS product ion to multistage MS/MS (MS3) in a quadrupole ion trap mass spectrometer, or by energy resolved "pseudo" MS3 in a triple quadrupole mass spectrometer. The general principles underlying this fixed charge derivatization approach are demonstrated here by MS/MS, MS3 and "pseudo" MS3 analysis of side chain fixed-charge sulfonium ion derivatives of peptides containing methionine formed by reaction with phenacylbromide. Incorporation of "light" and "heavy" isotopically encoded labels into the fixed-charge derivatives facilitates the application of this method to the quantitative analysis of differential protein expression, via measurement of the relative abundances of the neutral loss product ions generated by dissociation of the light and heavy labeled peptide ions. This approach, termed "selective extraction of labeled entities by charge derivatization and tandem mass spectrometry" (SELECT), thereby offers the potential for significantly improved sensitivity and selectivity for the identification and quantitative analysis of peptides or proteins containing selected structural features, without requirement for extensive fractionation or otherwise enrichment from a complex mixture prior to analysis.     

Development of novel guanidino-labelling derivatisation (GLaD) reagents for liquid chromatography/matrix-assisted laser desorption/ionisation analysis

A new generation of guanidino-labelling (GLaD) reagents were developed for quantitative proteomics using offline microcapillary liquid chromatography (LC) matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). In order to reduce the unwanted overlapping between the isotopic envelopes of the two differentially labelled peptide ions, a novel synthetic route was described for production of both (13)C- and (15)N-containing isotopomers of N,O-dimethylisourea. The use of these types of isotopes has no deleterious effect on the retention times of both differentially labelled peptides during offline microbore reversed-phase LC. In addition, the possibility to incorporate a mass difference of 4 Da can be exploited during post-source decay analysis to generate product ion spectra in which fragment ions containing the modifications appear as doublets in the corresponding product ion spectra, thus facilitating identification of the C-terminal fragment ions.     

On-line electrogeneration of copper-peptide complexes in microspray mass spectrometry

The interaction of copper ions with peptides was investigated by electrospray mass spectrometry. Two electrospray micro-emitters were compared, the first one with a platinum electrode using a copper(II) electrolyte solution containing a peptide sample, and the second one with a sacrificial copper anode in a water/methanol solution containing only a peptide (i.e., angiotensin III, bradykinin, or Leu-enkephalin). The former yielded mainly Cu(2+) complexes either with histidine residues or with the peptide backbone (Cu(+) complexes can be also formed due to gas-phase reactions), whereas the latter can generate a mixture of both Cu(+) and Cu(2+) aqueous complexes that yield different complexation patterns. This study shows that electrospray emitters with soluble copper anodes enable the study of Cu(I)-peptide complexes in solution.     

Isotope edited product ion assignment by α-N labeling of peptides with [2H3(50%)]2,4-Dinitrofluorobenzene

An isotopic modification of Sanger's method for identifying peptide N-termini has been developed to assist peptide sequencing by tandem mass spectrometry. Tryptic peptides, such as Val-His-Leu-Thr-Pro-Val-Glu-Lys, are derivatized with an equimolar mixture of 2,4-dinitrofluorobenzene and [2H3]2,4-dinitrofluorobenzene. Under optimized derivatization conditions, the alpha-amino group could be derivatized while the epsilon-amine of the lysine side chain and the imidazole of histidine remained underivatized. The alpha-dinitrophenyl modified peptides were characterized by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) and liquid chromatography (LC)-ESI-MS. The [M + H]+ ions showed a doublet pattern with a delta m/z of 3 and the [M + 2H]2+ ions were recognized as doublets with a delta m/z of 1.5. MS/MS was employed where both isotopic [M + 2H]2+ ions were alternately subjected to collision-induced dissociation in the second quadrupole. Fragmentation in the ionization source generated identical product ion patterns that were observed during fragmentation in the second quadrupole. In the product ion mass spectra, the N-terminal a and b ions (no c ion observed) are doublets with a delta m/z of 3 or 1.5, while the C-terminal y and z ions (no x ion observed) are singlets appearing at identical masses. Thus, the product ions containing the N-terminus derivatized with a dinitrophenyl group are unequivocally distinguished from the product ions containing the C-terminus. The dinitrophenyl modification generally enhanced the production of a and b ions without diminishing y and z ion yields.     

