Differentiation of Positional Isomers of Hybrid Peptides Containing Repeats of β-Nucleoside Derived Amino Acid (β-Nda-) and L-Amino Acids by Positive and Negative Ion Electrospray Ionization Tandem Mass Spectrometry (ESI-MS<Superscript><Emphasis Type="Italic">n</Emphasis></Superscript>)

A new class of positional isomeric pairs of -Boc protected oligopeptides comprised of alternating nucleoside derived β-amino acid (β-Nda-) and L-amino acid residues (alanine, valine, and phenylalanine) have been differentiated by both positive and negative ion electrospray ionization ion-trap tandem mass spectrometry (ESI-MS ⁿ ). The protonated dipeptide positional isomers with β-Nda- at the N-terminus lose CH₃OH, NH₃, and C₂H₄O₂, whereas these processes are absent for the peptides with L-amino acids at the N-terminus. Instead, the presence of L-amino acids at the N-terminus results in characteristic retro-Mannich reaction involving elimination of imine. A good correlation has been observed between the conformational structure of the peptides and the abundance of y_n⁺ and b_n⁺ ions in MS ⁿ spectra. In the case of tetrapeptide isomers that are reported to form helical structures in solution phase, no y_n⁺ and b_n⁺ ions are observed when the corresponding amide -NH- participates in the helical structures. In contrast, significant y_n⁺ and b_n⁺ ions are formed when the amide -NH- is not involved in the H-bonding. In the case of tetra- and hexapeptides, it is observed that abundant b_n⁺ ions are formed, presumably with stable oxazolone structures when the C-terminus of the b_n⁺ ions possessed L-amino acid and the β-Nda- at the C-terminus appears to prevent the cyclization process leading to the absence of corresponding b_n⁺ ions.     

A Systematic Study of Acidic Peptides for b-Type Sequence Scrambling

A systematic study was carried out to examine the effects of acidic amino acid residues and the position of the acidic group on the cyclization of b ions. The study utilized the model C-terminal amidated peptides XAAAAAA, AXAAAAA, AAXAAAA, AAAXAAA, AAAAXAA, AAAAAXA, AAAAAAX, XXAAAAAA, AAXXAAAA, AAAAXXAA, and AAAAAAXX, where X is a glutamic acid (E) or aspartic acid (D) residue. The CID mass spectra of b _n (where n = 7 and 8) ions derived from XAAAAAA, AAAXAAA, AAAAAAX and XXAAAAAA, AAXXAAAA, AAAAXXAA, and AAAAAAXX exhibited very similar fragmentation patterns for both the glutamic and the aspartic acid peptide series. The CID mass spectra of MH⁺ derived from model peptides presented substantial direct and non-direct sequence b ions. The results indicate that b ions produced from acidic peptides can also undergo head-to-tail cyclization, which is the reason for the formation of the non-direct sequence b ions. The b ion spectra derived from the peptides became more complex as the number of acidic residues in the peptides increased. Side chains of glutamic and aspartic acid did not inhibit the cyclization of the b ions. Substantial water elimination was observed in all CID spectra of b ₇ and b ₈ ions. Finally, the preferential cleavage of glutamic or aspartic acid residues from macrocyclic structures of b ions was also investigated under various collision energy conditions.     

Stereospecific control of peptide gas‐phase ion chemistry with cis and trans cyclo ornithine residues

We report non‐chiral amino acid residues cis‐ and trans‐1,4‐diaminocyclohexane‐1‐carboxylic acid (cyclo‐ornithine, cO) that exhibit unprecedented stereospecific control of backbone dissociations of singly charged peptide cations and hydrogen‐rich cation radicals produced by electron‐transfer dissociation. Upon collision‐induced dissociation (CID) in the slow heating regime, peptide cations containing trans‐cO residues undergo facile backbone cleavages of amide bonds C‐terminal to trans‐cO. By contrast, peptides with cis‐cO residues undergo dissociations at several amide bonds along the peptide ion backbone. Diastereoisomeric cO‐containing peptides thus provide remarkably distinct tandem mass spectra. The stereospecific effect in CID of the trans‐cO residue is explained by syn‐facially directed proton transfer from the 4‐ammonium group at cO to the C‐terminal amide followed by neighboring group participation in the cleavage of the CO―NH bond, analogous to the aspartic acid and ornithine effects. Backbone dissociations of diastereoisomeric cO‐containing peptide ions generate distinct [b_n]⁺‐type fragment ions that were characterized by CID‐MS³ spectra. Stereospecific control is also reported for electron‐transfer dissociation of cis‐ and trans‐cO containing doubly charged peptide ions. The stereospecific effect upon electron transfer is related to the different conformations of doubly charged peptide ions that affect the electron attachment sites and ensuing N―C_α bond dissociations.     

