Fragmentation reactions of deprotonated peptides containing proline. The proline effect

The collision-induced dissociation (CID) fragmentation reactions of a variety of deprotonated peptides containing proline have been studied in detail using MS(2) and MS(3) experiments, deuterium labelling and accurate mass measurements when necessary. The [M--H--CO(2)](-) (a(2)) ion derived from H-Pro-Xxx-OH dipeptides shows an unusual fragmentation involving loss of C(2)H(4); this fragmentation reaction is not observed for larger peptides. The primary fragmentation reactions of deprotonated tripeptides with an N-terminal proline are formation of a(3) and y(1) ions. When proline is in the central position of tripeptides, a(3), y(2) and y(1) ions are the primary fragmentation products of [M--H](-), while when the proline is in the C-terminal position, a(3)and y(1) ions are the major primary products. In the latter case, the a(3) ion fragments primarily to the 'b(2) ion; further evidence is presented that the 'b(2) ions have a deprotonated oxazolone structure. Larger deprotonated peptides having at least two amino acid residues N-terminal to proline show a distinct preference for cleavage of the amide bond N-terminal to proline to form, mainly, the appropriate y ion. This proline effect is compared and contrasted with the similar proline effect observed in the fragmentation of protonated peptides containing proline.     

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.     

Amide bond cleavage in deprotonated tripeptides: a newly discovered pathway to "b2 ions

The fragmentation reactions of the [M-H](-) ions of the tripeptides H-Gly-Leu-Sar-OH, H-Leu-Gly-Pro-OH and H-Gly-Leu-Gly-OH have been investigated in detail using energy-resolved mass spectrometry, isotopic labelling and MS(3) experiments. It is shown that the major route to the "b(2) ions involves loss of a neutral amine from the a(3) ([M-H-CO(2)](-)) ion rather than being formed directly by fragmentation of the [M-H](-) ion. When there is no C-terminal amidic hydrogen (Sar, Pro), loss of a neutral amine is the dominant primary fragmentation reaction of the a(3) ion. However, when there is a C-terminal amidic hydrogen (Gly), elimination of the N-terminal amino acid residue is the major fragmentation reaction of the a(3) ion and formation of the "b(2) ion is greatly reduced in importance. It is proposed that the "b(2) ions are deprotonated oxazolones.     

Characterization of alpha- and gamma-glutamyl dipeptides by negative ion collision-induced dissociation

The low-energy CID mass spectra of the [M-H](-) ions of a variety of dipeptides containing glutamic acid have been obtained using cone-voltage collisional activation. Dipeptides with the gamma-linkage, H-Glu(Xxx-OH)-OH, are readily distinguished from those with the alpha-linkage, H-Glu-Xxx-OH, by the much more prominent elimination of H-Xxx-OH from the [M-H](-) ions of the former isomers, resulting in formation of m/z 128, presumably deprotonated pyroglutamic acid. Dipeptides with the reverse linkage, H-Xxx-Glu-OH, show distinctive fragmentation reactions of the [M-H](-) ions including enhanced elimination of CO(2) and formation of deprotonated glutamic acid. Exchange of the labile hydrogens for deuterium has shown that there is considerable interchange of C-bonded hydrogens with labile (N- and O-bonded) hydrogens prior to most fragmentation reactions. All dipeptides show loss of H(2)O from [M-H](-). MS(3) studies show that the [M-H-H(2)O](-) ion derived from H-Glu-Gly-OH has the structure of deprotonated pyroglutamylglycine while the [M-H-H(2)O](-) ions derived from H-Glu(Gly-OH)-OH and H-Gly-Glu-OH show a different fragmentation behaviour indicating distinct structures for the fragment ions.     

