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The effect of charge state and the localization of charge on the collision-induced dissociation of peptide ions
Abstract
The effect that charge state has on the collision-induced dissociation (CID) of peptide ions is examined in detail for several
representative peptides under high-energy collision conditions. The CID spectra of singly and doubly charged precursor ions
(generated by fast-atom bombardment and electrospray ionization, respectively) are compared for several peptides with similar
primary structure. It is shown that for peptides that contain highly basic amino acids, the dissociation of doubly charged
ions is strongly influenced by the position of these residues within the peptide and the general observations reported concerning
the dissociation of singly charged ions can be extended to precursors with higher charge states. Based on the dissociation
behavior of the doubly charged ions of these peptides, it is demonstrated that two charges can reside in close proximity in
the precursor ions, overcoming possible repulsion effects, when favored by a high concentration of basic sites. In addition)’
this work illustrates that in the case of doubly charged ions..the charge state of some fragment ions can be determined directly
from the mass-to-charge ratio assignments of the CID spectrum.
... Most of the peaks at lower masses have been clearly assigned to the b n -series, due to peptide fragment ions from multistep cleavage of bonds along the peptide backbone [ 12 , 13 ]. The amide nitrogen connecting the two Glu residues is one of the most basic one, thereby facilitating its protonation [ 14 ]. This resulted in a weakening of the peptide bond and cleavage into a b 5 ion, exhibiting a signal at m / z 715. ...
The FEE cyclic hexapeptide (cFEE) is an investigational new drug added to the insemination medium in order to improve the in vitro fertilization rate. The pharmacological activity of small peptides is highly dependent on the conservation of the amino acid sequence and of the structural conformation of the active site. To enhance the scientific knowledge required for the clinical use of cFEE, a comprehensive determination of its chemical stability in solution was realized in accelerated conditions. Degradation products have been detected and identified by liquid chromatography/Qtrap(®) mass spectrometry. The main degradation products highlighted during the product shelf life were produced by hydrolysis and only certain sites were involved. In most cases, the cyclic conformation was lost and regarding the major degradation pathway, the sequence representing the active site was affected.
The use of smaller column diameters in liquid chromatography (LC) is often associated with capillary LC. Although there are many analytical benefits gained by adapting this format, routine use continues to be challenging due to column fragility and extra column dispersion. Bridging the gap between routinely used 2.1 mm columns and capillary bore columns allows for a sequential but far from insignificant increase in performance without the need for specialized equipment associated with very low dispersion LC systems. Moreover, an incremental decrease in column internal diameter (i.d.) allows for similar mass load (avoiding column overload that may be observed in much larger decreases in i.d. without trapping) and thus an increase in measured signal. As such, 1.5 mm i.d. columns provide an alternative intermediate dimension between the more regularly used 2.1 mm i.d. columns and 1 mm i.d. columns. These columns balance an increase in sensitivity compared to 2.1 mm i.d. columns (theoretically doubling the time-domain peak area in mass sensitive detectors for the same mass load), while mitigating the efficiency losses due to extra-column dispersion effects that are commonly observed with 1.0 mm i.d. columns. Here, the use of 1.5 mm i.d. columns was applied to LC/UV analysis of small molecules and LC/MS methods for the analysis of monoclonal antibodies. With equivalent mass load on column, the 1.5 mm i.d. columns provide two-to-threefold improvement in analyte peak area signal for small molecules as well as intact, subunit, and peptide levels of antibody analysis. Peak height was also increased using the 1.5 mm i.d. column, although the scale of increase varies between isocratic and gradient modes, likely due to differences in system dispersion effects and variation in electrospray ionization efficiency at different flow rates.
Advances in protein mass spectrometry have provided the ability to identify and sequence proteins with unprecedented speed, sensitivity and accuracy. These benefits now offer advantages for studies of protein evolution and phylogeny avoiding the need to generate and align DNA sequences which can prove time consuming, costly and difficult in the case of large genomes and for highly diverse organisms. The methods of phylogenetic analysis using protein mass spectrometry can be classified into three categories: (1) de novo protein sequencing followed by multiple sequence alignment for classical phylogenetic reconstruction, (2) direct phylogenetic reconstruction using expressed protein mass profiles exploited in microbial biotyping applications, and (3) the construction of trees using proteolytic peptide mass map or fingerprint data. This review describes the three approaches together with the relevant tools and algorithms required to implement them. It also compares each of these alternative protein based methods alongside conventional gene sequence based phylogenetics.
Charge transfer dissociation mass spectrometry (CTD-MS) has been shown to induce high energy fragmentation of biological ions in the gas phase and provide fragmentation spectra similar to extreme ultraviolet photodissociation (XUVPD). To date, CTD has typically employed helium cations with kinetic energies between 4-10 keV to initiate radical-directed fragmentation of analytes. However, as a reagent, helium has recently been listed as a critical mineral that is becoming scarcer and more expensive, so this study explored the potential for using cheaper and more readily available reagent gases. A model peptide, bradykinin, and a model oligosaccharide, κ-carrageenan with a degree of polymerization of 4, were fragmented using a variety of CTD reagent gases, which included helium, hydrogen, oxygen, nitrogen, argon and lab air. The CTD results were also contrasted with low-energy collision-induced dissociation (LE-CID), which were collected on the same 3D ion trap. Using constant reagent ion fluxes and kinetic energies, all five alterative reagent gases generated remarkably consistent sequence coverage and fragmentation efficiencies relative to He-CTD, which suggests that the ionization energy of the reagent gas has a negligible effect on the activation of the biological ions. The CTD efficiencies of all the gases ranged from 11-13% for bradykinin and 7-8% for κ-carrageenan. Within these tight ranges, the abundance of the CTnoD peak of bradykinin and the efficiency of CTD fragmentation of bradykinin both correlated with the ionization energy of the CTD reagent gas, which suggests that resonant charge transfer plays a small role in the activation of this peptide. The majority of the excitation energy for bradykinin and for κ-carrageenan comes from an electron stopping mechanism, which is described by long-range interactions between the reagent cations and electrons in the highest occupied molecular orbitals (HOMOs) of the biological ions. The CTD spectra do not provide any evidence for covalently bound products between the biological ions and the more-reactive gases like hydrogen, oxygen and nitrogen, which implies that the high kinetic energies of the reagent ions make them unavailable for covalent reactions. This work demonstrates that any of the substitute reagent gases tested are viable options for future CTD-MS experiments.
Discovery of a new drug is time-consuming, laborious, and expensive. Herein, a novel integrative strategy for discovering potential new lead compounds has been developed, which was based on the characteristics of mass spectrometry (MS). MS was used to predict the potential forced degradation products (DPs) and metabolites of drugs by electrospray ionization and collision-induced dissociation (CID). Special rearrangement ions representing unique predicted DPs and metabolites were identified. The consistency between the predicted and the measured results was proven by in vitro metabolism and forced degradation of a commercial drug, respectively. From this, new chemical scaffold rearrangement ions named (aza)-biphenylenes, as potent anticancer agents, were discovered. As a representative aza-biphenylene analogue, 2-azabiphenylene was proven in vitro to induce apoptosis and inhibit the growth of various human cancer cells in a dose-dependent manner. Surprisingly, 2-azabiphenylene exhibited the best comparable bioactivity with the positive control sorafenib, but showed significantly lower in vitro cytotoxicity than sorafenib (at least a 5-fold decrease in cytotoxicity) because it could be targeted to the tumor microenvironment at low pH. A biradical mechanism accompanied by a mitochondrion-dependent oxidative stress mechanism was proposed to explore its anticancer mechanism. The highly reactive intermediate aza-biphenylenediyl worked as an active pharmaceutical ingredient and induced apoptosis of cancer cells. This provided the basis for the potential applications of CID-induced special rearrangement ions in developing new lead compounds.
The ability to target discrete features within tissue using liquid surface extractions enables the identification of proteins while maintaining the spatial integrity of the sample. Here we present a liquid extraction surface analysis (LESA) workflow, termed microLESA, that allows proteomic profiling from discrete tissue features of ~110 μm in diameter by integrating non-destructive autofluorescence microscopy and spatially targeted liquid droplet micro-digestions. Autofluorescence microscopy provides the visualization of tissue foci without the need for chemical stains or the use of serial tissue sections. Tryptic peptides are generated from tissue foci by applying small volume droplets (~250 pL) of enzyme onto the surface prior to LESA. The microLESA workflow reduced the diameter of the sampled area almost 5-fold compared to previous LESA approaches. Experimental parameters such as tissue thickness, trypsin concentration, and enzyme incubation duration were tested to maximize proteomics analysis. The microLESA workflow was applied to the study of fluorescently-labeled Staphylococcus aureus infected murine kidney in order to identify unique proteins related to host defense and bacterial pathogenesis. Proteins related to nutritional immunity and host immune response were identified by performing microLESA at the infectious foci and surrounding abscess. These identifications were then used to annotate specific proteins observed in infected kidney tissue by MALDI FT-ICR IMS through accurate mass matching.
