Biomedical subjects

Sharon J Pitteri

Publications and source records attributed to Sharon J Pitteri.

13 recordsLinked to original sources

PubMed2006

Ion/molecule reactions of cation radicals formed from protonated polypeptides via gas-phase ion/ion electron transfer.

Cation radicals formed via gas-phase electron transfer to multiply protonated polypeptides have been found to react with molecular oxygen. Such cation radicals are of interest within the context of electron transfer dissociation, a phenomenon with high utility for the characterization of peptide and protein primary structures. Most of the cation radicals show the attachment of O(2) under room temperature storage conditions in an electrodynamic ion trap. At higher temperatures and under conditions of collisional activation, the oxygen adduct species lose O(2), HO(*), or HO(2)(*), depending upon the identity of the side chain at the radical site. The fragments containing the C-terminus, the so-called z-ions, which are predominantly radical species, engage in reactions with molecular oxygen. This allows for the facile distinction between z-ions and their complementary even-electron c-ion counterparts. Such a capability has utility in protein identification and characterization via mass spectrometry. Intact electron transfer products also show oxygen attachment. Subsequent activation of such adducts show dissociation behavior very similar to that noted for z-ion adducts. These observations indicate that ion/radical reactions can be used to probe the locations of radical sites in the undissociated electron transfer products as well as distinguish between c- and z-type ions.

Amino Acid Sequence↗

PubMed2006

Parallel ion parking of protein mixtures.

The multiple charging phenomenon resulting from electrospray ionization of proteins, while useful for the ability to make several mass measurements on a single component, can lead to highly complex spectra when mixtures are analyzed, as each component can generate multiple ions of distinct mass-to-charge ratio. Ion/ion proton-transfer reactions can overcome this problem by reduction of all components to the +1 charge state, but this typically requires the ability to extend the mass range of the instrument well beyond that available in most commercial instruments. Furthermore, reduction of protein charge to +1 also results in a reduction in detector response. Here it is shown that application of a relatively high amplitude, low-frequency auxiliary ac signal to the end cap electrodes of a 3-D ion trap during an ion/ion reaction can slow the ion/ion reaction rates of ions over a broad m/z range, in a process termed HALF parallel ion parking. Adjustment of the frequency and amplitude of the applied voltage allows the mass range into which the initial ion signal is moved to be controlled, allowing for the simplification of multicomponent mixtures within a mass range that is more commonly available on commercial systems. In addition to decreasing spectral complexity, this is advantageous for mixtures with low-abundance components, as there is less compromise with detector response than in reduction to the +1 charge state. Preliminary evidence also suggests that the ion collision cross section may play an important role in determining which charge states are most significantly inhibited from further ion/ion reactions under a given set of ion parking conditions.

Ions↗

PubMed2005

Differentiation of aspartic and isoaspartic acids using electron transfer dissociation.

Electron-transfer dissociation allows differentiation of isoaspartic acid and aspartic acid residues using the same c + 57 and z - 57 peaks that were previously observed with electron capture dissociation. These peaks clearly define both the presence and the position of isoaspartic acid residues and they are relatively abundant. The lower resolution of the ion trap instrument makes detection of the aspartic acid residue's diagnostic peak difficult because of interference with side-chain fragment ions from arginine residues, but the aspartic acid residues are still clearly observed in the backbone cleavages and can be inferred from the absence of the isoaspartic acid diagnostic ions.

Aspartic Acid↗

PubMed2005

Electron transfer versus proton transfer in gas-phase ion/ion reactions of polyprotonated peptides.

The ion/ion reactions of several dozen reagent anions with triply protonated cations of the model peptide KGAILKGAILR have been examined to evaluate predictions of a Landau-Zener-based model for the likelihood for electron transfer. Evidence for electron transfer was provided by the appearance of fragment ions unique to electron transfer or electron capture dissociation. Proton transfer and electron transfer are competitive processes for any combination of anionic and cationic reactants. For reagent anions in reactions with protonated peptides, proton transfer is usually significantly more exothermic than electron transfer. If charge transfer occurs at relatively long distances, electron transfer should, therefore, be favored on kinetic grounds because the reactant and product channels cross at greater distances, provided conditions are favorable for electron transfer at the crossing point. The results are consistent with a model based on Landau-Zener theory that indicates both thermodynamic and geometric criteria apply for electron transfer involving polyatomic anions. Both the model and the data suggest that electron affinities associated with the anionic reagents greater than about 60-70 kcal/mol minimize the likelihood that electron transfer will be observed. Provided the electron affinity is not too high, the Franck-Condon factors associated with the anion and its corresponding neutral must not be too low. When one or the other of these criteria is not met, proton transfer tends to occur essentially exclusively. Experiments involving ion/ion attachment products also suggest that a significant barrier exists to the isomerization between chemical complexes that, if formed, lead to either proton transfer or electron transfer.