Fragmentation of deprotonated cyclic dipeptides by electrospray ionization mass spectrometry

The fragmentation pathways of deprotonated cyclic dipeptides have been studied by electrospray ionization multi‐stage mass spectrometry (ESI‐MSⁿ) in negative mode. The results showed that the fragmentation pathways of deprotonated cyclic dipeptides depended significantly on the different substituents, the side chains of amino acid residues at the diketopiperazine ring. In the spectra of deprotonated cyclic dipeptides, the ion [M? H? substituent radical]^? was firstly observed in the ESI mode. The characteristic fragment ions [M? H? substituent radical]^? and [M? H? (substituent? H)]^? could be used as the symbols of particular cyclic dipeptides. The hydrogen/deuterium (H/D) exchange experiment, the high‐resolution mass spectrometry (Q‐TOF) and theoretical calculations were used to rationalize the proposed fragmentation pathways and to verify the differences between the fragmentation pathways. The relative Gibbs free energies (ΔG) of the product ions and possible fragmentation pathways were estimated using the B3LYP/6–31++G(d, p) model. The results have some potential applications in the structural elucidation and interpretation of the mass spectra of homologous compounds and will enrich the gas‐phase ESI‐MS ion chemistry of cyclic dipeptides. Copyright © 2009 John Wiley & Sons, Ltd.     

Fragmentation reactions of protonated peptides containing glutamine or glutamic acid

A variety of protonated dipeptides and tripeptides containing glutamic acid or glutamine were prepared by electrospray ionization or by fast atom bombardment ionization and their fragmentation pathways elucidated using metastable ion studies, energy-resolved mass spectrometry and triple-stage mass spectrometry (MS(3)) experiments. Additional mechanistic information was obtained by exchanging the labile hydrogens for deuterium. Protonated H-Gln-Gly-OH fragments by loss of NH(3) and loss of H(2)O in metastable ion fragmentation; under collision-induced dissociation (CID) conditions loss of H-Gly-OH + CO from the [MH - NH(3)](+) ion forms the base peak C(4)H(6)NO(+) (m/z 84). Protonated dipeptides with an alpha-linkage, H-Glu-Xxx-OH, are characterized by elimination of H(2)O and by elimination of H-Xxx-OH plus CO to form the glutamic acid immonium ion of m/z 102. By contrast, protonated dipeptides with a gamma-linkage, H-Glu(Xxx-OH)-OH, do not show elimination of H(2)O or formation of m/z 102 but rather show elimination of NH(3), particularly in metastable ion fragmentation, and elimination of H-Xxx-OH to form m/z 130. Both the alpha- and gamma-dipeptides show formation of [H-Xxx-OH]H(+), with this reaction channel increasing in importance as the proton affinity (PA) of H-Xxx-OH increases. The characteristic loss of H(2)O and formation of m/z 102 are observed for the protonated alpha-tripeptide H-Glu-Gly-Phe-OH whereas the protonated gamma-tripeptide H-Glu(Gly-Gly-OH)-OH shows loss of NH(3) and formation of m/z 130 as observed for dipeptides with the gamma-linkage. Both tripeptides show abundant formation of the y(2)' ion under CID conditions, presumably because a stable anhydride neutral structure can be formed. Under metastable ion conditions protonated dipeptides of structure H-Xxx-Glu-OH show abundant elimination of H(2)O whereas those of structure H-Xxx-Gln-OH show abundant elimination of NH(3). The importance of these reaction channels is much reduced under CID conditions, the major fragmentation mode being cleavage of the amide bond to form either the a(1) ion or the y(1)' ion. Particularly when Xxx = Gly, under CID conditions the initial loss of NH(3) from the glutamine containing dipeptide is followed by elimination of a second NH(3) while the initial loss of H(2)O from the glutamic acid dipeptide is followed by elimination of NH(3). Isotopic labelling shows that predominantly labile hydrogens are lost in both steps. Although both [H-Gly-Glu-Gly-OH]H(+) and [H-Gly-Gln-Gly-OH]H(+) fragment mainly to form b(2) and a(2) ions, the latter also shows elimination of NH(3) plus a glycine residue and formation of protonated glycinamide. Isotopic labelling shows extensive mixing of labile and carbon-bonded hydrogens in the formation of protonated glycinamide.     

