Precursor Ion Charge State Research Articles

Ion Trap Collision-Induced Dissociation of Locked Nucleic Acids

Gas-phase dissociation of model locked nucleic acid (LNA) oligonucleotides and functional LNA-DNA chimeras have been investigated as a function of precursor ion charge state using ion trap collision-induced dissociation (CID). For the model LNA 5 and 8 mer, containing all four LNA monomers in the sequence, cleavage of all backbone bonds, generating a/w-, b/x-, c/y-, and d/z-ions, was observed with no significant preference at lower charge states. Base loss ions, except loss of thymine, from the cleavage of N-glycosidic bonds were also present. In general, complete sequence coverage was achieved in all charge states. For the two LNA-DNA chimeras, however, dramatic differences in the relative contributions of the competing dissociation channels were observed among different precursor ion charge states. At lower charge states, sequence information limited to the a-Base/w-fragment ions from cleavage of the 3'C-O bond of DNA nucleotides, except thymidine (dT), was acquired from CID of both the LNA gapmer and mixmer ions. On the other hand, extensive fragmentation from various dissociation channels was observed from post-ion/ion ion trap CID of the higher charge state ions of both LNA-DNA chimeras. This report demonstrates that tandem mass spectrometry is effective in the sequence characterization of LNA oligonucleotides and LNA-DNA chimeric therapeutics.

Metastable atom‐activated dissociation mass spectrometry: leucine/isoleucine differentiation and ring cleavage of proline residues

Extensive backbone fragmentation resulting in a-, b-, c-, x-, y- and z-type ions is observed of singly and doubly charged peptide ions through their interaction with a high kinetic energy beam of argon or helium metastable atoms in a modified quadrupole ion trap mass spectrometer. The ability to determine phosphorylation-sites confirms the observation with previous reports and we report the new ability to distinguish between leucine and isoleucine residues and the ability to cleave two covalent bonds of the proline ring resulting in a-, b-, x-, y-, z- and w-type ions. The fragmentation spectra indicate that fragmentation occurs through nonergodic radical ion chemistry akin to electron capture dissociation (ECD), electron transfer dissociation (ETD) and electron ionization dissociation mechanisms. However, metastable atom-activated dissociation mass spectrometry demonstrates three apparent benefits to ECD and ETD: (1) the ability to fragment singly charged precursor ions, (2) the ability to fragment negatively charged ions and (3) the ability to cleave the proline ring that requires the cleavage of two covalent bonds. Helium metastable atoms generated more fragment ions than argon metastable atoms for both substance P and bradykinin regardless of the precursor ion charge state. Reaction times less than 250 ms and efficiencies approaching 5% are compatible with on-line fragmentation, as would be desirable for bottom-up proteomics applications.

Charge state dependent fragmentation of gaseous α-synuclein cations via ion trap and beam-type collisional activation

Ions derived from nano-electrospray ionization (nano-ESI) of α-synuclein, a 14.5 kDa, 140 amino acid residue protein that is a major component of the Lewy bodies associated with Parkinson's disease, have been subjected to ion trap and beam-type collisional activation. The former samples products from fragmentation at rates generally lower than 100 s −1 whereas the latter samples products from fragmentation at rates generally greater than 10 3 s −1. A wide range of protein charge states spanning from as high as [M+17H] 17+ to as low as [M+4H] 4+ have been formed either directly from nano-ESI or via ion/ion proton transfer reactions involving the initially formed protein cations and have been subjected to both forms of collision-induced dissociation (CID). The extent of sequence information (i.e., number of distinct amide bond cleavages) available from either CID method was found to be highly sensitive to protein precursor ion charge state. Furthermore, the relative contributions of the various competing dissociation channels were also dependent upon precursor ion charge state. The qualitative trends in the changes in extent of amide bond cleavages and identities of bonds cleaved with precursor ion charge state were similar for two forms of CID. However, for every charge state examined, roughly twice the primary sequence information resulted from beam-type CID relative to ion trap CID. For example, evidence for cleavage of 86% of the protein amide bonds was observed for the [M+9H] 9+ precursor ion using beam-type CID whereas 41% of the bonds were cleaved for the same precursor ion using ion trap CID. The higher energies required to drive fragmentation reactions at rates necessary to observe products in the beam experiment access more of the structurally informative fragmentation channels, which has important implications for whole protein tandem mass spectrometry.