Electrons↗

PubMed2005

Electron-transfer ion/ion reactions of doubly protonated peptides: effect of elevated bath gas temperature.

In this study, the electron-transfer dissociation (ETD) behavior of cations derived from 27 different peptides (22 of which are tryptic peptides) has been studied in a 3D quadrupole ion trap mass spectrometer. Ion/ion reactions between peptide cations and nitrobenzene anions have been examined at both room temperature and in an elevated temperature bath gas environment to form ETD product ions. From the peptides studied, the ETD sequence coverage tends to be inversely related to peptide size. At room temperature, very high sequence coverage (approximately 100%) was observed for small peptides (< or =7 amino acids). For medium-sized peptides composed of 8-11 amino acids, the average sequence coverage was 46%. Larger peptides with 14 or more amino acids yielded an average sequence coverage of 23%. Elevated-temperature ETD provided increased sequence coverage over room-temperature experiments for the peptides of greater than 7 residues, giving an average of 67% for medium-sized peptides and 63% for larger peptides. Percent ETD, a measure of the extent of electron transfer, has also been calculated for the peptides and also shows an inverse relation with peptide size. Bath gas temperature does not have a consistent effect on percent ETD, however. For the tryptic peptides, fragmentation is localized at the ends of the peptides suggesting that the distribution of charge within the peptide may play an important role in determining fragmentation sites. A triply protonated peptide has also been studied and shows behavior similar to the doubly charged peptides. These preliminary results suggest that for a given charge state there is a maximum size for which high sequence coverage is obtained and that increasing the bath gas temperature can increase this maximum.

Amino Acid Sequence↗

PubMed2005

Parallel ion parking: improving conversion of parents to first-generation products in electron transfer dissociation.

Electron-transfer dissociation (ETD) in a tandem mass spectrometer is an analytically useful ion/ion reaction technique for deriving polypeptide sequence information, but its utility can be limited by sequential reactions of the products. Sequential reactions lead to neutralization of some products, as well as to signals from products derived from multiple cleavages that can be difficult to interpret. A method of inhibiting sequential ETD fragmentation in a quadrupole ion trap is demonstrated here for the reaction of a triply protonated peptide with nitrobenzene anions. A tailored waveform (in this case, a filtered noise field) is applied during the ion/ion reaction time to accelerate simultaneously first-generation product ions and thereby inhibit their further reaction. This results in a approximately 50% gain in the relative yield of first-generation products and allows for the conversion of more than 90% of the original parent ions into first-generation products. Gains are expected to be even larger when higher charge-state cations are used, as the rates of sequential reaction become closer to the initial reaction rate.

Angiotensin I↗

PubMed2005

Electron transfer ion/ion reactions in a three-dimensional quadrupole ion trap: reactions of doubly and triply protonated peptides with SO2*-.

Ion-ion reactions between a variety of peptide cations (doubly and triply charged) and SO2 anions have been studied in a 3-D quadrupole ion trap, resulting in proton and electron transfer. Electron transfer dissociation (ETD) gives many c- and z-type fragments, resulting in extensive sequence coverage in the case of triply protonated peptides with SO2*-. For triply charged neurotensin, in which a direct comparison can be made between 3-D and linear ion trap results, abundances of ETD fragments relative to one another appear to be similar. Reactions of doubly protonated peptides with SO2*- give much less structural information from ETD than triply protonated peptides. Collision-induced dissociation (CID) of singly charged ions formed in reactions with SO2*- shows a combination of proton and electron transfer products. CID of the singly charged species gives more structural information than ETD of the doubly protonated peptide, but not as much information as ETD of the triply protonated peptide.

Electron Transport↗

PubMed2005

SO2-* electron transfer ion/ion reactions with disulfide linked polypeptide ions.