Identification of degradation products of aspartyl tripeptides by capillary electrophoresis-tandem mass spectrometry

Capillary electrophoresis-electrospray tandem mass spectrometry (CE-MS/MS) has been used to identify degradation products of the aspartyl tripeptides Phe-Asp-GlyNH(2) and Gly-Asp-PheNH(2) following incubation of the peptides in acidic and alkaline solution. At pH 2, the dominant decomposition products resulted from cleavage of the peptide backbone amide bonds to yield the respective dipeptides and amino acids. In addition, the cyclic aspartyl succinimide intermediate was identified by its [M+H](+) at m/z = 319 and the MS/MS spectrum exhibiting a simple fragmentation pattern with the [C(8)H(10)N](+)-ion as the principal daughter ion (a(1) of Phe-Asp-GlyNH(2)). Deamidation of the C-terminal amide as well as isomerization and enantiomerization of the Asp residue occurred upon incubation at pH 10. alpha-Asp and the isomeric beta-Asp and most of the diastereomeric forms (corresponding to D/L-Asp) could be separated by CE. All isomers could be identified based on their MS/MS spectra. Peptides with the amino acid sequence Phe-Asp-Gly containing the regular alpha-Asp bond displayed a highly intense b(2) fragment ion and a low abundant y(2) ion. In contrast, the y(2) and a(1) fragment were high abundant daughter ions in the mass spectra of beta-Asp peptides while the b(2) ion exhibited a lower abundance. Differences in the MS/MS spectra of the isomers of the peptides with the sequence Gly-Asp-Phe were obvious but less pronounced. In conclusion, CE-MS/MS proved to be a useful tool to study the decomposition and enantiomerization of peptides including the isomerization of Asp residues, due to the combination of efficient separation of isomers by CE and their identification by MS/MS.     

Intramolecular condensation reactions in protonated dipeptides: Carbon monoxide,water, and ammonia losses in competition

The elimination of carbon monoxide and water from a series of protonated dipeptides, [XxxYyy + H](+), is investigated by tandem mass spectrometry experiments and density functional theory. The combined results show that CO loss occurs on the a(1)-y(1) pathway, which begins by rearrangement of the added proton to the amide N-atom and creates the proton-bound dimer of an amino acid (Yyy) and an imine (that from Xxx residue). The loss of H(2)O is initiated from a tautomer in which the added proton has migrated to the hydroxyl group of the C-terminus, thereby promoting the formation of an ion with protonated oxazolone structure (a nominal b(2) ion). The highest yields of [XxxYyy + H - CO](+) and [XxxYyy + H - H(2)O](+) are observed at threshold energies. As the internal energy of the protonated dipeptides increases, these primary products are depleted by consecutive dissociations yielding mostly backbone fragments. Specifically, [XxxYyy + H - CO](+) decomposes to y(1) (protonated Yyy) and a(1) (immonium ion of Xxx residue), while [XxxYyy + H - H(2)O](+) produces a(2) and the immonium ions of residues Xxx (a(1)) and Yyy ("internal" immonium ion). Water loss takes place more efficiently when the more basic residue is at the C-terminal position. Increasing the basicity of the N-terminal residue enhances the extent of CO versus H(2)O loss and introduces the competitive elimination of NH(3). The dissociations leading to eliminations of small neutrals (CO, H(2)O, etc.) generally proceed over transition states that lie higher in energy than the corresponding dissociation products. The excess energy is disposed of either in translational or rovibrational modes of the products, depending on the stability of the incipient noncovalent assemblies emerging during the cleavage of the small neutrals.     