Studies on Fragmentation of Peptide by Nano-electrospray Ionization Fourier Transform Ion Cyclotron Resonance Multi-stage Tandem Mass Spectrometry

The sequence analysis of peptides was performed by nano-electrospray ionization Fourier transform ion cyclotron resonance tandem mass spectrometry(Nano-ESI-FT-ICR-MSn) and several peptides were chosen as examples. With the aid of the collision induced dissociation(CID), FT-ICR provides not only precise mass/charge ratio, but also structure information of the selected peptides. The fragment ions were identified according to the observed molecular weights and peptide sequence was determined successfully. So Nano-ESI-FT-ICR-MSn is a useful tool for identification of the amino acid sequence of peptides with high confidence. Besides, a pathway for the dehydration of y ions without amino acids containing carboxylic acid under sustained off-resonance irradiation collision-induced dissociation(SORI-CID) condition was proposed.     

Electrospray ionization mass spectrometric analysis of complexes between peptide‐derived Amadori products and borate ions

Hexose‐modified peptides, products of the enzymatic hydrolysis of glycated proteins, could be used as markers of diabetes mellitus, the aging process and other diseases. The main difficulty in this approach is the detection of glycated peptides in the complex mixtures of compounds. In this study we investigated the formation of borate complexes of the peptide‐derived Amadori products by high‐resolution mass spectrometry (HRMS) and tandem mass spectrometry (MS/MS) experiments. It was found that the formation of a complex with the borate ion stabilizes the sugar moiety, resulting in the simplification of the fragmentation patterns of peptide‐derived Amadori products. The level of dehydration, as well as the elimination of formaldehyde from the precursor ions of borate complexes, was lower as compared to the free peptide. On the other hand the intensity of the b‐ and y‐type ions for borate complexes is significantly higher in comparison to the free peptide‐derived Amadori product. Moreover, the elimination of a whole hexose moiety was not detected in the examined peptides. Copyright © 2009 John Wiley & Sons, Ltd.     

Effect of mutated transporters associated with antigen-processing 2 on characteristic major histocompatibility complex binding peptides: analysis using electrospray ionization tandem mass spectrometry

A novel allele of transporters associated with the antigen-processing (TAP) 2 gene, TAP2*Bky2 (Val(577)), is significantly increased in Japanese patients with Sj?gren's syndrome (SS), and has a strong association with SS-A/Ro autoantibody production in SS and autoantibody including anti-SS-A/Ro and anti-U1 RNP antibody in systemic lupus erythematosus (SLE). To determine the influence of this natural mutated TAP on peptides loaded onto MHC class I, we analyzed the repertoire of peptides loaded onto MHC class I on transfectants with TAP1 and TAP2 or mutated TAP2 by electrospray ionization tandem mass spectrometry (ESI-MS/MS). After comparison of the peptide profiles we identified three peptides from only mutated TAP transfectants. Moreover, one of these peptides is derived from snRNP A, which is a target for anti-U1 RNP antibody. To our knowledge this is the first report to show that the natural mutation of TAP2 changes the peptide profile loaded onto MHC class I molecules.     

Dynamic flexibility of a peptide-binding groove of human HLA-DR1 class II MHC molecules: normal mode analysis of the antigen peptide-class II MHC complex

Class II major histocompatibility complex (MHC) has tolerance for binding longer antigen peptides than those bound by class I MHC. In this paper, a normal mode analysis on HLA-DR1 class II MHC involving an antigen peptide indicated that the peptide-binding groove had some different dynamic characteristics from that of HLA-A2 class I MHC. The dynamic changes in the class I groove with removal of the bound peptide were limited primarily to the central region and the C-terminal side (corresponding to the C-terminal side of the bound peptide) of the groove, while the dynamic changes in the class II groove with removal of the bound peptide extended to the whole of the groove, and were especially remarkable around a strand located in the N-terminal side (corresponding to the N-terminal side of the bound peptide) of the groove. These results suggest that the N-terminal side of the class II groove is more flexible than the same side of the class I groove, and this flexibility may allow some N-terminal residues of the bound peptide to extend outside the class II groove. Definite anti-correlative motions with removal of the bound peptide appeared between two alpha-helical regions of class II MHC as in the case of class I MHC. These motions of the class II groove may play an important role in obtaining "a flexible dynamic fit" against diverse longer peptides both of whose terminals extend outside the groove.     