Evaluation of Gas-Phase Rearrangement and Competing Fragmentation Reactions on Protein Phosphorylation Site Assignment Using Collision Induced Dissociation-MS/MS and MS3

The development of strategies directed toward comprehensive analysis of the phosphoproteome have undoubtedly been facilitated by recent advances in the application of ion trap tandem mass spectrometry-based techniques for routine phosphopeptide identification. However, when multiple potential sites of phosphorylation exist within a phosphorylated peptide sequence, unambiguous characterization of the site of phosphorylation remains a significant challenge. Here, the gas-phase fragmentation reactions of a series of 33 synthetic phospho-serine, -threonine, and -tyrosine peptides containing multiple potential phosphorylation sites have been examined using collision induced dissociation (CID) and multistage tandem mass spectrometry (MS/MS and MS(3)) in a linear quadrupole ion trap. From this study, 15 of the peptides (45%) gave rise to product ions that were formed following initial transfer of a phosphate group from the phosphorylated residue to an unmodified hydroxyl-containing amino acid residue upon CID-MS/MS. The propensity for this rearrangement was found to be highly dependent on the precursor ion charge state and amino acid composition (i.e, proton mobility) of the peptide and was observed predominantly for peptides under "nonmobile" or "partially mobile" protonation conditions. The observation of these rearrangement reactions and/or the lack of product ions that provided definitive evidence for the correct site of phosphorylation, limited the ability to unambiguously assign the correct site of phosphorylation to only 12 of the 33 peptides (36%). Furthermore, the observation of competing fragmentation reactions for the neutral loss of 98 Da from these precursor ions (i.e., the loss of H(3)PO(4) versus the combined losses of HPO(3) and H(2)O) indicates that CID-MS(3) of [M + nH - 98](n+) ions may not be used for unambiguous phosphorylation site localization.

Collision-Induced Dissociation of Intact Duplex and Single-Stranded siRNA Anions

A tandem mass spectrometry approach is demonstrated for complete sequencing of a model small interfering RNA (siRNA) based on ion trap collisional activation of intact single-stranded anions. Various charge states of the siRNA duplex and the individual strands were generated by nanoelectrospray (nano-ESI). The siRNA duplex anions were predominantly dissociated into the sense and antisense strands by collisional activation. The characteristic fragment ions (c/y- and a-B/w-ion series) from both strands were observed when higher activation amplitude was applied and when beam-type collisional activation was examined; however, the coexistence of fragment ions from both strands complicated spectral interpretation. The effect of precursor ion charge state on the dissociation of the individual sense and antisense strand siRNA anions was studied using ion trap collision-induced dissociation under various activation amplitudes. Through the activation of relatively low charge state precursor ions at relatively low excitation energy, selective backbone dissociation predominantly via the c/y channels was achieved. By applying relatively high excitation energy, the a-B/w channels also became prominent; however, the increase in spectral complexity made complete peak assignment difficult. In order to simplify the product ion spectra, proton-transfer reactions were applied, and complete sequencing of each strand was achieved. The application of tandem mass spectrometry to intact single-stranded anions demonstrated in this study can be adapted for the rapid identification of other noncoding RNAs in RNomics studies.

Ion Trap Collision-Induced Dissociation of Multiply Deprotonated RNA: c/y-Ions versus (a-B)/w-Ions

The dissociation of model RNA anions has been studied as a function of anion charge state and excitation amplitude using ion trap collisional activation. Similar to DNA anions, the precursor ion charge state of an RNA anion plays an important role in directing the preferred dissociation channels. Generally, the complementary c/y-ions from 5' P-O bond cleavage dominate at low to intermediate charge states, while other backbone cleavages appear to a limited extent but increase in number and relative abundance at higher excitation energies. The competition between base loss, either as a neutral or as an anion, as well as the preference for the identity of the lost base are also observed to be charge-state dependent. To gain further insight into the partitioning of the dissociation products among the various possible channels, model dinucleotide anions have been subjected to a systematic study. In comparison to DNA, the 2'-OH group on RNA significantly facilitates the dissociation of the 5' P-O bond. However, the degree of excitation required for a 5' base loss and the subsequent 3' C-O bond cleavage are similar for the analogous RNA and DNA dinucleotides. Data collected for protonated dinucleotides, however, suggest that the 2'-OH group in RNA can stabilize the glycosidic bond of a protonated base. Therefore, base loss from low charge state oligonucleotide anions, in which protonation of one or more bases via intramolecular proton transfer can occur, may also be stabilized in RNA anions relative to corresponding DNA anions.