Progress in proteomics is mainly driven by advances in mass spectrometric (MS) technologies. Here we benchmarked the performance of the latest MS instrument in the benchtop Orbitrap series, the Q Exactive HF-X, against its predecessor for proteomics applications. A new peak picking algorithm, a brighter ion source and optimized ion transfers enable productive MS/MS acquisition above 40 Hz at 7500 resolution. The hardware and software improvements collectively resulted in improved peptide and protein identifications across all comparable conditions, with an increase of up to fifty percent at short LC-MS gradients, yielding identification rates of more than one thousand unique peptides per minute. Alternatively, the Q Exactive HF-X is capable of achieving the same proteome coverage as its predecessor in approximately half the gradient time or at 10-fold lower sample loads. The Q Exactive HF-X also enables rapid phosphoproteomics with routine analysis of more than five thousand phosphopeptides with short single-shot 15-minute LC-MS/MS measurements, or 16,700 phosphopeptides quantified across ten conditions in six gradient hours using TMT10-plex and offline peptide fractionation. Finally, exciting perspectives for data independent acquisition are highlighted with reproducible identification of 55,000 unique peptides covering 5900 proteins in half-an-hour of MS analysis.
Full text at https://pubs.acs.org/doi/10.1021/acs.jproteome.7b00602
The first systematic and comprehensive study of the charging behaviour and effect of charge on the conformation of specifically constructed arginine-rich peptides and its significance to the N- and C-terminal basic tail regions of histone proteins was conducted using ion mobility mass spectrometry (IM-MS). Among the basic amino acids, arginine has the greatest impact on the charging behaviour and structures of gas phase ions by virtue of its high proton affinity. A close linear correlation was found between either the maximum charge, or most abundant charge state, that the peptides support and their average collision cross section (CCS) values measured by ion mobility mass spectrometry. The calculated collision cross sections for the lowest energy solution state models predicted by the PEP-FOLD algorithm using a modified MOBCAL trajectory method were found to best correlate with the values measured by IM-MS. In the case of the histone peptides, more compact folded structures, supporting less than the maximum number of charges, were observed. These results are consistent with those previously reported for histone dimers where neutralization of the charge at the basic residues of the tail regions did not affect their CCS values.
Collision energy and fragmentation pathways of doubly-and triply-protonated peptide RRMKWKK upon low-energy collision-induced dissociation (CID) were investigated with ESI-MS/MS. Triply-protonated ion dissociated more easily than the doubly-protonated ion, with a dependence of the number of charges vs. the number of the arginine residues in the peptide. The two basic Arg residues were the primary charge location, resulted in a high dissociation energy for Arg-contained peptides. The protonated ions with one more proton than the number of Arg residues in the peptides were suggested when Arg-containing peptides were studied by using mass spectroscopy. Secondary structures of the protonated RRMKWKK calculated by AM1 method indicate that the two protons locating at the basic side chains of the two adjacent Arg were separated in space, so that Coulomb repulsion did not affect the proton location.
The use of double focussing and tandem sector instruments has decreased during the 1990s, owing to the difficulty of interfacing electrospray and matrix-assisted laser desorption ionizations with them. Nevertheless, their high dynamic range, resolving power in the 10,000 range, exact-mass-measurement and high-energy-collisional-activation capabilities continue to recommend their use and improvement. This chapter is a review of analyses for which sectors are well, or even uniquely, qualified and of instrumentation advances that incorporate array detectors and other mass analyzers to give a new generation of hybrid instruments.
VIGNY P., Professeur des Universités détaché au CNRS : Président LANGE C., Professeur, Université de Rouen : Co-Directeur DELMAS A., Directeur de recherche CNRS Orléans :Co-Directeur TORTAJADA J., Directeur de recherche CNRS Université d'Evry Val d'Essonne : Rapporteur LAPREVOTE O., Directeur de recherche CNRS Gif sur Yvette : Rapporteur LAFOSSE M., Professeur, Universitéd'Orléans : Examinateur
Selective cleavage effect of basic residues in the fragmentation of short peptides has been studied intensively. In contrast, the role of basic residues in the degradation of large peptides, such as cell-penetrating peptides, is largely unknown. In this work, the fragmentation of a 21 residues cell-penetrating peptide TP10 containing four lysine residues was studied by collision-induced dissociation mass spectrometry and computation methods. The influence of lysine residues on amide bond cleavage and fragmentation products was investigated. The results revealed that the selective cleavage effect of lysine residue did not present when the adjacent lysine residues in TP10 were both protonated. The localized high positive charge density might be the reason of preventing the mobile proton from migrating to the amide bonds in this part of the peptide. In contrast, the mobile proton preferred to reside in the N-terminal part of TP10 which had less positive charge. This preference gave more information of the peptide sequence in the mass spectrometry study and was helpful for stabilizing the C-terminal part of TP10, in which the basic lysine residues were preserved and crucial to the cell-penetrating process. Copyright © 2015 John Wiley & Sons, Ltd.
Copyright © 2015 John Wiley & Sons, Ltd.
The dissociation of singly or multiply protonated peptide ions by using low-energy collisional activation (CA) is highly dependent on the sites of protonation. The presence of strongly basic amino acid residues in the peptide primary structure dictates the sites of protonation, which generates a precursor ion population that is largely homogeneous with respect to charge sites. Attempts to dissociate this type of precursor ion population by low-energy CA result in poor fragmentation via few pathways. The work described here represents a systematic investigation of the effects of charge heterogeneity in the precursor ion population of a series of model peptides in low-energy CA experiments. Incorporation of acidic residues in the peptide RLC*IFSC*FR (where C* indicates a cysteic acid residue), for example, balances the charge on the basic arginine residues, which enables the ionizing protons to reside on a number of less basic sites along the peptide backbone. This results in a precursor ion population that is heterogeneous with respect to charge site. Low-energy CA of these ions results in diverse and efficient fragmentation. Molecular modeling has been utilized to demonstrate that energetically preferred conformations incorporate an intraionic interaction between arginine and cysteic acid residues.
The tandem mass spectrometry fragmentation reactions of 21 protonated N-acyl amino acid methyl esters are examined as models for more complicated peptides. Four main types of reactions are observed: loss of CH2CO from the N-terminal acetyl group; loss of CH3OH from the C-terminal ester group to yield a model system for a b2 ion structure; loss of water from amino acids without an OH side chain group; fragmentation of the side chain by way of small molecule loss (e.g. H2O, NH3, and CH3SH). CH3OH loss is the only common reaction observed for all systems. The resultant [M+H−CH3OH]+ ions were examined in further detail by way of MS3 experiments because previous studies have shown that the oxozolone structures liberate CO. Only lysine and arginine do not fragment by way of CO, which is suggestive of alternative cyclic structures involving the side chain. Ab initio calculations (at the MP2/6-31G∗//HF/6-31G∗) were carried out on isomeric b2 ions of both types (oxazolone and that involving side-chain interaction) derived from arginine, histidine, lysine, methionine, asparagine, glutamine, and serine. For arginine, histidine, and lysine the cyclic structures involving the side chain are more stable than the oxozolone structures. Finally, solution phase data relevant to the gas phase processes are highlighted.
Clusters, Detection, Ion trap, Photon impact, Review, Structure confirmation, Surface.
The extent of charging (or protonation) during the electrospray ionization process has been examined for a series of specifically constructed arginine-rich peptides, which differ in structure by the length of the peptide chain and the number and proximity of arginine residues. It has been found that although a small peptide of the series will protonate fully, supporting a charge on each arginine side-chain, the same charging behavior is not observed for larger peptides with the same repeating primary structure. Furthermore, no significant increase in the extent of charging was observed as the length of the peptide chain, or the distance between potential charge-bearing sites, was increased. The apparent sites of protonation in the [M + nH]n+ peptide ions have been examined for several representative peptides based on the extent of protonation compared to that of structurally related peptides, and their dissociation behavior. Despite the potential for proton migration during the collisional activation event, the fragmentation pattern of the peptide ions studied suggests that the charge-bearing protons are reasonably localized at the time of dissociation commensurate with our previous observations for singly and multiply charged peptide ions. The charging behavior of the model peptides is discussed in the context of a reported mechanism for the electrospray ionization process.