Role of the sulfhydryl group on the gas phase fragmentation reactions of protonated cysteine and cysteine containing peptides

The gas phase fragmentation reactions of protonated cysteine and cysteine-containing peptides have been studied using a combination of collisional activation in a tandem mass spectrometer and ab initio calculations [at the MP2(FC)/6-31G*//HF/6-31G* level of theory]. There are two major competing dissociation pathways for protonated cysteine involving: (i) loss of ammonia, and (ii) loss of the elements of [CH₂O₂]. MS/MS, MS/MS of selected ions formed by collisional activation in the electrospray ionization source as well as ab initio calculations have been carried out to determine the mechanisms of these reactions. The ab initio results reveal that the most stable [M + H − NH₃]⁺ isomer is an episulfonium ion (A), whereas the most stable [M + H − CH₂O₂]⁺ isomer is an immonium ion (B). The effect of the position of the cysteine residue on the fragmentation reactions of the [M + H]⁺ ions of all the possible simple dipeptide and tripeptide methyl esters containing one cysteine (where all other residues are glycine) has also been investigated. When cysteine is at the N-terminal position, NH₃ loss is observed, although the relative abundance of the resultant [M + H − NH₃]⁺ ion decreases with increasing peptide size. In contrast, when cysteine is at any other position, water loss is observed. The proposed mechanism for loss of H₂O is in competition with those channels leading to the formation of structurally relevant sequence ions.     

Fragmentation chemistry of [M + Cu]+ peptide ions containing an N-terminal arginine

[M + Cu]+ peptide ions formed by matrix-assisted laser desorption/ionization from direct desorption off a copper sample stage have sufficient internal energy to undergo metastable ion dissociation in a time-of-flight mass spectrometer. On the basis of fragmentation chemistry of peptides containing an N-terminal arginine, we propose the primary Cu+ ion binding site is the N-terminal arginine with Cu+ binding to the guanidine group of arginine and the N-terminal amine. The principal decay products of [M + Cu]+ peptide ions containing an N-terminal arginine are [a(n) + Cu - H]+ and [b(n) + Cu - H]+ fragments. We show evidence to suggest that [a(n) + Cu - H]+ fragment ions are formed by elimination of CO from [b(n) + Cu - H]+ ions and by direct backbone cleavage. We conclude that Cu+ ionizes the peptide by attaching to the N-terminal arginine residue; however, fragmentation occurs remote from the Cu+ ion attachment site involving metal ion promoted deprotonation to generate a new site of protonation. That is, the fragmentation reactions of [M + Cu]+ ions can be described in terms of a "mobile proton" model. Furthermore, proline residues that are adjacent to the N-terminal arginine do not inhibit formation of [b(n) + Cu - H]+ ion, whereas proline residues that are distant to the charge carrying arginine inhibit formation of [b(n) + Cu - H]+ ions. An unusual fragment ion, [c(n) + Cu + H]+, is also observed for peptides containing lysine, glutamine, or asparagine in close proximity to the Cu+ carrying N-terminal arginine. Mechanisms for formation of this fragment ion are also proposed.     

'Top-down' characterization of site-directed mutagenesis products of Staphylococcus aureus dihydroneopterin aldolase by multistage tandem mass spectrometry in a linear quadrupole ion trap