Fragmentation Reactions of Methionine-Containing Protonated Octapeptides and Fragment Ions Therefrom: An Energy-Resolved Study

The fragmentation reactions of the MH⁺ ions as well as the b₇, a₇, and a₇* ions derived therefrom have been studied in detail for the octapeptides MAAAAAAA, AAMAAAAA, AAAAMAAA, and AAAAAAMA. Ionization was by electrospray using a QqToF mass spectrometer, which allowed a study of the evolution of the fragmentation channels as a function of the collision energy. Not surprisingly, the product ion mass spectra for the b₇ ions are independent of the original precursor sequence, indicating macrocyclization and reopening to the same mixture of protonated oxazolones prior to fragmentation. The results show that this sequence scrambling results in a distinct preference to place the Met residue in the C-terminal position of the protonated oxazolones. The a₇ and a₇* ions also produce product ion mass spectra independent of the original peptide sequence. The results for the a₇ ions indicate that fragmentation occurs primarily from an amide structure analogous to that observed for a₄ ions (Bythell et al. in J Am Chem Soc 132:14766–14779, 2010). Clearly, the rearrangement reaction they have proposed applies equally well to a_n ions as large as a₇. The major fragmentation modes of the MH⁺ ions at low collision energies produce b₇, b_6, and b₅ ions. As the collision energy is increased further fragmentation of these primary products produces, in part, non-direct sequence ions, which become prominent at lower m/z values, particularly for the peptides with the Met residue near the N-terminus.

Can <Emphasis Type="Italic">α</Emphasis>- and <Emphasis Type="Italic">β</Emphasis>-alanine containing peptides Be distinguished based on the CID spectra of their protonated ions?

The fragmentation reactions of isomeric dipeptides containing α- and β-alanine residues (αAla-αAla, αAla-βAla, βAla-αAla, and βAla-βAla) were studied using a combination of low-energy and energy resolved collision induced dissociation (CID). Each dipeptide gave a series of different fragment ions, allowing for differentiation. For example, peptides containing an N-terminal β-Ala residue yield a diagnostic imine loss, while lactam ions at m/z 72 are unique to peptides containing β-Ala residues. In addition, MS³ experiments were performed. Structure-specific fragmentation reactions were observed for y₁ ions, which help identify the C-terminal residue. The MS³ spectra of the b₂ ions are different suggesting they are unique for each peptide. Density functional theory (DFT) calculations predict that b₂ ions formed via a neighboring group attack by the amide are thermodynamically favored over those formed via neighboring group attack by the N-terminal amine. Finally, to gain further insight into the unique fragmentation chemistry of the peptides containing an N-terminal β-alanine residue, the fragmentation reactions of protonated β-Ala-NHMe were examined using a combination of experiment and DFT calculations. The relative transition-state energies involved in the four competing losses (NH₃, H₂O, CH₃NH₂, and CH₂=NH) closely follow the relative abundances of these as determined via CID experiments.     

Cyclization of peptide b9 ions

The product ion mass spectra obtained by CID of the b₉ ions derived by loss of neutral alanine from the MH⁺ ion of the peptides Tyr(Ala)₉, (Ala)₄Tyr(Ala)₅, and (Ala)₈TyrAla are essentially identical, indicative of full cyclization reaction to a common intermediate before fragmentation. This leads to abundant nondirect sequence ions in the product ion mass spectra of the b₉ ions. The product ion mass spectra of the b₈ ions from the first two peptides also are essentially identical. The fragmentation of the MH⁺ ions also leads to low intensity nondirect sequence ions in the product ion mass spectra. N-terminal acetylation blocks the cyclization and eliminates nondirect sequence fragment ions in the product ion mass spectra.     