Multiply-deprotonated, single-strand products of mono and heteronucleotides, prepared in a nanospray external source coupled to an ion trap mass spectrometer, were studied under low-energy collision conditions. Fragmentations enabled the negative charge carried by the selected anions to be located. Comparison of multi-deprotonated 5'-dephosphorylated and 5'-phosphorylated nucleotides showed that the former led to competitive BH and B- losses via isomerization into ion-dipole complexes prior to dissociation, which contrasts with the 5'-phosphorylated nucleoside yielding the competitive HPO3 and nucleobase eliminations. The proposed mechanisms are discussed according to: (i) the charge state of the precursor studied and (ii) the thermochemical gas-phase properties such as the relative acidities of neutrals, which formally constitute the ion-dipole. The formation of various fragment ions from doubly- and triply-deprotonated precursor ions enabled the negative charge to be located, specifically at the terminal phosphodiester groups of the single-strand oligonucleotides. The potential energy is, therefore, minimal for distant charged phosphorylated groups. These findings suggest that a specific conformation is favored in order to minimize coulomb repulsion during the desolvation process. This hypothesis is consistent with the assumed behavior of triply- and quadruply-deprotonated, mono-cationized oligonucleotides, which involves a specific location of the sodium cation, as previously shown.
It has been shown that neighboring group participation plays an important role in the fragmentation of protonated amides;
the attachment of an adjacent functional group capable of accepting a proton provides alternative pathways of low energy for
the formation of the inevitable N-protonated species in the fragmentation of the amide bond. Under methane chemical ionization (CI) conditions, protonated
aniline (m/z 94) is only 1. 6% of the base peak MH+ ion for acetanilide; the abundance of the m/z 94 ion is increased to 15% for acetoacetanilide and protonated o-methoxyaniline reaches a relative intensity of 49% for N-acetyl-o-methoxyaniline. A more striking difference in ease of the formation of protonated anilines is found for acetanilides bearing
a nitro group at different positions. Protonated nitroaniline (m/z 139) is the base peak in the methane CI spectrum of N-acetyl-o-nitroaniline; the m/z 139 ion drops to only 0.7% for the para isomer, and this ion is increased to 31.5% in the spectrum of N-acetoaceto-p-nitroaniline. By employing low energy collision-induced dissociation, it has been found that the fragmentation of protonated
amides proceeds by way of ion-neutral complexes. In the case of acetanilide, for example, the cleavage of the amide bond gives
rise to an acetylium ion and neutral aniline, which are bound together as a complex. An α-hydrogen of the acetylium ion, which
is activated by the positive charge, is captured by aniline due to its higher proton affinity as compared with ketene. For
those compounds having mobile protons other than the amidic hydrogen, it is indicated that such proton has the priority to
be transferred in the reaction. Thus, the proton on the free carboxyl group of N-phenyl succinic and maleic monoamides is transferred in the fragmentation, leading to anhydrides as the neutral species in
the formation of protonated aniline.
We report on the formation and collision-induced dissociation of hydrogen deficient peptide radical cations of the type [M+nH](n+1)+·. These ions are formed from multiply protonated precursors (i.e. [M+nH]n+) by collisional electron transfer in high energy collisions with dioxygen. Increasing the charge state of doubly protonated bradykinin ([BK+2H]2+), results in a selective enhancement of b type ions with an especially dominant b6 ion. Further, we observe a c4 ion that is ascribed to the radical character of the triply charged ion. The charge reversal spectrum of deprotonated bradykinin [BKH] provides additional information with the appearance of z type ions, probably formed from the hydrogen-deficient [BKH]+· radical cation which is also observed. The fragmentation pattern of the triply charged gramicidin S radical ([GS+2H]3+·) also differs from those of the singly and doubly protonated peptide and reveals structure-indicative acylium ions such as VOLFPVOL and OLFP (O = Ornithine).
Recent experiments on biomolecules in accelerators and storage rings are reviewed. Topics included are measurements of geometrical cross sections of macromolecular ions, fragmentation of peptide and oligonucleotide ions after collisional electron transfer, lifetimes of excited ions, and radiative cooling. Statistical versus non-statistical decay after excitation in collisions or by photon absorption is discussed.
The tandem mass spectrometry fragmentation reactions of 21 protonated N-acyl amino acid methyl esters are examined as models for more complicated peptides. Four main types of reactions are observed: loss of CH2CO from the N-terminal acetyl group; loss of CH3OH from the C-terminal ester group to yield a model system for a b2 ion structure; loss of water from amino acids without an OH side chain group; fragmentation of the side chain by way of small molecule loss (e.g. H2O, NH3, and CH3SH). CH3OH loss is the only common reaction observed for all systems. The resultant [M+H−CH3OH]⁺ ions were examined in further detail by way of MS³ experiments because previous studies have shown that the oxozolone structures liberate CO. Only lysine and arginine do not fragment by way of CO, which is suggestive of alternative cyclic structures involving the side chain. Ab initio calculations (at the MP2/6-31G∗//HF/6-31G∗) were carried out on isomeric b2 ions of both types (oxazolone and that involving side-chain interaction) derived from arginine, histidine, lysine, methionine, asparagine, glutamine, and serine. For arginine, histidine, and lysine the cyclic structures involving the side chain are more stable than the oxozolone structures. Finally, solution phase data relevant to the gas phase processes are highlighted.
The analysis of organic and biological substances by secondary ion mass spectrometry (SIMS) has greatly benefited from the use of cluster ions as primary bombarding species. Thereby, depth profiling and three-dimensional (3D) imaging of such systems became feasible. Large Arn (+) cluster ions may constitute a further improvement in this direction.
To explore this option, large Arn (+) cluster ions (with n ~1500 Ar atoms per cluster) were used to investigate the emission of positive secondary ions from two peptide specimens (angiotensin I and bradykinin) by orthogonal time-of-flight SIMS using bombarding energies 6, 10 and 14 keV.
For both peptides, the protonated molecular ion is observed in the mass spectra. In addition, distinct fragmentation patterns were observed; these indicate that fragment ions under Ar cluster irradiation form primarily via cleavage of bonds along the peptide backbone whereas the rapture of side chains occurs much less frequently. These features appear to be similar to low-energy collision-induced dissociation pathways.
Tentatively, these findings can then be ascribed to the concerted action of the large number of Ar atoms in the impact zone of cluster at the surface: these low-energy Ar species (with an average energy of few eV) may effect the cleavage of the peptide bonds and lead, eventually, to the emission of the fragment ions. Copyright © 2013 John Wiley & Sons, Ltd.
Singly, doubly, and triply charged bradykinin ions have been dissociated thermally in a quadrupole ion trap operated with a bath gas comprised principally of helium at 1 mTorr. Dissociation rates as a function of bath gas temperature were measured along with product ion spectra. Studies were conducted to show that dissociation rates were independent of ion storage conditions and bath gas pressure. These studies indicate that good approximations to true Arrhenius parameters could be determined from Arrhenius plots using the bath gas temperature as a measure of the internal energy distribution of the parent ion population. Arrhenius parameters derived in this work agree with those reported recently using blackbody-induced radiative dissociation for the singly and doubly charged ions within experimental error. The spectrum of dissociation products were also very similar, reflecting the equivalency of the two methods for deriving Arrhenius parameters. Arrhenius parameters for the triply charged ion (Ea= 0.79 ± 0.03 eV; log A = 9.3 ± 0.36 s-1) are reported for the first time. Comparison of the Arrhenius parameters for the doubly and triply charged parent ions, along with dissociation reactions of the ions formed via water loss from the two parent ions indicate that water loss reactions from doubly protonated bradykinin and from triply protonated bradykinin proceed via distinctly different mechanisms.