The gas-phase fragmentation reactions of a series of site-directed mutagenesis products of Staphylococcus aureus dihydroneopterin aldolase have been examined by multistage tandem mass spectrometry (MS/MS and MS(3)) in a linear quadrupole ion trap in order to explore the utility of this instrumentation for routine 'top-down' recombinant protein characterization. Following a rapid low resolution survey of the fragmentation behavior of the precursor ions from the wild type (WT) protein, selected charge states were subjected to detailed structural characterization by using high resolution 'zoom' and 'ultrazoom' resonance ejection MS/MS product ion scans. Dissociation of the [M + 18H](18+) charge state yielded a range of product ions from which extensive sequence information could be derived. In contrast, dissociation of the [M + 20H](20+) charge state resulted in a single dominant y(96) product ion formed by fragmentation between adjacent Ile/Gly residues, with only limited sequence coverage. Further extensive sequence information was readily obtained however, by MS(3) dissociation of this initial product. From the combined MS/MS and MS(3) spectra an overall sequence coverage of 66.9%, with fragmentation of 85 of the 127 amide bonds within the WT protein, was obtained. MS/MS and MS(3) of three of the four site-directed mutagenesis products (E29A), (Y61F) and (E81A) were found to yield essentially identical product ion spectra to the WT protein, indicating that these modifications had no significant influence on the fragmentation behavior. The specific site of modification could be unambiguously determined in each case by characterization of product ions resulting from fragmentation of amide bonds on either side of the mutation site. In contrast, MS/MS and MS(3) of the K107A mutant led to significantly different product ion spectra dominated by cleavages occurring N-terminal to proline, which restricted the ability to localize the modification site to within only an 8 amino acid region of the sequence. This work highlights the need for further studies to characterize the charge state, sequence and structural dependence to the low energy collision induced dissociation reactions of multiply protonated intact protein ions.     

Influences of peptide side chains on the metal ion binding site in metal ion-cationized peptides: Participation of aromatic rings in metal chelation

Aromatic side chains on amino acids influence the fragmentations of cationic complexes of doubly charged metal ions and singly deprotonated peptides. The metal ion interacts with an aromatic side chain and binds to adjacent amide nitrogens. When fragmentation occurs, this bonding leads to the formation of abundant metal-containing a-type ions by reactions that occur at the sites of amino acids that contain the aromatic side chain. Furthermore, formation of metal-containing immonium ions of the amino acids that contain the aromatic side chain also are formed. The abundant a-type ions may be useful in interpretation strategies in which it is necessary to locate in a peptide the position of an amino acid that bears an aromatic side chain.     

Occurrence of C-Terminal Residue Exclusion in Peptide Fragmentation by ESI and MALDI Tandem Mass Spectrometry

By screening a data set of 392 synthetic peptides MS/MS spectra, we found that a known C-terminal rearrangement was unexpectedly frequently occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTOF and TOF/TOF mass analyzer configuration, respectively. Any residue localized at the C-terminal carboxylic acid end, even a basic one, was lost, provided that a basic amino acid such arginine and to a lesser extent histidine and lysine was present in the sequence leading to a fragment ion, usually depicted as (b_n-1 + H₂O) ion, corresponding to a shortened non-scrambled peptide chain. Far from being an epiphenomenon, such a residue exclusion from the peptide chain C-terminal extremity gave a fragment ion that was the base peak of the MS/MS spectrum in certain cases. Within the frame of the mobile proton model, the ionizing proton being sequestered onto the basic amino acid side chain, it is known that the charge directed fragmentation mechanism involved the C-terminal carboxylic acid function forming an anhydride intermediate structure. The same mechanism was also demonstrated from cationized peptides. To confirm such assessment, we have prepared some of the peptides that displayed such C-terminal residue exclusion as a C-terminal backbone amide. As expected in this peptide amide series, the production of truncated chains was completely suppressed. Besides, multiply charged molecular ions of all peptides recorded in ESI mass spectrometry did not undergo such fragmentation validating that any mobile ionizing proton will prevent such a competitive C-terminal backbone rearrangement. Among all well-known nondirect sequence fragment ions issued from non specific loss of neutral molecules (mainly H₂O and NH₃) and multiple backbone amide ruptures (b-type internal ions), the described C-terminal residue exclusion is highly identifiable giving raise to a single fragment ion in the high mass range of the MS/MS spectra. The mass difference between this signal and the protonated molecular ion corresponds to the mass of the C-terminal residue. It allowed a straightforward identification of the amino acid positioned at this extremity. It must be emphasized that a neutral residue loss can be misattributed to the formation of a y_m-1 ion, i.e., to the loss of the N-terminal residue following the a₁-y_m–1 fragmentation channel. Extreme caution must be adopted when reading the direct sequence ion on the positive ion MS/MS spectra of singly charged peptides not to mix up the attribution of the N- and C-terminal amino acids. Although such peculiar fragmentation behavior is of obvious interest for de novo peptide sequencing, it can also be exploited in proteomics, especially for studies involving digestion protocols carried out with proteolytic enzymes other than trypsin (Lys-N, Glu-C, and Asp-N) that produce arginine-containing peptides.     