Charge-separation reactions of doubly-protonated peptides: Effect of peptide chain length

The collision induced dissociation of doubly-protonated (Ala)_xHis (x=5, 6, 7, 8, 10) peptides have been studied. The major fragmentation reactions observed are symmetrical amide bond cleavages to give the complementary b_m and y_N-m ions, where N is the total number of residues in the peptide. Minor asymmetric cleavage to give doubly-protonated y ions also is observed, involving cleavage near the N-terminus. The shorter peptides (x=5, 6, 7) show major cleavage of the second amide bond to yield b₂ and y_N-2 ions, while (Ala)₁₀His shows major symmetrical cleavage at the fourth and fifth amide bonds. (Ala)₈His appears to be a transitional peptide in showing substantial symmetrical cleavage at the second, fourth, and fifth amide bonds. The results are in general agreement with the bifurcating nature of charge separation noted by Zubarev (J. Am. Soc. Mass Spectrom. 2008, 19, 1755–1763) from a statistical analysis of a large body of doubly-protonated tryptic peptide CID mass spectra. It is shown that the b₂ ion derived from doubly-protonated (Ala)₅His has a protonated oxazolone structure.     

Investigations of the Mechanism of the “Proline Effect” in Tandem Mass Spectrometry Experiments: The “Pipecolic Acid Effect”

The fragmentation behavior of a set of model peptides containing proline, its four-membered ring analog azetidine-2-carboxylic acid (Aze), its six-membered ring analog pipecolic acid (Pip), an acyclic secondary amine residue N-methyl-alanine (NMeA), and the D stereoisomers of Pro and Pip has been determined using collision-induced dissociation in ESI-tandem mass spectrometers. Experimental results for AAXAA, AVXLG, AAAXA, AGXGA, and AXPAA peptides are presented, where X represents Pro, Aze, Pip, or NMeA. Aze- and Pro-containing peptides fragment according to the well-established “proline effect” through selective cleavage of the amide bond N-terminal to the Aze/Pro residue to give y_n ⁺ ions. In contrast, Pip- and NMA-fragment through a different mechanism, the “pipecolic acid effect,” selectively at the amide bond C-terminal to the Pip/NMA residue to give b_n ⁺ ions. Calculations of the relative basicities of various sites in model peptide molecules containing Aze, Pro, Pip, or NMeA indicate that whereas the “proline effect’ can in part be rationalized by the increased basicity of the prolyl-amide site, the “pipecolic acid effect” cannot be justified through the basicity of the residue. Rather, the increased flexibility of the Pip and NMeA residues allow for conformations of the peptide for which transfer of the mobile proton to the amide site C-terminal to the Pip/NMeA becomes energetically favorable. This argument is supported by the differing results obtained for AAPAA versus AA(D-Pro)AA, a result that can best be explained by steric effects. Fragmentation of pentapeptides containing both Pro and Pip indicate that the “pipecolic acid effect” is stronger than the “proline effect.” Figure

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Protonated Dipeptide Losses from b 5 and b 4 Ions of Side Chain Hydroxyl Group Containing Pentapeptides

In this study, C-terminal protonated dipeptide eliminations were reported for both b ₅ and b ₄ ions of side chain hydroxyl group (–OH) containing pentapeptides. The study utilized the model C-terminal amidated pentapeptides having sequences of XGGFL and AXVYI, where X represents serine (S), threonine (T), glutamic acid (E), aspartic acid (D), or tyrosine (Y) residue. Upon low-energy collision-induced dissociation (CID) of XGGFL (where X?=?S, T, E, D, and Y) model peptide series, the ions at m/z 279 and 223 were observed as common fragments in all b ₅ and b ₄ ion (except b ₄ ion of YGGFL) mass spectra, respectively. By contrast, peptides, namely S_MeGGFL-NH₂ and E_OMeGGFL-NH₂, did not show either the ion at m/z 279 or the ion at m/z 223. It is shown that the side chain hydroxyl group is required for the possible mechanism to take place that furnishes the protonated dipeptide loss from b ₅ and b ₄ ions. In addition, the ions at m/z 295 and 281 were detected as common fragments in all b ₅ and b ₄ ion (except b ₄ ion of AYVYI) mass spectra, respectively, for AXVYI model peptide series. The MS⁴ experiments exhibited that the fragment ions at m/z 279, 223, 295, and 281 entirely reflect the same fragmentation behavior of [M?+?H]⁺ ion generated from commercial dipeptides FL-OH, GF-OH, YI-OH, and VY-OH. These novel eliminations reported here for b ₅ and b ₄ ions can be useful in assigning the correct and reliable peptide sequences for high-throughput proteomic studies.