Gas-phase fragmentation patterns of poly(propylene imine) dendrimers DAB-dendr-(NH2)n with n = 4, 8, 16, 32, and 64 are described as a result of MSn electrospray ionization mass spectrometry (ESI-MS) studies. The generally accepted on-the-average globular shape of dendrimers is supported here by the linear relationship found between the extent of protonation and molecular mass (M2/3). Unique fragmentation patterns are observed due to this globular shape. Most fragment ions are readily accounted for when, in addition to nucleophilic displacement reactions, proton shifts and rearrangements are taken into consideration. There is a striking difference in fragmentation behavior between singly charged dendrimers and multiply charged dendrimers. Therefore, it is proposed that the site of protonation within the dendritic framework has a large impact on the fragments observed. The position of the proton is largely dependent on both the relative proton affinities and the Coulomb repulsion between neighboring protonated sites. In case of singly charged dendrimers, electrostatic interactions are absent and the fragmentation is predominantly governed by the relative differences in proton affinity between the basic sites present in the dendrimer. In the gas phase the nitrogen atoms in the core of the dendrimer are the most basic ones present, leading to [M + H]+ ions carrying a protonated diaminobuthane core in all cases. This triggers a cascade of nucleophilic displacement reactions, starting with a bond cleavage of the central dendritic core and finishing with the formation of bis(1,1-propylamine)azetidinium (m/z 172) and fragments derived thereof. In the case of multiply charged dendrimers, Coulomb repulsions lead primarily to protonation of the peripheral tertiary amines, leading to the direct formation of bis(1,1-propylamine)azetidinium. These gas-phase reactions were subsequently utilized as a model for reactions in solution. The gas-phase reactions in ESI-MS thus provided a unique “fingerprint” area of dendrimer fragmentation adducts in the lower m/z range (m/z < 180), which matches perfectly with that obtained from dendrimers after prolonged heating in aqueous solution.
Relative energetics of fragmentation of protonated peptides are investigated by using electrospray ionization/surface-induced dissociation (ESI/SID) tandem mass spectrometry. ESI/SID fragmentation efficiency curves (percent fragmentation versus laboratory collision energy) are presented for 20 oligopeptides and are a measure of how easily a peptide fragments. The relative positions of the ESI/SID fragmentation efficiency curves depend on several parameters which include peptide composition (e.g., presence/absence of a basic amino acid residue) and peptide size. The ESI/SID fragmentation efficiency curves, in combination with quantum chemical calculations, provide a unique approach to substantiate and refine the mobile proton model for peptide fragmentation. Selected peptides are also investigated to further test and confirm the mobile proton model; these include doubly-protonated peptides and chemically-modified peptides (i.e., acetylated and fixed-charge derivatized peptides). Doubly-protonated peptides fragment more easily than the singly-protonated forms of the same peptides, with a sequence dependence for the difference in energy required for the fragmentation of singly- vs doubly-protonated peptides. Acetylation at the amino terminus and arginine side chain leads to a decrease in basicity and a corresponding lower energy onset for fragmentation than for the unmodified form of the peptide. Fixing the site of charge by addition of trimethylammonium acetyl to the amino terminus, i.e., eliminating the mobile proton, results in a higher energy onset than that for the protonated form of the same peptide. Curves for doubly protonated peptides with two adjacent basic residues (Arg, Arg) suggest the localization of the two protons at the two basic side chains rather than at opposite termini of the peptide.
Six years after coining the term ‘mass spectrometric (MS) peptide charting’ for the component analysis of peptide mixtures in a whole tissue, body fluid, or an extract thereof, we offer our current perspective of this field. Matrixassisted laser desorption/ionization and electrospray ionization have replaced plasma desorption and fast atom bombardment as ionization methods of choice. At the same time, the upper mass range has been extended to now include most peptides and proteins of interest to research on cell-cell communication. In addition to qualitative aspects, quantitative applications of MS charting have become important. In combination with new database search algorithms, on-line liquid chromatography-tandem mass spectrometry promises greater dividends from MS charting than are achievable with molecular mass matching alone. We discuss what is and is not yet possible, and consider foreseeable means for overcoming current limitations. Our intent is to encourage researchers in the biological and medical sciences to take advantage of this powerful methodology in their various fields of endeavor.
Soft-landing of singly and doubly protonated peptide ions onto three self-assembled monolayer surfaces (SAMs) was performed using a novel ion deposition instrument constructed in our laboratory and a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially designed for studying collisions of large ions with surfaces. Modified surfaces were analyzed using in situ 2 keV Cs+ secondary ion mass spectrometry or ex situ 15 keV Ga+ time-of-flight-secondary ion mass spectrometry (ToF-SIMS). The results demonstrate that a fraction of multiply protonated peptide ions retain more than one proton following soft-landing on the FSAM surface. It is shown that the [M+2H]2+ ions observed in FT-ICR SIMS spectra are produced by desorption of multiply charged ions from the surface, while re-ionization of singly protonated ions or neutral peptides is a source of [M+2H]2+ ions in ToF-SIMS spectra. Differences in neutralization efficiency of soft-landed ions following exposure of surfaces to laboratory air has a measurable effect on the results of ex situ ToF-SIMS analysis of soft-landed ions on SAM surfaces.
The occurrence of charge-separation reactions in tandem mass spectrometry of doubly protonated angiotensin II is demonstrated by the use of mass-analyzed ion kinetic energy spectrometry (MIKES) and kinetic energy release distributions (KERDs). Linked scans at a constant B/E severely discriminate against product ions formed by charge-separation reactions. Although the products are significantly more abundant in MIKES experiments, instrumental discrimination still makes quantitation of relative product ion abundances highly inaccurate. The most probable KERs (T
m. p.) and the average KERs (T
ave.) of the reactions are determined from the KERDs, and these values are compared to the KERs determined from the peak widths at half-height (T
0. 5). The measurement of T
0. 5 is a poor approximation to T
m. p. and T
ave.. The T
m. p. is used to calculate a most probable intercharge distance, which is compared to results from molecular dynamics calculations. The results provide evidence with regard to the mechanisms of fragmentation of multiply charged ions and the location of the charge site in relation to the decomposition reactions.
Proton transfer properties were studied for doubly-protonated ions from bradykinin and seven smaller peptides whose sequences have C-terminal residues successively removed from bradykinin. Ions were generated by electrospray ionization (ESI) and their deprotonation reactions were investigated in a Fourier transform ion cyclotron resonance mass spectrometer. Sustained off-resonance irradiation (SORI)-collision-induced dissociation (CID) was used to obtain information on sites of protonation, while molecular dynamics calculations provided information on peptide ion conformations and Coulomb energies. As the number of amino acid residues decreases the peptide ions undergo proton transfer more readily. Apparent gas-phase acidities (GAapps), for the doubly-protonated ions decreased as the peptide size decreased, with values spanning the range of 225.8±4.2 kcal/mol for the nonapeptide bradykinin to 196.6±4.4 kcal/mol for the tripeptide Arg1Pro2Pro3. The magnitude of the drop in GAapp as a residue was removed varied from 10.9 kcal/mol for removal of the highly basic Arg9 to 0 kcal/mol for removal of Phe8 (whose phenylalanine ring juts away from the peptide backbone and does not participate in hydrogen bonding). Hydrogen bonding in the modeled structures decreased as the peptide chain length decreased. The lowest energy structure for doubly-protonated bradykinin contained nine hydrogen bonds, while only one hydrogen bond was found for the tripeptide ions. In addition, the tripeptide ions had an extended structure, while the larger peptide ions were compact. There is no single reason that the peptide ions become more acidic (i.e., more reactive to proton transfer) as the peptide chain length is decreased. Depending upon the situation, factors that play a role in the proton transfer reactivity include intrinsic basicity of the protonation sites, intramolecular hydrogen bonding (particularly involving the protonated residue), Coulombic repulsion of charge sites, and conformational considerations such as accessibility/shielding of the protonation site.
Mass-analyzed ion kinetic energy (MIKE) spectrometry has been employed to measure intercharge distances in the doubly protonated octapeptide PPGFSPFR. The interpretation of the experimental observations, aided by macromolecular mechanics calculations, suggests that two isomeric forms of the doubly charged ions are produced by both FAB and electrospray, which differ from each other by the location of one of the protons. Different fragmentation pathways are shown to occur in each of these isomers. This is interpreted following the mechanism of Adams et al., J. Am. Soc. Mass Spectrom., 7 (1996) 30.