Multiply-charged peptide cations comprised of two polypeptide chains (designated A and B) bound via a disulfide linkage have been reacted with SO2-* in an electrodynamic ion trap mass spectrometer. These reactions proceed through both proton transfer (without dissociation) and electron transfer (with and without dissociation). Electron transfer reactions are shown to give rise to cleavage along the peptide backbone, loss of neutral molecules, and cleavage of the cystine bond. Disulfide bond cleavage is the preferred dissociation channel and both Chain A (or B)-S* and Chain A (or B)-SH fragment ions are observed, similar to those observed with electron capture dissociation (ECD) of disulfide-bound peptides. Electron transfer without dissociation produces [M + 2H]+* ions, which appear to be less kinetically stable than the proton transfer [M + H]+ product. When subjected to collision-induced dissociation (CID), the [M + 2H]+* ions fragment to give products that were also observed as dissociation products during the electron transfer reaction. However, not all dissociation channels noted in the electron transfer reaction were observed in the CID of the [M + 2H]+* ions. The charge state of the peptide has a significant effect on both the extent of electron transfer dissociation observed and the variety of dissociation products, with higher charge states giving more of each.

Amino Acid Sequence↗

PubMed2003

Phosphorylation site identification via ion trap tandem mass spectrometry of whole protein and peptide ions: bovine alpha-crystallin A chain.

Tandem mass spectrometry was applied both to ions of a tryptic fragment and intact protein of bovine alpha-crystallin A chain to localize the single site of phosphorylation. The [M + 19H](19+) to [M + 11H](11+) charge states of both phosphorylated and unphosphorylated bovine alpha-crystallin A chain whole protein ions were subjected to collisional activation in a quadrupole ion trap. Ion parking was used to increase the number of parent ions over that yielded by electrospray. Ion-ion proton-transfer reactions were used to reduce the product ion charge states largely to +1 to simplify spectral interpretation. In agreement with previous studies on whole protein ion fragmentation, both protein forms showed backbone cleavages C-terminal to aspartic acid residues at lower charge states. The phosphorylated protein showed competitive fragmentation between backbone cleavage and the neutral loss of phosphoric acid. Analysis of which backbone cleavage products did or did not contain the phosphate was used to localize the site of phosphorylation to one of two possible serine residues. A tryptic digest of the bovine alpha-crystallin A chain yielded a phosphopeptide containing one missed cleavage site. The peptide provided information complementary to that obtained from the intact protein and localized the modified serine to residue 122. Fragmentation of the triply charged phosphopeptide yielded five possible serine phosphorylation sites. Fragmentation of the doubly charged phosphopeptide, formed by ion/ion proton-transfer reactions, positively identified the phosphorylation site as serine-122.

Amino Acid Sequence↗

PubMedDate not supplied

Recent developments in the ion/ion chemistry of high-mass multiply charged ions.

The ability to form multiply charged high-mass ions in the gas-phase, most notably via electrospray ionization (ESI), has allowed the study of many different combinations of positively and negatively charged ions. The charged products are directly amenable to study with mass spectrometry. Ion/ion reactions have proved to be "universal" in the sense that the high exothermicities and large rate constants associated with essentially any combination of oppositely charged ions lead to reaction regardless of the chemical functionalities associated with the ions. These characteristics make ion/ion reactions potentially analytically useful provided reagent ion densities and spatial overlap of the oppositely charged ions are high. These conditions can be readily met by several instrumental configurations. The focus of this review is to highlight developments in this field since 1998. Novel instrumentation has been developed to study ion/ion reactions, such as atmospheric pressure ion/ion reactors followed by mass analysis, or electrodynamic ion trap mass spectrometers, which are used as reaction vessels at sub-atmospheric pressures. A wide variety of reaction phenomenologies have been observed in various ion/ion reactions, with proton transfer being the most common. New phenomenologies have been observed in the reactions of multiply charged positive ions with singly charged negative ions, including cation transfer and cation exchange. A new series of reactions between multiply charged positive ions and multiply charged negative ions have been made possible by recent instrumentation developments. These reactions have led to the observation of proton transfer and complex formation. These observations have provided new insights into ion/ion reaction dynamics and a bound orbit model appears to best account for experimental results. New applications are also discussed for a several ion/ion reaction.

Algorithms↗

PubMedDate not supplied

Affecting proton mobility in activated peptide and whole protein ions via lysine guanidination.