A non-covalent dimer formed in electrospray ionisation mass spectrometry behaving as a precursor for fragmentations

A non-covalent-bonded dimer was detected in the positive ion electrospray ionisation (ESI) mass spectra of a synthetic impurity. In tandem mass spectrometry (MS/MS) experiments using collision-induced dissociation (CID), the ion was found to behave as a [M+H]+-type precursor ion for fragmentation until MS5. The dimer was probably formed through multi-hydrogen bonds over a proton bridge. When the fragmentation occurred at the center of the bridge, the dimer was broken apart to give monomer fragments at MS6. However, no corresponding deprotonated dimer [2M-H]- was found in the negative ion ESI spectra. The dimer was extremely stable, and it could still be observed when a fragmentation voltage of up to 50 V was applied in the ionisation source. The formation of the non-covalent dimer was also found to be instrument-dependent, but independent of sample concentration. Accurate mass measurements of the [2M+H]+ and [M+H]+ ions, and their MSn product ions, provided the basis for assessing the fragmentation mechanism proposed for [2M+H]+. The fragmentation pathway was also illustrated for the deprotonated molecule [M-H]-.     

Metastable decomposition of peptide [M + H]+ ions via rearrangement involving loss of the C-terminal amino acid residue

A novel fragmentation of metastable peptide [M + H]⁺ ions is described. Loss of the C-terminal amino acid residue is accomqanied by retention of one of the carboxyl oxygens, as judged by ¹⁸O-labeling. The retained ⁸O label is located at the new C-terminus. Sequential mass spectrometric analyses indicate that the structure of the first-generation product ion is indistinguishable from that of the [M + H]⁺ ion of the peptide with one fewer amino acid residues. Thus, for example, the metastable decompositions of ions of m/z 904 are similar whether they correspond to des-Arg⁹-bradykinin [M + H]⁺ ions or to fragments derived from bradykinin [M + H]⁺ ions. No corresponding rearrangements have been observed for peptides with C-terminal amide or ester functions. The mechanism of this fragmentation may be considered to be analogous to that previously suggested for fragmentations of [M + alkali metal cation]⁺ ions. For the examples of bradykinin and related peptides, the rearrangement is strongly promoted when arginine is the amino acid residue lost. The same fragmentation is also favored by the presence of an arginine residue at or near the N-terminus. The strong influence of peptide amino acid composition, including residues remote from the C-terminus, on the prevalence of this fragmentation suggests mechanistic complexities that require further elucidation.

     

Formation of c1 fragment ions in collision-induced dissociation of glutamine-containing peptide ions: a tip for de novo sequencing

A c1 ion was observed with significant yield in the tandem mass (MS/MS) spectra of peptide ions containing glutamine as the second amino acid residue from the N-terminus. The c1 fragment was generated independently of the N-terminal residue of the peptide, but its abundance was strongly dependent on the side-chain identity. This ion is not a common fragmentation product in low-energy collision-induced dissociation of peptide ions, but it assists in identification of the first two amino acid residues, often difficult due to a low or absent signal from the heaviest y ion. A consecutive fragmentation mechanism is proposed, involving a b2 ion with a six-membered ring as an intermediate, to explain the exceptional stability of the c1 fragment ion. The utility of this information is discussed, especially in de novo sequencing of peptide ions.     