Liquid chromatography and electrospray mass spectrometric mapping of peptides from human plasma filtrate

We present a multidimensional approach to map the composition of complex peptide mixtures obtained as crude extract from biological liquids by (1) cation exchange chromatography and (2) subsequent microbore reversed-phase liquid chromatography and electrospray mass spectrometry coupling (LC-MS). Human hemofiltrate is an equivalent to blood and is used to obtain peptide material in large quantities from patients with chronic renal failure. The upper exclusion limit of the filtration membranes used results in a protein-free filtrate containing peptides in a range up to 20 ku. Using this unique peptide source, several thousand peptides were detected and an LC-MS data base of circulating human peptides was created. The search for known peptides by their molecular mass is a reliable method to guide peptide purification.     

New method to study the effects of peptide sequence on the dissociation energetics of peptide ions

A new method has been developed to study the dissociation patterns of singly protonated peptides by using a quadrupole ion trap mass spectrometer. The new approach involves using boundary-activated dissociation to characterize the ease of dissociation of peptide ions. Insight can be gained into the effect of specific peptide sequences on the dissociation energetics of protonated peptides. Increased knowledge of the effects of specific sequences on the dissociation patterns of peptide ions should improve the ability to interpret complex spectra from tandem mass spectrometry (MS/MS) experiments. This method has confirmed the previously observed increase in the energy needed for the dissociation of peptide ions containing basic residues. In addition, this technique has revealed the effect of the location of proline residues on the dissociation energetics of peptides with this amino acid.     

Positive and negative ion electrospray tandem mass spectrometry (ESI MS/MS) of Boc-protected peptides containing repeats of L-Ala-gamma4Caa/gamma4Caa-L-Ala: differentiation of some positional isomeric peptides

High-resolution electrospray ionization (ESI) quadrupole time-of-flight and ion trap tandem mass spectrometry has been used to distinguish the positional isomers of a new class of N-blocked hybrid peptides containing repeats of the amino acids, L-Ala-gamma(4)Caa ((l))/gamma(4)Caa((l))-L-Ala [Caa((l)) = Carbo (lyxose) amino acid, derived from D-mannose]. Both MS/MS and MS(3) of protonated isomeric peptides produce characteristic fragmentation involving the peptide backbone, Boc-group, and the side-chain. It is interesting to observe that the abundant y(n)(+) ions are formed when the corresponding amide -NH does not participate in the helical structures in solution phase and relatively low abundance y(n)(+) ions resulted when the amide -NH involves in the H-bonding. Thus, it was possible to identify the amide -NH hydrogens that participate in the helical structures through H-bonding in solution phase. Further, negative ion ESI MS/MS has also been found to be useful for differentiating these isomeric peptide acids.     

Sequence-specific fragmentation of deprotonated peptides containing H or alkyl side chains