We have evaluated the effect of lysine guanidination in peptides and proteins on the dissociation of protonated ions in the gas phase. The dissociation of guanidinated model peptide ions compared to their unmodified forms showed behavior consistent with concepts of proton mobility as a major factor in determining favored fragmentation channels. Reduction of proton mobility associated with lysine guanidination was reflected by a relative increase in cleavages occurring C-terminal to aspartic acid residues as well as increases in small molecule losses. To evaluate the effect of guanidination on the dissociation behavior of whole protein ions, bovine ubiquitin was selected as a model. Essentially, all of the amide bond cleavages associated with the +10 charge state of fully guanidinated ubiquitin were observed to occur C-terminal to aspartic acid residues, unlike the dissociation behavior of the +10 ion of the unmodified protein, where competing cleavage N-terminal to proline and nonspecific amide bond cleavages were also observed. The +8 and lower charge states of the guanidinated protein showed prominent losses of small neutral molecules. This overall fragmentation behavior is consistent with current hypotheses regarding whole protein dissociation that consider proton mobility and intramolecular charge solvation as important factors in determining favored dissociation channels, and are also consistent with the fragmentation behaviors observed for the guanidinated model peptide ions. Further evaluation of the utility of condensed phase guanidination of whole proteins is necessary but the results described here confirm that guanidination can be an effective strategy for enhancing C-terminal aspartic acid cleavages. Gas phase dissociation exclusively at aspartic acid residues, especially for whole protein ions, could be useful in identifying and characterizing proteins via tandem mass spectrometry of whole protein ions.

Guanidine↗

PubMedDate not supplied

Complementary structural information from a tryptic N-linked glycopeptide via electron transfer ion/ion reactions and collision-induced dissociation.

Glycosylation is an important post-translational modification. Analysis of glycopeptides is difficult using collision-induced dissociation, as it typically yields only information about the glycan structure, without any peptide sequence information. We demonstrate here how a 3D-quadrupole ion trap, using the complementary techniques of collision induced dissociation (CID) and electron-transfer dissociation (ETD), can be used to elucidate the glycan structure and peptide sequence of the N-glycosylated peptide from a fractionated tryptic digest of the lectin from the coral tree, Erythina cristagalli. CID experiments on the multiply protonated glycopeptide ions yield, almost exclusively, cleavage at glycosidic bonds, with little peptide backbone fragmentation. ETD reactions of the triply charged glycopeptide cations with either sulfur dioxide or nitrobenzene anions yield cleavage of the peptide backbone with no loss of the glycan structure. These results show that a 3D-quadrupole ion trap can be used to provide glycopeptide amino acid sequence information as well as information about the glycan structure.

Electron Transport↗

PubMedDate not supplied

Effects of single amino acid substitution on the collision-induced dissociation of intact protein ions: Turkey ovomucoid third domain.

Expanded understanding of the factors that direct polypeptide ion fragmentation can lead to improved specificity in the use of tandem mass spectrometry for the identification and characterization of proteins. Like the fragmentation of peptide cations, the dissociation of whole protein cations shows several preferred cleavages, the likelihood for which is parent ion charge dependent. While such cleavages are often observed, they are far from universally observed, despite the presence of the residues known to promote them. Furthermore, cleavages at residues not noted to be common in a variety of proteins can be dominant for a particular protein or protein ion charge state. Motivated by the ability to study a small protein, turkey ovomucoid third domain, for which a variety of single amino acid variants are available, the effects of changing the identity of one amino acid in the protein sequence on its dissociation behavior were examined. In particular, changes in amino acids associated with C-terminal aspartic acid cleavage and N-terminal proline cleavage were emphasized. Consistent with previous studies, the product ion spectra were found to be dependent upon the parent ion charge state. Furthermore, the fraction of possible C-terminal aspartic acid cleavages observed to occur for this protein was significantly larger than the fraction of possible N-terminal proline cleavages. In fact, very little N-terminal proline cleavage was noted for the wild-type protein despite the presence of three proline residues in the protein. The addition/removal of proline and aspartic acids was studied along with changes in selected residues adjacent to proline residues. Evidence for inhibition of proline cleavage by the presence of nearby basic residues was noted, particularly if the basic residue was likely to be protonated.

Amino Acid Sequence↗