Electron transfer dissociation of iTRAQ labeled peptide ions.

Triply and doubly charged iTRAQ ( isobaric tagging for relative and absolute quantitation) labeled peptide cations from a tryptic peptide mixture of bovine carbonic anhydrase II were subjected to electron transfer ion/ion reactions to investigate the effect of charge bearing modifications associated with iTRAQ on the fragmentation pattern. It was noted that electron transfer dissociation (ETD) of triply charged or activated ETD (ETD and supplemental collisional activation of intact electron transfer species) of doubly charged iTRAQ tagged peptide ions yielded extensive sequence information, in analogy with ETD of unmodified peptide ions. That is, addition of the fixed charge iTRAQ tag showed relatively little deleterious effect on the ETD performance of the modified peptides. ETD of the triply charged iTRAQ labeled peptide ions followed by collision-induced dissociation (CID) of the product ion at m/ z 162 yielded the reporter ion at m/ z 116, which is the reporter ion used for quantitation via CID of the same precursor ions. The reporter ion formed via the two-step activation process is expected to provide quantitative information similar to that directly produced from CID. A 103 Da neutral loss species observed in the ETD spectra of all the triply and doubly charged iTRAQ labeled peptide ions is unique to the 116 Da iTRAQ reagent, which implies that this process also has potential for quantitation of peptides/proteins. Therefore, ETD with or without supplemental collisional activation, depending on the precursor ion charge state, has the potential to directly identify and quantify the peptides/proteins simultaneously using existing iTRAQ reagents.

Influence of Charge State and Sodium Cationization on the Electron Detachment Dissociation and Infrared Multiphoton Dissociation of Glycosaminoglycan Oligosaccharides

Electron detachment dissociation (EDD) Fourier transform mass spectrometry has recently been shown to be a useful method for tandem mass spectrometry analysis of sulfated glycosaminoglycans (GAGs). EDD produces abundant glycosidic and cross-ring fragmentations that are useful for localizing sites of sulfation in GAG oligosaccharides. Although EDD fragmentation can be used to characterize GAGs in a single tandem mass spectrometry experiment, SO3 loss accompanies many peaks and complicates the resulting mass spectra. In this work we demonstrate the ability to significantly decrease SO3 loss by selection of the proper ionized state of GAG precursor ions. When the degree of ionization is greater than the number of sulfate groups in an oligosaccharide, a significant reduction in SO3 loss is observed in the EDD mass spectra. These data suggested that SO3 loss is reduced when an electron is detached from carboxylate groups instead of sulfate. Electron detachment occurs preferentially from carboxylate versus sulfate for thermodynamic reasons, provided that carboxylate is in its ionized state. Ionization of the carboxylate group is achieved by selecting the appropriate precursor ion charge state, or by the replacement of protons with sodium cations. Increasing the ionization state by sodium cation addition decreases, but does not eliminate, SO3 loss from infrared multiphoton dissociation of the same GAG precursor ions.

Protein identification via ion‐trap collision‐induced dissociation and examination of low‐mass product ions

A whole-protein tandem mass spectrometry approach for protein identification based on precursor ion charge state concentration via ion/ion reactions, ion-trap collisional activation, ion/ion proton-transfer reactions involving the product ions, and mass analysis over a narrow m/z range (up to m/z 2000) is described and evaluated. The experiments were carried out with a commercially available electrospray ion-trap instrument that has been modified to allow for ion/ion reactions. Reaction conditions and the approach to searching protein databases were developed with the assumption that the resolving power of the mass analyzer is insufficient to distinguish charge states on the basis of the isotope spacings. Ions derived from several charge states of cytochrome c, myoglobin, ribonuclease A, and ubiquitin were used to evaluate the approach for protein identification and to develop a two-step procedure to database searching to optimize specificity. The approach developed with the model proteins was then applied to whole cell lysate fractions of Saccharomyces cerevisiae. The results are illustrated with examples of assignments made for three a priori unknown proteins, each selected randomly from a lysate fraction. Two of the three proteins were assigned to species present in the database, whereas one did not match well any database entry. The combination of the mass measurement and the product ion masses suggested the possibility for the oxidation of two methionine residues of a protein in the database. The examples show that this limited whole-protein characterization approach can provide insights that might otherwise be lacking with approaches based on complete enzymatic digestion.