Neutral peptides of natural amino acids of the type (X)(n)-Y (n = 1,2,3) are prepared in the gas phase by laser desorption and supersonic cooling. Local ionization is performed by resonant laser excitation in aromatic amino acids (Y) located at the C-terminal end. In a one-color experiment, subsequent UV photofragmentation of the cation is shown to directly reflect the prior charge migration in these large molecules. Peptides are engineered, which show either fragment ions originating from the chromophore or from the opposite N-terminal side (X). The results show that by changing local ionization energies and thus absolute positions of ionic dissociation energies, one has complete control over different paths of chemical reactivity. The length dependence of the process shows, that charge mobility seems to be not the bottleneck for dissociation pathways at high internal energies: charge transfer over more than 10 sigma-bonds is shown. When we apply a local picture and estimate local ionization potentials, we find, for the peptides used here, that after localized ionization, positive charge is statically localized at the initial prepared site. In a two-color experiment (UV + VIS) we observe indications that in the photoexcited tripeptide cation Leu-Leu-Tyr charge transfer can occur at internal energies of about 2.2 eV, an energy at which no dissociation occurs. We interpret the process in terms of direct photoexcitation into a charge transfer (CT) band or by a photoexcitation to a localized state followed by nonradiative relaxation to a CT state. For the charge-transfer process we propose a through bond HOMO electron transfer (hole transfer) as the relevant mechanim. Consequences of our findings for charge migration and fragmentation processes in peptides are discussed.
A new tandem time-of-flight mass spectrometer with an electrospray ionization ion source 'ESI-TOF/quadTOF' was designed and constructed to achieve the desired aim of structural elucidation via high-energy collision-induced dissociation (CID), and the simultaneous detection of all fragment ions. The instrument consists of an orthogonal acceleration-type ESI ion source, a linear TOF mass spectrometer, a collision cell, a quadratic-field ion mirror and a microchannel plate detector. High-energy CID spectra of doubly protonated angiotensin II and bradykinin were obtained. Several fragment ions such as a-, d-, v- and w-type ions, characteristic of high-energy CID, were clearly observed in these spectra. These high-energy CID fragment ions enabled confirmation of the complete sequence, including leucine-isoleucine determinations. It was demonstrated that high-energy CID of multiply protonated peptides could be achieved in the ESI-TOF/quadTOF.
MALDI in combination with high-energy collision-induced dissociation (CID) performed by tandem time-of-flight mass spectrometry (TOF/RTOF) is a relatively new technology for the structural analysis of various classes of biomolecules as e.g., peptides, carbohydrates, glycoconjugate drugs and lipids. Fragmentation mechanisms for these classes of compounds as well as corresponding fragment ion nomenclatures based mainly on data from tandem magnetic sector mass spectrometers are summarized in this article. The major instrumental differences between the present commercially available TOF/RTOFs are compiled (e.g., ion gate, gas-collision cell, type of reflectron, etc.). Whereas peptides have been investigated by MALDI-TOF/RTOF and their CID spectra are well understood, other classes of compounds (e.g., carbohydrates or lipids) are far less well investigated. By comparing data from two different MALDI-TOF/RTOF-instruments, it becomes evident that as they are operated at rather different collision energies for CID (1 versus 20 keV) strong differences in corresponding CID spectra for the same analyte are observed, causing problems with library searches in databases as e.g., abundant peptide side-chain fragmentations mainly occurring in the 8 to 20 keV collision regime are not considered. In contrast, differences in CID spectra of carbohydrates among different TOF/RTOF instruments are less clear-cut, because the required collision energy is spread across a wide range. Especially, carbohydrate cross-ring cleavages require less collision energy in the keV-range than the corresponding peptide side-chain fragmentations. Some of these carbohydrate cross-ring fragmentations are even observed by very low energy CID (< 1 eV fragmentation amplitude). Similar observations can also be made for glycoconjugates (e.g., the drug tylosin A). The lipid class triacylglycerol needs rather high collision energies for dissociating carbon-carbon bonds based upon classical charge-remote fragmentation mechanisms. Comparison of high-energy CID-data of ESI generated triacylglycerol precursors with CID spectra from MALDI generated precursors shows different mechanisms for charge-remote fragmentations. MALDI-TOF/RTOF-instruments operated in the elevated high-energy CID mode exhibit a strong potential in structural analysis of natural and synthetic biomolecules with information often not obtainable by low energy CID.
This review is intended to give readers a snapshot of current mass spectrometry for proteomics research. It covers a brief history of mass spectrometry proteomic research, peptidomics and proteomics for biomarker search, quantitative proteomics, proteomics with post-translational modification and future perspective of proteomics.
Single-frequency collisional activation of multiply charged peptides has been studied via electrospray ionization in an ion trap storage/reflectron time-of-flight device (IT/reTOF). Several peptides with molecular weights ranging from 600 to 1700 were used to demonstrate that sequence information can be obtained with this hybrid instrument with a sensitivity in the low picomole level. Further, a resolution of nearly 1000 can be obtained for the fragment products, due to the cooling effects of the buffer gas in the trap before analysis by the reTOF. The influence of the primary structure of the peptides on the observed collision-induced fragmentation patterns is discussed. Although the current study is limited by the electronics, in terms of its ion isolation capabilities, it is demonstrated that the device has the potential for obtaining sequence information for peptides with excellent sensitivity and relatively high resolution.
A statistical study of the fragmentation behaviour of 138 model peptides, containing 3-9 amino acid residues (n = 3-9) under high-energy collision conditions is presented. The aim was to identify characteristic patterns of ions in the spectra of peptides which can be translated into general rules to be used in the spectral interpretation and provide a better insight into their fragmentation behaviour. It was found that both number and nature of the amino acids are important factors directing the fragmentation behaviour. The spectra of tri- and tetrapeptides exhibit a comparable probability for the formation of B2- and Y"n-2 ions, whereas larger peptides show a preference for the formation of Bn-1 ions. This generally observed fragmentation pattern of peptides is changed significantly when basic amino acid residues (Arg, Lys and His) and/or Pro are present Arginine appears to have the most pronounced influence on the fragmentation behaviour and overrules that of the other amino acid residues.
Sequencing conditions for postsource decay and collision-induced dissociation/postsource decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry have been optimized to elucidate the structure of polyglycylation of tubulin. This posttranslational modification involves the linkage of multiple glycine residues through the gamma-carboxyl of glutamic acid residues in the carboxyl termini of the protein. Individual alpha- and beta-tubulin polypeptides contain respectively three and four potential glycylation sites. The sample preparation we used was the thin-layer preparation of the target specimen in the presence of alpha-cyano-4-hydroxycinnamic acid and nitrocellulose. The study of different synthetic polyglycylated peptides fragmentation (modified peptides with the linear sequence DATAEEEGEFEEEGEQ) shows that the peptides fragment regularly to form major fragments of b- and y-type ions with negligible side-chain fragmentation. The rules were applied to the structural elucidation of a Paramecium beta-tubulin hexaglycylated peptide available in the subpicomole range. Polyglycylation was identified on the last four glutamic acid residues.
The hexakis (4-diemthylaminopyridyl) benzene hexacation of 1 is investigated as an extreme example of the ability of electrospray ionization to allow transfer of small multivalent ions to the gas phase. The hexacationized benzene ring ions are stabilized by forming gas phase complexes with two to five trifluoromethanesulfonate counterions. MS/MS analysis reveals that their fragmentation takes place by loss of neutrals such as trifluoromethanesulfonic acid and 4-dimethylaminopyridine; no rupture of the benzene or pyridine rings was observed in spite of accumulation of positive charge in a restricted geometry.
A series of truncated Herpes simplex virion peptides studied by fast atom bombardment mass spectrometry under high and low energy collision induced dissociation conditions showed preferential fragmentation of the aspartyl-proline amide bond, compared to other peptide bonds. Electrospray ionization investigation proved that this favoured fragmentation can not be attributed to only the known proline effect, as a change from Asp to Asn in the peptide yielded an Asn-Pro bond which was found to be stable under the same ionization conditions. This mass spectrometric behaviour is in good agreement with the observation that DP bonds are sensitive to acidic conditions.
A method has been developed for de novo peptide sequencing using matrix-assisted laser desorption ionization mass spectrometry. This method will facilitate biological studies that require rapid determination of peptide or protein sequences, e.g., determination of posttranslational modifications, identification of active compounds isolated from combinatorial peptide libraries, and the selective identification of proteins as part of proteome studies. The method involves fast, one-step addition of a sulfonic acid group to the N terminus of tryptic peptides followed by acquisition of postsource decay (PSD) fragment ion spectra. The derivatives are designed to promote efficient charge site-initiated fragmentation of the backbone amide bonds and to selectively enhance the detection of a single fragment ion series that contains the C terminus of the molecule (y-ions). The overall method has been applied to pmol quantities of peptides. The resulting PSD fragment ion spectra often exhibit uninterrupted sequences of 20 or more amino acid residues. However, fragmentation efficiency decreases considerably at amide bonds on the C-terminal side of Pro. The spectra are simple enough that de novo sequence tagging is routine. The technique has been successfully applied to peptide mixtures, to high-mass peptides (up to 3,600 Da) and to the unambiguous identification of proteins isolated from two-dimensional gel electrophoresis. The PSD spectra of these derivatized peptides often allow far more selective protein sequence database searches than those obtained from the spectra of native peptides.