The [M - H]- ions of a variety of di- to pentapeptides containing H or alkyl side chains have been prepared by electrospray ionization and low-energy collision-induced dissociation (CID) of the deprotonated species carried out in the interface region between the atmospheric pressure source and the quadrupole mass analyzer. Using the nomenclature applied to the fragmentation of protonated peptides, deprotonated dipeptides fragment to give a2 ions (CO2 loss) and y1 ions, where the y1 ion has two fewer hydrogens than the y"1 ions formed from protonated peptides. Deprotonated tri- and tetrapeptides fragment to give primarily y1, c1, and "b2 ions, where the "b2 ion has two fewer hydrogens than the b2 ion observed for protonated peptides. More minor yields of y2, c2, and a2 ions also are observed. The a ion formed by loss of CO2 from the [M - H]- ion shows loss of the N-terminal residue for tripeptides and sequential loss of two amino acid residues from the N-terminus for tetrapeptides. The formation of c(n) ions and the sequential loss of N-terminus residues from the [M - H - CO2]- ion serves to sequence the peptide from the N-terminus, whereas the formation of y(n) ions serves to sequence the peptide from the C-terminus. It is concluded that low-energy CID of deprotonated peptides provides as much (or more) sequence information as does CID of protonated peptides, at least for those peptides containing H or alkyl side chains. Mechanistic aspects of the fragmentation reactions observed are discussed.     

Complexation between the fungicide tebuconazole and copper(II) probed by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is used to probe the complex formation between tebuconazole (1) and copper(II) salts, which both are commonly used fungicides in agriculture. Experiments with model solutions containing 1 and CuCl(2) reveal the initial formation of the copper(II) species [(1)CuCl](+) and [(1)(2)CuCl](+) which undergo reduction to the corresponding copper(I) ions [(1)Cu](+) and [(1)(2)Cu](+) under more drastic ionization conditions in the ESI source. In additional experiments, copper/tebuconazole complexes were also detected in samples made from soil solutions of various origin and different amount of mineralization. The direct sampling of such solutions via ESI-MS is thus potentially useful for understanding of the interactions between copper(II) salts and tebuconazole in environmental samples.     

MS/MS of protonated polyproline peptides: The influence of N-terminal protonation on dissociation

A unique collision-induced dissociation pattern was observed for protonated polyproline peptides of length n in which y(n-2) and/or y(n-4) ions were formed in much higher abundance than any other product ions. Cleavage occurs only at every other amide bond, such that product ions are formed only from the losses of even numbers of proline residues. Exclusive losses of even numbers of proline residues were not observed from sodiated peptides. Further study of the tandem mass spectrometry (MS/MS) patterns of protonated proline-rich peptides showed that the substitution of alanine in the second position of polyproline peptides did not prevent the dominant formation of y(n-2) and y(n-4) ions. The loss of ProAla to form the y(8) ion from (ProAlaPro(8)NH(2)+H)(+) was as abundant as the loss of ProPro from (Pro(10)NH(2)+H)(+). However, modification of the peptides that presumably affected the location of the proton on the peptide did alter the MS/MS spectra. Pro(10) and Pro(5) with blocked N-termini or with arginine substituted for the first proline residue did not form abundant y(n-2) or y(n-4) ions. MS(3) and double resonance experiments showed that dissociation of intermediate y(n) product ions can produce y(n-2) ions, but are not necessary dissociation pathway intermediates. This analysis suggests that the ionizing proton must be located at the N-terminus for the peptide ion to dissociate in this manner.     

Glycan side chains on naturally presented MHC class II ligands

The molecular characterization of unknown naturally presented major histocompatibility complex (MHC) class II glycopeptides carrying complex glycans has so far not been achieved, reflecting the different fragmentation characteristics of sugars and peptides in mass spectrometric analysis. Human leukocyte antigen (HLA)-DR-bound peptides were isolated by affinity purification, separated via high performance liquid chromatography and analyzed by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. We were able to identify two naturally processed MHC class II ligands, CD53(122-136) and CD53(121-136), carrying complex N-linked glycan side chains by a combination of in-source and collision-induced fragmentation on a quadrupole time-of-flight tandem mass spectrometer.     