Effect of N-terminal glutamic acid and glutamine on fragmentation of peptide ions

A prominent dissociation path for electrospray generated tryptic peptide ions is the dissociation of the peptide bond linking the second and third residues from the ammo-terminus. The formation of the resulting b₂ and y _n−2 fragments has been rationalized by specific facile mechanisms. An examination of spectral libraries shows that this path predominates in diprotonated peptides composed of 12 or fewer residues, with the notable exception of peptides containing glutamine or glutamic acid at the N-terminus. To elucidate the mechanism by which these amino acids affect peptide fragmentation, we synthesized peptides of varying size and composition and examined their MS/MS spectra as a function of collision voltage in a triple quadrupole mass spectrometer. Loss of water from N-terminal glutamic acid and glutamine is observed at a lower voltage than any other fragmentation, leading to cyclization of the terminal residue. This cyclization results in the conversion of the terminal amine group to an imide, which has a lower proton affinity. As a result, the second proton is not localized at the N-terminus but is readily transferred to other sites, leading to fragmentation near the center of the peptide. Further confirmation was obtained by examining peptides with N-terminal pyroglutamic acid and N-acetyl peptides. Peptides with N-terminal proline maintain the trend of forming b₂ and y _n−2 because their ring contains an imine rather than imide and has sufficient proton affinity to retain the proton at the N-terminus.     

Fragmentation Reactions of b<Subscript>5</Subscript> and a<Subscript>5</Subscript> Ions Containing Proline—The Structures of a<Subscript>5</Subscript> Ions

A detailed study has been made of the b₅ and a₅ ions derived from the amides H-Ala-Ala-Ala-Ala-Pro-NH₂, H-Ala-Ala-Ala-Pro-Ala-NH₂, and H-Ala-Ala-Pro-Ala-Ala-NH₂. From quasi-MS³ experiments it is shown that the product ion mass spectra of the three b₅ ions are essentially identical, indicating macrocyclization/reopening to produce a common mixture of intermediates prior to fragmentation. This is in agreement with numerous recent studies of sequence scrambling in b ions. By contrast, the product ion mass spectra for the a₅ ions show substantial differences, indicating significant differences in the mixture of structures undergoing fragmentation for these three species. The results are interpreted in terms of a mixture of classical substituted iminium ions as well as protonated C-terminal amides formed by cyclization/rearrangement as reported recently for a₄ ions (Bythell, Maître , Paizs, J . Am. Chem. Soc. 2010, 132, 14761–14779). Novel fragment ions observed upon fragmentation of the a₅ ions are protonated H-Pro-NH₂ and H-Pro-Ala-NH₂ which arise by fragmentation of the amides. The observation of these products provides strong experimental evidence for the cyclization/rearrangement reaction to form amides and shows that it also applies to a₅ ions.     

Specific rearrangement reactions of acetylated lysine containing peptide bn (n = 4–7) ion series