Substituent effects on the gas‐phase fragmentation reactions of sulfonium ion containing peptides

The multistage mass spectrometric (MS/MS and MS3) gas-phase fragmentation reactions of methionine side-chain sulfonium ion containing peptides formed by reaction with a series of para-substituted phenacyl bromide (XBr where X=CH2COC6H4R, and R=--COOH, --COOCH3, --H, --CH3 and --CH2CH3) alkylating reagents have been examined in a linear quadrupole ion trap mass spectrometer. MS/MS of the singly (M+) and multiply ([M++nH](n+1)+) charged precursor ions results in exclusive dissociation at the fixed charge containing side chain, independently of the amino acid composition and precursor ion charge state (i.e., proton mobility). However, loss of the methylphenacyl sulfide side-chain fragment as a neutral versus charged (protonated) species was observed to be highly dependent on the proton mobility of the precursor ion, and the identity of the phenacyl group para-substituent. Molecular orbital calculations were performed at the B3LYP/6-31+G** level of theory to calculate the theoretical proton affinities of the neutral side-chain fragments. The log of the ratio of neutral versus protonated side-chain fragment losses from the derivatized side chain were found to exhibit a linear dependence on the proton affinity of the side-chain fragmentation product, as well as the proton affinities of the peptide product ions. Finally, MS3 dissociation of the nominally identical neutral and protonated loss product ions formed by MS/MS of the [M++H]2+ and [M++2H]3+ precursor ions, respectively, from the peptide GAILM(X)GAILK revealed significant differences in the abundances of the resultant product ions. These results suggest that the protonated peptide product ions formed by gas-phase fragmentation of sulfonium ion containing precursors in an ion trap mass spectrometer do not necessarily undergo intramolecular proton 'scrambling' prior to their further dissociation, in contrast to that previously demonstrated for peptide ions introduced by external ionization sources.

Charge state dependent ion trap collision-induced dissociation of reduced bovine and porcine trypsin cations

The quadrupole ion trap collision-induced dissociation (CID) behavior of reduced bovine and porcine trypsin cations, formed via nanospray ionization, is reported for the odd-numbered precursor ion charge states over the range of +9 to +21. Dissociation of precursor ions in both bovine and porcine trypsin yield predominantly b- and y-type ion backbone cleavages with strong evidence for preferential cleavages C-terminal to aspartic acid at intermediate to low charge states, C-terminal to lysine residues at intermediate charge states, and N-terminal to proline residues across all charge states examined. Both homologs showed similarly rich fragmentation within the first dozen residues at the N-terminus and both homologs proved to be resistant to fragmentation within the roughly 40 residues from the C-terminal end of the protein. Spectra derived from single precursor ion charge states showed fragmentation at 15% or less of the 222 amide linkages, while cleavage at roughly 30% of the bonds was observed when all charge states examined were considered. Significant differences in the identities of the observed fragmentation channels for the different charge state precursor ions accounts for the larger sequence coverage associated with the integration of fragmentation information across all charge states. In terms of individual fragmentation channels, many similarities between the two protein homologs were noted. Many of the major differences can be accounted for on the basis of the sequence differences associated with the homologs. However, some notable differences were noted for cleavage sites where residue identities were the same. Observations of this type highlight the fact that numerous factors, in addition to primary structure, affect protein ion dissociation under ion trap collisional activation conditions.

Charge-state dependent dissociation of a trypsin/inhibitor complex via ion trap collisional activation

Ion trap collision-induced dissociation (CID) of a non-covalent complex formed between porcine trypsin and bovine pancreatic trypsin inhibitor (BPTI) has been studied for charge states +10 to +5. Fragmentation of the +10 and +9 complexes formed directly from solution shows separation of the two subunits as the predominant dissociation channel. Lower charge states of the complex were formed by ion/ion (or, in one case, ion/molecule) proton transfer reactions. The +8 complex shows a mixture of fragmentation behavior, including subunit separation and losses of small neutral and charged species. The neutral loss is also a dominant pathway for the +7 to +5 charge states and the loss of a small cation is also common to the +7 and +6 charge states. The identity of the small cation lost was investigated and is likely to be the b 2 + ion from the BPTI subunit. This identification was supported by examination of fragmentation of various charge states of BPTI cations and a “fast” collisional activation experiment performed on the +7 complex. These results suggest that precursor ion charge state can play a dramatic role in the gas phase dissociation of protein–protein complexes such that covalent bond dissociation can come to dominate over subunit separation when Coulombic repulsion is decreased.