The low energy collision-induced dissociation of fixed-charge derivatives [tris(2,4,6-trimethoxyphenyl)phosphonium] of peptides was investigated using an electrospray ion trap mass spectrometer. The fixed charge directed the fragmentation pattern and generated solely N-terminal fragments with minimal internal rearrangement, regardless of the presence and position of basic amino acids in the peptide chain. Generally only b-type ions, accompanied by less intense a-type ions, were observed, depending on the collision energy. It was observed that the fixed charge controlled the fragmentation beyond typical MS/MS, and thus the capacity of the ion trap to perform multiple stage fragmentation (MS(n)) was found particularly useful for obtaining the complete sequence information of the peptides.
High-energy tandem mass spectrometry and molecular dynamics calculations are used to determine the locations of charge in metastably decomposing (M + 2H)2+ ions of human angiotensin II. Charge-separation reactions provide critical information regarding charge sites in mutiply charged ions. The most probable kinetic energy released (T
m.p.) from these decompositions are obtained using kinetic energy release distributions (KERDs) in conjunction with MS/MS (MS2), MS/MS/MS (MS3), and MS/MS/MS/MS (MS4) experiments. The most abundant singly and doubly charged product ions arise from precursor ion structures in which one proton is located on the arginine (Arg) side chain and the other proton is located on a distal peptide backbone carbonyl oxygen. The MS3 KERD experiments show unequivocally that neither the N-terminal amine nor the aspartic acid (Asp) side chain are sites of protonation. In the gas phase, protonation of the less basic peptide backbone instead of the more proximal and basic histidine (His) side chain is favored as a result of reduced coulomb repulsion between the two charge sites. The singly and doubly charged product ions of lesser abundance arise from precursor ion structures in which one proton is located on the Arg side chain and the other on the His side chain. This is demonstrated in the MS3 and MS4 mass-analyzed ion kinetic energy spectrometry experiments. Interestingly, (b
7″ + OH)2+ product ions, like the (M + 2H)2+ ions of angiotensin II, are observed to have at least two different decomposing structures in which charge sites have a primary and secondary location.
Charged derivatives of peptides are useful in obtaining simpler collision-activated dissociation (CAD) mass spectra. An N-terminal charge-derivatizing reagent capable of reacting with picomole levels of peptide has been recently reported (Huang et al. Anal. Chem.
1997, 69, 137–144) in the contexts of analyses by fast atom bombardment (FAB) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Electrospray ionization (ESI) mass spectrometric investigation of these tris(trimethoxyphenylphosphonium) acetyl derivatives are described in this article, including studies by in-source fragmentation (ISF) and tandem mass spectrometry (MS/MS). Results from ISF are compared with those from MS/MS. Similarities and differences between ESI-ISF, MALDI-post-source decay (PSD), and FAB-CAD data are presented. Differences in fragmentation of these charged derivatives in the triple quadrupole and ion trap mass spectrometers also are discussed. Application of this derivatizing procedure to tryptic digests and subsequent analysis by liquid chromatography-mass spectrometry is also shown.
The high resolution, mass range and sensitivity of Fourier transform mass spectrometry (FTMS) suggest that it could be a valuable tool for the quantitative analysis of biomolecules. To determine the applicability of electrospray ionization combined with FTMS to the quantitation of biomolecules in multi-component samples, mixtures of varying compositions and concentrations of cytochrome c, angiotensin II, insulin and chicken egg white lysozyme were examined. The instrument used has an electrospray source with a hexapole trap to accumulate ions for injection into an ion cyclotron resonance mass analyzer. Linear responses for single component samples of angiotensin II and insulin were in the range 0.031-3 microM and those of both cytochrome c and lysozyme were between 0.031 and 1 microM. In examining various mixtures of the proteins with angiotensin II, it was found that the presence of the large molecules suppresses the signal of the smaller molecules. This is suggested to be a result of ion-ion interactions producing selective ion loss from either the hexapole trap or the ion cyclotron resonance mass analyzer trap. More massive, more highly charged ions can collisionally transfer large amounts of translational energy to smaller, less highly charged ions, ejecting the smaller ions from the trap. Mass discrimination effects resulting from the trapping voltage were also examined. It was found that relative signal intensities of ions of different masses depend on trapping voltage for externally produced ions. The effect is most significant for spectra including masses that differ by 30% or more. This suggests that for quantitation all samples and standards be run at a constant trapping potential.
Prophenin 1 (PF-1) is a 79-residue polypeptide originally isolated from porcine leukocytes. Its amino acid sequence has been determined by a combination of mass spectrometry and Edman degradation (Harwig SSL. et al. FEBS Lett. 1995; 362: 65). Prophenin (PF) and variants thereof are also found in organic extracts of porcine pulmonary tissue (Wang Y. et al. FEBS Lett. 1999; 460: 257). In the present study we have characterised the variant forms of PF found in these extracts using nano-electrospray (nano-ES) high resolution and tandem mass spectrometry. The major forms of PF found in these extracts by nano-ES mass spectrometry are the 80-residue polypeptides prophenin-2-Pyr (PF-2-Pyr) and prophenin-2-Gln (PF-2-Gln). Prophenin-2-Pyr is refractory to Edman degradation due to the presence of an N-terminal pyroglutamic residue. In PF-2-Gln the N-terminal residue is glutamine and the C-terminus is amidated. In porcine pulmonary extracts PF-1 is present to only a minor extent. Other shorter polypeptides are also found in these extracts including 18- and 17-residue C-terminal fragments of PF. The primary structure of PF is highly unusual in that it shows four almost perfect decamer repeats of FPPPN(V/F)PGPR and, out of the 79/80 residues, 42 are proline and 14 are phenylalanine. Tryptic digestion of PF gives peptides containing the decamer repeat and collision-induced dissociation of these peptides provides an insight into the fragmentation mechanisms of proline-rich peptides. Facile cleavage within the Pro-Pro-Pro sequence of these peptides suggests the involvement of a cyclic peptide in the fragmentation mechanism. Fragmentation mechanisms that account for the formation of fragment ions at other cleavage sites are also discussed.
A strategy for determination of O-glycosylation site(s) in glycopeptides has been developed using model compounds obtained by enzymatic glycosylation (by human GaNTase-T2 isoform) on peptides derived from the human MUC5AC mucin tandem repeat motif. The beta-elimination-addition reaction (using dimethylamine and concomitantly ethanethiol) on the formerly glycosylated sites through a Michael-type condensation produced efficient deglycosylation with appropriate chemical modification. After N-terminal derivatization by a phosphonium group, peptide sequencing was then carried out by nanospray tandem mass spectrometry experiments. The highly predictable fragmentation pathways of these fixed-charge phosphonium derivatives enable straightforward recognition of glycosylation site(s) based on the mass increment of +44 Da for originally glycosylated threonine compared to the mass of fragments containing nonglycosylated residues.
The dependence upon ion mass of the loss of translational energy suffered by an ion with kiloelectron volt energy as a result of collision with helium has been investigated over the mass range 400 u to 1600 u. At masses below about 1500 u and an incident ion energy of 8 keV, there is forward-scattering in the centre-of-mass frame: at mass 1620 u at the same energy, there is backscattering in the centre-of-mass frame. The uptake of internal energy by an ion is at a maximum in the region in which the change from forward-scattering to back-scattering occurs.
Methodology for determining amino acid sequences of proteins by tandem mass spectrometry is described. The approach involves enzymatic and/or chemical degradation of the protein to a collection of peptides which are then fractionated by high-performance liquid chromatography. Each fraction, containing as many as 10-15 peptides, is then analyzed directly, without further purification, by a combination of liquid secondary-ion/collision-activated dissociation mass spectrometry on a multianalyzer instrument. Interpretation of collision-activated dissociation mass spectra is described, and results are presented from a study of soluble peptides produced by treatment of apolipoprotein B with cyanogen bromide and trypsin.
Acetylation of the animo groups (N-terminus and lysine) of proteins before enzymatic or chemical cleavage was explored as an approach to provide additional information in the course of the determination of amino acid sequences. The major advantage is the ability to differentiate glutamine from lysine, because only the latter is acetylated and thus increases in mass by 42 Da. Horse heart cytochrome c could be fully N-actetylated and even on prolonged digestion with chymotrypsin underwent very little tryptic cleavage, in contrast to the native protein where this side reaction is extensive. Sperm whale myoglobin is more difficult to acetylate, but even at 40%-50% average acetylation, all 19 lysines could be identified unambiguously. A proteolytic digest of acetylated protein is thus a useful component of strategies for the determination of the primary structure of proteins by tandem mass spectrometry.