Characterization of ε‐N‐acetylated lysine containing peptides, one of the most prominent post‐translational modifications of proteins, is an important goal for tandem mass spectrometry experiments. A systematic study for the fragmentation reactions of b ions derived from ε‐N‐acetyllysine containing model octapeptides (K_AcYAGFLVG and YAK_AcGFLVG) has been examined in detail. Collision‐induced dissociation (CID) mass spectra of b_n (n = 4–7) fragments of ε‐N‐acetylated lysine containing peptides are compared with those of N‐terminal acetylated and doubly acetylated (both ε‐N and N‐terminal) peptides, as well as acetyl‐free peptides. Both direct and nondirect fragments are observed for acetyl‐free and singly acetylated (ε‐N or N‐terminal) peptides. In the case of ε‐N‐acetylated lysine containing peptides, however, specific fragment ions (m/z 309, 456, 569 and 668) are observed in CID mass spectra of b_n (n = 4–7) ions. The CID mass spectra of these four ions are shown to be identical to those of selected protonated C‐terminal amidated peptides. On this basis, a new type of rearrangement chemistry is proposed to account for the formation of these fragment ions, which are specific for ε‐N‐acetylated lysine containing peptides. Consistent with the observation of nondirect fragments, it is proposed that the b ions undergo head‐to‐tail macrocyclization followed by ring opening. The proposed reaction pathway assumes that b_n (n = 4–7) of ε‐N‐acetylated lysine containing peptides has a tendency to place the K_Ac residue at the C‐terminal position after macrocyclization/reopening mechanism. Then, following the loss of CO, it is proposed that the marker ions are the result of the loss of an acetyllysine imine as a neutral fragment. Copyright © 2014 John Wiley & Sons, Ltd.     

Studies of peptide a- and b-type fragment ions using stable isotope labeling and integrated ion mobility/tandem mass spectrometry

The structures of peptide a- and b-type fragment ions were studied using synthetic peptides including a set of isomeric peptides, differing in the sequence location of an alanine residue labeled with ¹⁵N and uniformly with ¹³C. The pattern of isotope labeling of second-generation fragment ions derived via a _n and b _n ions (where n=4 or 5) suggested that these intermediates existed in part as macrocyclic structures, where alternative sites of ring opening gave rise to different linear forms whose simple cleavage might give rise to the observed final products. Similar conclusions were derived from combined ion mobility/tandem MS analyses where different fragmentation patterns were observed for isomeric a- or b-type ions that display different ion mobilities. These analyses were facilitated by a new approach to the processing of ion mobility/tandem MS data, from which distinct and separate product ion spectra are derived from ions that are incompletely separated by ion mobility. Finally, an example is provided of evidence for a macrocyclic structure for b _n ions where n=8 or 9.     

Characterization of N‐Boc/Fmoc/Z‐N′‐formyl‐gem‐diaminoalkyl derivatives using electrospray ionization multi‐stage mass spectrometry

N‐Boc/Fmoc/Z‐N′‐formyl‐gem‐diaminoalkyl derivatives, intermediates particularly useful in the synthesis of partially modified retro‐inverso peptides, have been characterized by both positive and negative ion electrospray ionization (ESI) ion‐trap multi‐stage mass spectrometry (MSⁿ). The MS² collision induced dissociation (CID) spectra of the sodium adduct of the formamides derived from the corresponding N‐Fmoc/Z‐amino acids, dipeptide and tripeptide acids show the [M + Na‐NH₂CHO]⁺ ion, arising from the loss of formamide, as the base peak. Differently, the MS² CID spectra of [M + Na]⁺ ion of all the N‐Boc derivatives yield the abundant [M + Na‐C₄H₈]⁺ and [M + Na‐Boc + H]⁺ ions because of the loss of isobutylene and CO₂ from the Boc protecting function. Useful information on the type of amino acids and their sequence in the N‐protected dipeptidyl and tripeptidyl‐N′‐formamides is provided by MS² and subsequent MSⁿ experiments on the respective precursor ions. The negative ion ESI mass spectra of these oligomers show, in addition to [M‐H]^?, [M + HCOO]^? and [M + Cl]^? ions, the presence of in‐source CID fragment ions deriving from the involvement of the N‐protecting group. Furthermore, MSⁿ spectra of [M + Cl]^? ion of N‐protected dipeptide and tripeptide derivatives show characteristic fragmentations that are useful for determining the nature of the C‐terminal gem‐diamino residue. The present paper represents an initial attempt to study the ESI‐MS behavior of these important intermediates and lays the groundwork for structural‐based studies on more complex partially modified retro‐inverso peptides. Copyright © 2013 John Wiley & Sons, Ltd.     