Fragmentation of the deprotonated ions of peptides containing cysteine, cysteine sulfinic acid, cysteine sulfonic acid, aspartic acid, and glutamic acid

We examined the fragmentation of the electrospray-produced [M-H]- and [M-2H]2- ions of a number of peptides containing two acidic amino acid residues, one being aspartic acid (Asp) or glutamic acid (Glu), and the other being cysteine sulfinic acid [C(SO2H)] or cysteine sulfonic acid [C(SO3H)], on an ion-trap mass spectrometer. We observed facile neutral losses of H2S and H2SO2 from the side chains of cysteine and C(SO2H), respectively, whereas the corresponding elimination of H2SO3 from the side chain of C(SO3H) was undetectable for most peptides that we investigated. In addition, the collisional activation of the [M-H]- ions of the C(SO2H)-containing peptides resulted in the cleavage of the amide bond on the C-terminal side of the C(SO2H) residue. Moreover, collisional activation of the [M-2H]2- ions of the above Asp-containing peptides led to the cleavage of the backbone N-Calpha bond of the Asp residue to give cn and/or its complementary [zn-H2O] ions. Similar cleavage also occurred for the singly deprotonated ions of the otherwise identical peptides with a C-terminal amide functionality, but not for the [M-H]- ions of same peptides with a free C-terminal carboxylic acid. Furthermore, ab initio calculation results for model cleavage reactions are consistent with the selective cleavage of the backbone N-Calpha bond in the Asp residue.     

A study of b<Subscript>1</Subscript>+H<Subscript>2</Subscript>O and b<Subscript>1</Subscript>-ions in the product ion spectra of dipeptides containing N-terminal basic amino acid residues

The product ion spectra of approximately 200 dipeptides were acquired under low-energy conditions using a triple quadrupole mass spectrometer. The spectra of dipeptides containing an N-terminal arginine (R), histidine (H), or lysine (K) were observed to yield a b(1) + H(2)O ion corresponding to the protonated basic amino acid. This was equivalent to the y(1)-ion in the corresponding C-terminal isomer. The formation of a b(1) + H(2)O ion was not a significant fragmentation channel in any dipeptides analyzed including those containing a C-terminal basic amino acid unless they also contained an N-terminal basic amino acid. Occurring simultaneously and under equal energy conditions an apparent b(1)-ion was formed, which has its corresponding C-terminal equivalent in the y(1)-H(2)O ion. Energy resolved mass spectrometry (ERMS), deuterium labeling, and accurate mass experiments as well as data reported were used to show the relationships between the b(1)+H(2)O and b(1)-ions in the dipeptides containing an N-terminal basic amino acid and the y(1) and y(1)-H(2)O ions in the corresponding C-terminal isomers.     

Electrospray tandem mass spectrometry of new porphyrin amino acid conjugates

Porphyrin amino acid conjugates with one or two porphyrin units were analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The ESI-MS spectra of all the porphyrins studied, obtained in positive ion mode, show the presence of the corresponding protonated molecule [M+H]+; ESI-MS spectra of diporphyrinyl compounds also show the doubly charged ions [M+2H]2+. The fragmentations of these ions induced by collision with argon were studied (ESI-MS/MS). ESI-MS/MS gives detailed structural information about the amino acids associated with the porphyrin. Cleavage of the bonds in the vicinity of the porphyrin moiety and those involving the side chain of amino acid residues gives structural information about this type of association. A fragmentation common to all derivatives corresponds to the cleavage of the phenyl-CO bond. The expected cleavage of the amide bond, that links the porphyrin to the amino acid moiety, is a minor fragmentation, which in some cases is even absent. The MS/MS spectra of the monoporphyrinyl derivatives show product ions characteristic of the amino acid linked to the porphyrin; the fragmentation also indicates when the amino acids has a terminal carboxylic group or a terminal ester group. The fragmentations of the diporphyrinyl compounds occur mainly by the cleavage of the spacer, leading, in the case of the doubly charged ions, to predominantly mono-charged ions, indicating a preferential location of the two protons in separated porphyrinic units.     

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

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