The recent commercial implementation of an electrospray source on a four-sector mass spectrometer has allowed the study of
high-energy collisional activation of multiply charged cations. With this configuration, higher mass-to-charge ratios can
be accommodated in both precursor ion selection and fragment ion detection. Good mass accuracy facilitates analysis of fragment
ions and allows more reliable mechanistic correlation of these fragments. A convenient scheme was devised to permit the use
of kilovolt potentials in both MS-I and MS-II, with precursors of varying charge states. Algorithms were devised to assign
masses of different types of multiply charged fragment ions. Nine polypeptides were studied in the mass range 2000–5000 Da.
Through this entire mass range, fragment ions were observed to be amply formed by cleavages in both the backbone and side
chains, analogous to high-energy collisional activation of singly charged ions. This stands in sharp contrast to the patterns
reported with low-energy, multiple collisions. Abundances of sequence ion series are influenced by the positions of basic
residues. Analysis of charge distributions in fragment ions also indicates that the charges tend to be spread out across the
peptides.
Tryptic digests were analyzed by means of online microbore liquid chromatography combined with mass spectrometry (LC/MS) for
some common proteins. Following conventional enzymatic digestion with trypsin, the freeze-dried residues were dissolved in
high-performance liquid chromatography (HPLC) eluent and subjected to gradient reversed-phase microbore HPLC separation with
mass spectrometric detection. The latter was done in the full-scan single or tandem (MS/MS) mass spectrometry mode. The formation
of gas-phase ions from dissolved analytes was accomplished at atmospheric pressure by pneumatically assisted electrospray
(ion spray) ionization. This produced field-assisted ion evaporation of dissolved ions, which could then be mass-analyzed
for molecular mass or structure. In the full-scan LC/MS mode, the masses for the peptide fragments in the tryptic digests
can be determined as either their singly or multiply charged ions. When the molecular weights of the peptides lie outside
the mass range of the mass spectrometer, the multiply charged feature of these experimental conditions still provides reliable
molecular weight determinations. In addition, collision-activated dissociation (CAD) on selected peptide precursor ions provides
online LC/MS/MS sequence information for the tryptic fragments. Results are shown for the tryptic digests of horse heart cytochrome
c, bovine β-lactoglobulin A, and bovine β-lactoglobulin B.
The field desorption mass spectrum of bradykinin
and collision-induced decomposition spectra of ions formed from bradykinin during field desorption
are reported. Low-energy fragmentation pathways of bradykinin
are identified. It is proposed that bradykinin can
undergo rearrangement to a novel ion structure, characterized by a propensity
for cleavage at one specific site within the peptide.
A scan law is derived for the detection of fragment ions formed by collisional activation (CA) of a multiply charged precursor in a floated collision cell of a tandem mass spectrometer. Comparisons of the CA spectra of multiply charged ions obtained in either a floated or a grounded collision cell demonstrate the benefits associated with raising the collision cell above ground potential. In addition to the advantages observed for singly charged ions, floating the collision cell increases the transmission of multiply charged ions through the first mass spectrometer by permitting higher source potentials to be used. This technique also increases the detection efficiency for products of charge separation reactions, which may prove useful in the charge state assignment of the fragment ions.
Solutions passed through a small capillary at 2-10 kV relative to ground are electrosprayed into a bath gas to form a gaseous dispersion of ions that is expanded into vacuum in a small supersonic free jet. A portion of the jet is passed through a skimmer to form a molecular beam that contains a variety of ionic species. Mass spectrometric analysis reveals that these species include solute cations in aggregation with solvent molecules and/or nonionized solute species. The nature of the product ions depends upon the composition and feed rate of the original solution, the temperature and composition of the bath gas, and the voltage applied to the capillary. The exploratory experiments reported here suggest that this novel ion source may be useful for producing in vacuo a wide variety of cluster ions for examination by various spectroscopic techniques. Also inviting is the prospect of extending the applicability of mass spectrometric analysis to large organic molecules that are too complex, too fragile, or too nonvolatile for ionization by more conventional methods. Another intriguing possibility is to use the technique on probing the microscopic structure and properties of solutions.
We describe two algorithms that extract molecular mass information from spectra showing sequences of peaks due to ions with varying numbers of charges. The first, called here the "averaging algorithm", unambiguously assigns charge numbers to the ions associated with the m/z value for each peak in the sequence and then averages the resulting values of M to give a best estimate of the molecular mass. The second, identified as the "deconvolution algorithm", mathematically transforms a spectrum of several peaks for multiply charged ions into one peak corresponding to a singly charged ion. The procedures can be readily implemented with a personal computer and are here applied to representative spectra of small proteins generated by electrospray mass spectrometry. These algorithms are now routinely used in our laboratory for the interpretation of such spectra. They both are fast and convenient, discriminate against background, and take advantage of much of the information contained in a sequence of peaks. Achievable accuracy and sources of error are discussed.
The mass spectra of biological molecules, whose molecular mass exceeds 10 kDa, invariably contain multiply charged ions. For example, a survey scan of a small protein will produce singly, doubly and triply protonated molecules, the intensity of the doubly charged species often being greater than that of the singly charged entity. Although the spectra resulting from doubly charged peptides have not previously been studied, collisional activation of such doubly charged species may result in significant additional information pertaining to molecular structure. The techniques employed to study ions originating from multiply charged species were linked scanning of constant B/E and tandem mass spectrometry, namely low collision energy spectra acquired on a BEQQ hybrid instrument. The methodology was applied to model compounds whose tandem mass spectrometry characteristics are well known, e.g. gramicidin S and angiotensin I. The results for the product ions of the [M + 2H]2+ species of the models were obtained which highlight the methodology required for high-mass materials.
Mass analyses have been carried out on ions produced by an Electrospray (ES) source from dilute solutions of protein molecules with molecular weights (M) in the range from 5000 to nearly 40000. Each spectrum comprises a sequence of peaks corresponding to multiply charged intact parent species. The ions of each peak differ from those of their adjacent neighbors by one unit charge, H+ in these experiments. The maximum number of charges per ion generally increases with the molecular weight of the parent molecule, reaching a value of 45 in the case of alcohol dehydrogenase, atM=39830 the largest species in this study. Thus the resulting values ofm/z are within reach of a simple quadrupole mass filter whose nominal upper mass limit is 1500 daltons! The immediate application for the ES source is in mass spectrometric analysis of large fragile molecules of biochemical importance. But the multiply charged ions it produces are newcomers to the laboratory scene that constitute interesting subjects for study.
For the differentiation of leucine and isoleucine in a peptide by high energy collision-induced dissociation (CID), it is generally required that there is a basic amino acid present at or near either the C-terminus or the N-terminus of the peptide. In these cases, fragmentation or the β,γ bond of the side chain occurs, generating ions designated wn or dn that permit the differentiation of these isomeric amino acids. While trypsin and Endo Lys-C generate peptides with a basic C-terminal amino acid, other enzymes cleave at neutral or acidic amino acids and may thus produce proteolytic peptides that do not contain any basic amino acids. For these, a microderivatization method has been developed that places a fixed positive charge at the C-terminus. It involves exposure of the peptide(s) deposited on the inner wall of a capillary tube, first to chloroacetyl chloride vapor and then to trimethylamine and water vapors. This two-step reaction attaches a trimethylammonium acetyl (TMA) moiety to the N-terminal amino group of the peptide. The CID spectra of these derivatives are very simple, exhibit the same characteristics (including abundant dn ions) as peptides bearing the strongly basic arginine at the N-terminus, and thus permit the differentiation of leucine from isoleucine. The reaction can be carried out at the sub-nanomole level.
The formation of ions from the charged droplets produced in the several spray ionization techniques is viewed as an activated rate process involving field-assisted desorption, in accord with the ideas first set forth by Iribarne and Thomson. The novel features of the present treatment are particularly relevant to the unique ability of electrospray ionization to transform large molecules in solution to free ions in the gas phase, with extensive multiple charging. These new features stem mainly from the realization that the spacing of charges on a desorbed ion must relate to the spacing of charges on the surface of the droplet whence it came. The consequences of this “rule” can account for the existence of maxima and minima in the number of charges on the ions of a particular species as well as the nature of the distribution of ions among the intervening charge states. They also explain the dependence of charge state on the configuration in solution of the parent molecule of the desorbed ion. In addition, they provide insight into the sequence in time at which ions in the various charge states leave an evaporating droplet.