Differentiation of Boc‐protected α,δ‐/δ,α‐ and β,δ‐/δ,β‐hybrid peptide positional isomers by electrospray ionization tandem mass spectrometry

Two new series of Boc‐N‐α,δ‐/δ,α‐ and β,δ‐/δ,β‐hybrid peptides containing repeats of L ‐Ala‐δ⁵‐Caa/δ⁵‐Caa‐L ‐Ala and β³‐Caa‐δ⁵‐Caa/δ⁵‐Caa‐β³‐Caa (L ‐Ala = L ‐alanine, Caa = C‐linked carbo amino acid derived from D ‐xylose) have been differentiated by both positive and negative ion electrospray ionization (ESI) ion trap tandem mass spectrometry (MS/MS). MSⁿ spectra of protonated isomeric peptides produce characteristic fragmentation involving the peptide backbone, the Boc‐group, and the side chain. The dipeptide positional isomers are differentiated by the collision‐induced dissociation (CID) of the protonated peptides. The loss of 2‐methylprop‐1‐ene is more pronounced for Boc‐NH‐L ‐Ala‐δ‐Caa‐OCH₃ (1), whereas it is totally absent for its positional isomer Boc‐NH‐δ‐Caa‐L ‐Ala‐OCH₃ (7), instead it shows significant loss of t‐butanol. On the other hand, second isomeric pair shows significant loss of t‐butanol and loss of acetone for Boc‐NH‐δ‐Caa‐β‐Caa‐OCH₃ (18), whereas these are insignificant for its positional isomer Boc‐NH‐β‐Caa‐δ‐Caa‐OCH₃ (13). The tetra‐ and hexapeptide positional isomers also show significant differences in MS² and MS³ CID spectra. It is observed that ‘b’ ions are abundant when oxazolone structures are formed through five‐membered cyclic transition state and cyclization process for larger ‘b’ ions led to its insignificant abundance. However, b₁⁺ ion is formed in case of δ,α‐dipeptide that may have a six‐membered substituted piperidone ion structure. Furthermore, ESI negative ion MS/MS has also been found to be useful for differentiating these isomeric peptide acids. Thus, the results of MS/MS of pairs of di‐, tetra‐, and hexapeptide positional isomers provide peptide sequencing information and distinguish the positional isomers. Copyright © 2010 John Wiley & Sons, Ltd.     

Using Gas-Phase Guest–Host Chemistry to Probe the Structures of b Ions of Peptides

Middle-sized b _n (n????5) fragments of protonated peptides undergo selective complex formation with ammonia under experimental conditions typically used to probe hydrogen?Cdeuterium exchange in Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Other usual peptide fragments like y, a, a*, etc., and small b _n (n????4) fragments do not form stable ammonia adducts. We propose that complex formation of b _n ions with ammonia is characteristic to macrocyclic isomers of these fragments. Experiments on a protonated cyclic peptide and N-terminal acetylated peptides fully support this hypothesis; the protonated cyclic peptide does form ammonia adducts while linear b _n ions of acetylated peptides do not undergo complexation. Density functional theory (DFT) calculations on the proton-bound dimers of all-Ala b ₄ , b ₅ , and b ₇ ions and ammonia indicate that the ionizing proton initially located on the peptide fragment transfers to ammonia upon adduct formation. The ammonium ion is then solvated by N⁺-H??O H-bonds; this stabilization is much stronger for macrocyclic b _n isomers due to the stable cage-like structure formed and entropy effects. The present study demonstrates that gas-phase guest?Chost chemistry can be used to selectively probe structural features (i.e., macrocyclic or linear) of fragments of protonated peptides. Stable ammonia adducts of b ₉ , b ₉ -A, and b ₉ -2A of A₈YA, and b ₁₃ of A₂₀YVFL are observed indicating that even these large b-type ions form macrocyclic structures.     

The promotion of d-type ions during the low energy collision-induced dissociation of some cysteic acid-containing peptides

Low energy collisionally activated dissociations (CAD) of doubly protonated peptides incorporating cysteic acid and arginine residues have been studied. Deuterium labeling experiments have established that loss of the elements of H₂SO₃ occurs with cleavage of one CH bond and transfer of the hydrogen to a neutral fragment. Prominent d-type ions were observed corresponding to cleavage at the cysteic acid residue. The analysis of structural analogs suggested that the unexpectedly low energy requirement for this process is attributable to a charge-proximal process promoted by intra-ionic interaction of the arginine and cysteic acid side chains. CAD (in the collision hexapole of a tandem quadrupole instrument) of electrospray source-formed fragment ions established that the d-type ions can form via b-type ions; there was no evidence of formation via (a _n + 1) or (b _n — H₂SO₃) ions. The equivalent d-ion was observed, albeit with lesser abundance, when the cysteic acid residue was replaced by aspartic acid, but not by glutamic acid.     