Differentiating alpha- and beta-aspartic acids by electrospray ionization and low-energy tandem mass spectrometry

Spectra obtained by low-energy electrospray ionization tandem mass spectrometry (ESI-MS/MS) of 34 peptides containing aspartic acids at position n were studied and unambiguously differentiated. beta-Aspartic acid yields an internal rearrangement similar to that of the C-terminal rearrangements of protonated and cationized peptides. As a result of this rearrangement, two different ions containing the N- and the C-terminal ends of the original peptide are formed, namely, the bn-1 + H2O and y"l - n + 1 - 46 ions, respectively, where e is the number of amino acid residues in the peptide. The structure suggested for the y"l - n + 1 - 46 ion is identical to that proposed for the vn ions observed upon high-energy collision-induced dissociation (CID) experiments. The intensity of these ions in the low-energy MS/MS spectra is greatly influenced by the presence and position of basic amino acids within the sequences. Peptides with a basic amino acid residue at position n - 1 with respect to the beta-aspartic acid yield very intense bn-1 + H2O ions, while the y"l - n + 1 - 46 ion was observed mostly in tryptic peptides. Comparison between the high- and low-energy MS/MS spectra of several isopeptides suggests that a metastable fragmentation process is the main contributor to this rearrangement, whereas for long peptides (40 AA) CID plays a more important role. We also found that alpha-aspartic acid containing peptides yield the normal immonium ion at 88 Da, while peptides containing beta-aspartic acid yield an ion at m/z 70, and a mechanism to explain this phenomenon is proposed. Derivatizing isopeptides to form quaternary amines, and performing MS/MS on the sodium adducts of isopeptides, both improve the relative intensity of the bn + 1 + H2O ions. Based on the above findings, it was possible to determine the isomerization sites of two aged recombinant growth proteins.     

Analysis of peptides and proteins containing nitrotyrosine by matrix-assisted laser desorption/ionization mass spectrometry

Oxidative damage to proteins can occur under physiological conditions through the action of reactive oxygen species, including those containing nitrogen such as peroxynitrite (ONO2-). Peroxynitrite has been shown in vitro to target tyrosine residues in proteins through free radical addition to produce 3-nitrotyrosine. In this work, we show that mass spectral patterns associated with 3-nitrotyrosine containing peptides allow identification of peptides containing this modification. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to characterize a synthetic peptide AAFGY(m-NO2)AR and several peptides containing 3-nitrotyrosine derived from bovine serum albumin treated with tetranitromethane. A unique series of ions were found for these peptides in addition to the mass shift of +45 Da corresponding to the addition of the nitro group. Specifically, two additional ions were observed at roughly equal abundance that correspond to the loss of one and two oxygens, and at lower abundances, two ions are seen that suggest the formation of hydroxylamine and amine derivatives. These latter four components appear to originate by laser-induced photochemical decomposition. MALDI-MS analysis of the synthetic peptide containing 3-nitrotyrosine revealed this same pattern. Post-source decay (PSD) MALDI-time-of-flight (TOF) and collisional activation using a prototype MALDI quadrupole TOF yielded extensive fragmentation that allowed site-specific identification of 3-nitrotyrosine. Conversion of peptides containing 3-nitrotyrosine to 3-aminotyrosine with Na2S2O4 yielded a single molecular ion by MALDI with an abundant sidechain loss under PSD conditions. These observations suggest that MALDI can provide a selective method for the analysis and characterization of 3-nitrotyrosine-containing peptides.     

Identification and characterization by high-performance liquid chromatography/electrospray ionization mass spectrometry of a new variant hemoglobin, Mataro [beta134(H12) Val > Ala

This work illustrates the practical use of combined microbore reversed-phase high-performance liquid chromatography (RP-HPLC) with electrospray ionization mass spectrometry (ESI-MS) in protein identification. The approach consisted of the detection of the abnormal beta-globin chain by ESI-MS analysis of mixtures of intact globins, which simultaneously provided their molecular masses. Separation of the polypeptide globin chains was carried out using microbore C4 RP-HPLC on-line with ESI-MS. Direct peptide-mapping ESI-MS without previous chromatographic separation was performed in order to identify tryptic peptides from whole blood. For the sequence confirmation of the abnormal peptide containing the mutation point, C18 RP-HPLC tryptic separation of the globin mixture on-line with collision-induced dissociation (CID) fragmentation was done. The y series ions allowed the identification of the hemoglobin (Hb) variant as [beta134(H12) Val > Ala]. This new Hb was named Hb Mataró, after the city where it was detected.