The collision (10 keV)-induced decomposition (CID) of (M + H)+ ions of peptides generated by fast atom bombardment ionization has been studied in a tandem mass spectrometer consisting of two consecutive double-focussing analyzers. Two novel fragmentation processes are described; one (termed dn) leads to the formation of N-terminal ions that permit the differentiation of leucine isoleucine; the other leads to a new set of C-terminal ions (termed vn) and is related to the structure of the amino acid representing the N-terminus of the fragment. The mechanisms of formation are supported by B2/E and B/E scans, which define the precursor and product ions. These and other fragmentations of (M + H)+ ions under CID conditions and kilovolt collision energies seem to involve fragmentation at a site remote from the charge. The fragmentation processes which (M + H)+ ions of peptides undergo are related to the site of protonation and the degree to which the positive charge is fixed at that site.
Between 650 and 2000 different peptides are associated with the major histocompatibility complex class II molecule I-Ad. Sequences
for nine of these were obtained by a combination of automated Edman degradation and tandem mass spectrometry. All of the peptides
are derived from secretory or integral membrane proteins that are synthesized by the antigen-presenting cell itself. Peptides
were 16 to 18 residues long, had ragged NH2-and COOH-termini, and contained a six-residue binding motif that was variably
placed within the peptide chain. Binding data on truncated peptides suggest that the peptide binding groove on class II molecules
can be open at both ends.
We have examined the hypothesis that structural features which predispose to localization of charge at a strongly favored site are not conducive to the low-energy fragmentation of peptide ions via a multiplicity of pathways. Consistent with this proposal, it is demonstrated that the formation of N- or C-terminal pre-charged derivatives is detrimental to the formation of sequence-specific product ions following low-energy collisional activation. Protonation of pre-charged derivatives (yielding doubly charged ions) restores favorable fragmentation properties; the effect is attributed to the fragmentation-directing properties of the proton which may occupy one of several sites. Similarly, a doubly protonated peptide which incorporates a C-terminal arginine residue as a single strongly favored site of protonation exhibits favored low-energy fragmentations attributable to location of the second proton at one of several sites remote from the C-terminus.
Peptides formed as reaction products, of specific hydrolysis of proteins by trypsin, are characterized by a basic residue (Arg or Lys) at the C-terminus, which facilitates formation of abundant [M + 2H]2+ ions under electrospray or ionspray conditions. These doubly charged ions readily dissociate upon collisional activation to y" and b fragment ions which are mass complements of one another. The suggestion that these fragments are formed by direct charge-separation dissociations must contend with the observation that the y" intensities are generally appreciably larger than those of their b counterparts. However, it is shown that this can be accounted for by a greater susceptibility of the b ions to undergo further dissociation to smaller fragments such as immonium ions. In addition no evidence could be found to support alternative mechanisms, including dissociative electron capture, for which equal intensities of the two fragment ion series are not obligatory. Initial protonation at the N-terminus was shown to be required for formation of these [M + 2H]2+ ions via its suppression by mono-acetylation at the N-terminus. These findings, and others concerning formation of [y"']2+ fragments, are consistent with extensions of published mechanisms for formation of b and of y" fragments from singly protonated peptides, via charge-site-induced cleavages and intramolecular proton transfers between nitrogen atoms, respectively.
The mechanism for the formation of y ions in the collision-induced dissociation (CID) spectra of protonated peptides produced by fast-atom bombardment was investigated by tandem mass spectrometry and deuterium labelling studies. The results show that a hydrogen atom attached to nitrogen and not to carbon migrates during cleavage of the amide bond. A mechanism based on these results is presented.
Serum albumin proteins, Mr ∼66 kDa, from 10 different species (bovine, human, rat, horse, sheep, goat, rabbit, dog, porcine, and guinea pig) have been studied by electrospray ionization mass spectrometry (ESI-MS) and tandem MS using a triple-quadrupole instrument. The effectiveness of collisional activation for the multiply charged albumin ions greatly exceeds that for singly charged ions, allowing an extension by a factor of at least 20 to the molecular mass range for obtaining sequence-specific product ions by tandem MS. Efficient dissociation is largely attributed to "preheating" in the interface Coulombic instability and the large number of collisions. Increasing the electric field in the intermediate pressure region, between the nozzle-skimmer elements of the atmospheric pressure/vacuum interface, allows fragmentation of the multiply protonated (to 96+) molecules produced by ESI. The most abundant dissociation product ions assigned have a low charge state (2+ to 5+) and are attributed to "bn" mode species from cleavage of the -CO-N- peptide backbone bonds. Particularly abundant dissociation products originate from regions near residues n = 20-25 from the NH2 terminus for parent ions of moderate charge (∼50+). Collisionally activated dissociation (CAD) mass spectra from porcine serum albumin, in contrast to the other albumins, also gave prominent singly charged "yn" fragments formed from cleavages near the COOH terminus. Tandem mass spectrometry (MS/MS) of the multiply charged molecular Ions, and of fragment species produced by dissociation in the interface (i.e., effective MS/MS/MS), produced similar "bn" species and served to confirm spectral assignments. We also show that ESI mass spectra allow a qualitative assessment of protein microheterogeneity and, in some cases, resolution of major contributions. The physical and analytical implications of the results are discussed, including the identification of possible errors in previously published sequences.
With the advent of recombinant deoxyribonucleic acid (DNA) technology which allows a wide range of manipulation of genes and their expression in cell lines other than the natural ones, many aspects of protein structure have become more important than ever. In addition to the determination of the amino acid sequence questions relating to homogeneity, the nature of post-translational modifications, the verification of the structure of a protein produced by a synthetically modified gene or the detection of a natural mutant are all questions that are more and more frequently asked and with the demand for more detail. Mass spectrometry has emerged as an important contributor to this field, particularly since the advent of fast atom bombardment (FAB) ionization, which makes it possible to ionize directly large polar molecules such as peptides and small proteins. As such, FAB mass spectrometry provides mainly molecular weight information which, in itself, often suffices to answer certain questions, particularly because the mass of many peptides can be determined directly from a single mass spectrum of a mixture. However, in order to obtain detailed structural information, such as the amino acid sequence, fragmentation has to be induced by collision processes and the product ions separated, preferably in the second mass spectrometer of a tandem system. This approach is particularly suited for the determination of the sequence of N-blocked peptides and the nature of the blocking group; the type and location of modified (i.e. phosphorylated, sulfated, glycosylated) amino acids; detection or verification of amino acid replacements; confirmation of the structure of synthetic peptides; and last but not least, the determination of the primary structure of proteins.
The low-energy collision-induced dissociation reactions of a series of multiply-protonated peptides have been investigated by tandem mass spectrometry. It is known that doubly-protonated tryptic peptides undergo facile fragmentation yielding redundant sequence information. The present work has shown that this fortunate circumstance seems likely to be the exception rather than the rule. The presence of additional basic residues, at positions other than the C-terminus, complicates the spectra. The most important such complication discovered in the present work involves wholesale transfer of one or two residues from the C-terminal end of a doubly-charged b fragment to the side chain of a lysine residue located near the N-terminus, resulting in mass shifts of the products of subsequent second-stage fragmentations. Other examples of the participation of the flexible lysine side chain are suggested but could not be confirmed to the same extent. The role of Coulombic repulsion in facilitating fragmentation has been explored via investigations of triply- and quadruply-protonated basic peptides bearing one charge for every three or four amino acid residues. Such species yielded almost no sequence information under low-energy collision conditions, due to the localization of the ionizing protons on highly basic sites rather than on the peptide backbone. It is proposed that collisionally activated mobilization of protons from the basic sites, where they are originally located upon formation, to the backbone is a necessary condition for structurally useful fragmentation to occur. It was not possible, on the basis of the present work, to deduce mechanistic generalizations and predictive schemes which would permit structural interpretations of such fragment spectra for unknown peptides.
Electrospray ionization of carbonic anhydrase with ion dissociation yields a mass spectrum from which the masses of > 100 isotopic clusters are determined accurately, with the number of charges assigned directly from resolved isotopic peaks. Of these clusters, 80% correspond to fragmentation at or near the amino acid proline. The masses of combinations of two, three, and four of these clusters sum to the molecular mass with 0.1-Da accuracy, while further fragment ion dissociation provides additional sequence information. The capability of this methodology to detect sequence variations is illustrated with an isozyme having a single amino acid replacement.
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