Combined Use of Post-Ion Mobility/Collision-Induced Dissociation and Chemometrics for b Fragment Ion Analysis

Although structural isomers may yield indistinguishable ion mobility (IM) arrival times and similar fragment ions in tandem mass spectrometry (MS), it is demonstrated that post-IM/collision-induced dissociation MS (post-IM/CID MS) combined with chemometrics can enable independent study of the IM-overlapped isomers. The new approach allowed us to investigate the propensity of selected b type fragment ions from AlaAlaAlaHisAlaAlaAla-NH₂ (AAA(His)AAA) heptapeptide to form different isomers. Principle component analysis (PCA) of the unresolved post-IM/CID profiles indicated the presence of two different isomer types for b₄ ⁺, b₅ ⁺, and b₆ ⁺ and a single isomer type for b₇ ⁺ fragments of AAA(His)AAA. We employed a simple-to-use interactive self-modeling mixture analysis (SIMPLISMA) to calculate the total IM profiles and CID mass spectra of b fragment isomers. The deconvoluted CID mass spectra showed discernible fragmentation patterns for the two isomers of b₄ ⁺, b₅ ⁺, and b₆ ⁺ fragments. Under our experimental conditions, calculated percentages of the “cyclic” isomers (at the 95 % confidence level for n = 3) for b₄ ⁺, b₅ ⁺, and b₆ ⁺ were 61 (± 5) %, 36 (± 5) %, and 48 (± 2) %, respectively. Results from the SIMPLISMA deconvolution of b₅ ⁺ species resembled the CID MS patterns of fully resolved IM profiles for the two b₅ ⁺ isomers. The “cyclic” isomers for each of the two-component b fragment ions were less susceptible to ion fragmentation than their “linear” counterparts.

Gas-phase conformation-specific photofragmentation of proline-containing peptide ions

Singly-protonated proline-containing peptides with N-terminal arginine are photodissociated with vacuum ultraviolet (VUV) light in an ESI linear ion trap/orthogonal-TOF (LIT/o-TOF). When proline is the n^th residue from the N-terminus, unusual b _n + 2 and a _n + 2 ions are observed. Their formation is explained by homolytic cleavage of the C_α− C bond in conjunction with a rearrangement of electrons and an amide hydrogen. The latter is facilitated by a proline-stabilized gas-phase peptide conformation.     

Effect of the sarcosine residue on sequence scrambling in peptide b5 ions

The effect of N‐methylation on sequence scrambling in the fragmentation of b₅ ions has been investigated by studying a variety of peptides containing sarcosine (N‐methylglycine). The product ion mass spectra for the b₅ ions derived from Sar‐A‐A‐A‐Y‐A and Sar‐A‐A‐Y‐A‐A show only minor signals for non‐direct sequence ions the major fragmentation reactions occurring from the unrearranged structures. This is in contrast to the b₅ ions where the Sar residue is replaced by Ala and sequence scrambling occurs. The b₅ ion derived from Y‐Sar‐A‐A‐A‐A shows a product ion mass spectrum essentially identical to the spectrum of the b₅ ion derived from Sar‐A‐A‐A‐Y‐A, indicating that in the former case macrocyclization has occurred but the macrocyclic form shows a strong preference to reopen to put the Sar residue in the N‐terminal position. Similar results were obtained in the comparison of b₅ ions derived from A‐Sar‐A‐A‐Y‐A and Sar‐A‐A‐Y‐A‐A. The product ion mass spectra of the MH⁺ ions of Y‐Sar‐A‐A‐A‐A and A‐Sar‐A‐A‐Y‐A show substantial signals for non‐direct sequence ions indicating that fragmentation of the MH⁺ ions channels extensively through the respective b₅ ions and further fragmentation of these species. Copyright © 2014 John Wiley & Sons, Ltd.