Top-Down Proteomics for Rapid Identification of Intact Microorganisms

We apply MALDI-TOF/TOF mass spectrometry for the rapid and high-confidence identification of intact Bacillus spore species. In this method, fragment ion spectra of whole (undigested) protein biomarkers are obtained without the need for biomarker prefractionation, digestion, separation, and cleanup. Laser-induced dissociation (unimolecular decay) of higher mass (>5 kDa) precursor ions in the first TOF analyzer is followed by reacceleration and subsequent high-resolution mass analysis of the resulting sequence-specific fragments in a reflectron TOF analyzer. In-house-developed software compares an experimental MS/MS spectrum with in silico-generated tandem mass spectra from all protein sequences, contained in a proteome database, with masses within a preset range around the precursor ion mass. A p-value, the probability that the observed matches between experimental and in silico-generated fragments occur by chance, is computed and used to rank the database proteins to identify the most plausible precursor protein. By inference, the source microorganism is then identified on the basis of the identification of individual, unique protein biomarker(s). As an example, intact Bacillus atrophaeus and Bacillus cereus spores, either pure or in mixtures, were unambiguously identified by this method after fragmenting and identifying individual small, acid-soluble spore proteins that are specific for each species. Factors such as experimental mass accuracy and number of detected fragment ions, precursor ion charge state, and sequence-specific fragmentation have been evaluated with the objective of extending the approach to other microorganisms. MALDI-TOF/TOF-MS in a lab setting is an efficient tool for in situ confirmation/verification of initial microorganism identification.

Tandem mass spectrometric strategies for determination of sulfation positions and uronic acid epimerization in chondroitin sulfate oligosaccharides

Chondroitin sulfate (CS) is a glycosaminoglycan consisting of repeating (HexA-GalNAc sulfate) disaccharides, the functions of which depend on patterns of sulfation and uronic acid epimerization. The correlation of biological activities with structure requires a strategy to determine the sequences of CS oligosaccharides without the need for total isolation. Tandem mass spectrometry has enabled the development of proteomics, based on CID fragmentation of ions produced from complex mixtures of proteolytic peptides, and has the potential for rapid sequencing of CS and other glycosaminoglycan classes. The most challenging aspects of CS sequencing are to distinguish GalNAc residues sulfated at the 4- versus the 6-position and uronic acid epimers. This work describes the utility of (1) reducing terminal derivatives and (2) control of precursor ion charge state for tandem mass spectrometric strategies for determining GalNAc sulfation positional isomers of CS. The capability of tandem MS to differentiate uronic acid epimers is also shown, providing evidence that complete or nearly complete information on CS covalent structure may be obtained using tandem MS.

Tandem mass spectrometry of sulfated heparin-like glycosaminoglycan oligosaccharides.

The structural characterization of heparin-like glycosaminoglycans (HLGAGs) is a major challenge in glycobiology. These linear, sulfated oligosaccharides are expressed on animal cell surfaces, in extracellular matrixes, basement membranes, and mast cell granules and bind with varying degrees of specificity to families of proteases, growth factors, chemokines, and blood coagulation proteins. Cell surface HLGAGs bind growth factors and growth factor receptors and serve as coreceptors in these interactions. Understanding of the mechanism and regulation of growth factor-receptor binding requires efficient determination of cell surface HLGAG structures and the variations in their expression in response to the cellular environment. The solution to this problem entails rapid, sensitive structural analysis of these molecules. To date, HLGAG sequencing requires multistep processes that combine chemical and enzymatic degradation with gel-based or mass spectrometry-based detection systems. Although tandem mass spectrometry has revolutionized proteomics, the fragility of sulfate groups has limited its usefulness in the analysis of HLGAGs. This work demonstrates that tandem mass spectrometry can be effectively used to determine HLGAG structures while minimizing losses of SO3. First, collision-induced dissociation (CID) is shown to produce abundant backbone cleavage ions for HLGAG oligosaccharides, provided that most sulfate groups are deprotonated. Fragmentation of different precursor ion charge states produces complementary data on the structure of the HLGAG. Second, calcium ion complexation of HLGAGs stabilizes the sulfate groups, increases the relative abundances of backbone cleavage ions, and decreases the abundances of ions produced from SO3 losses.

Dissociation of multiple protein ion charge states following a single gas-phase purification and concentration procedure.

The formation of a range of precursor ion charge states from a single concentrated and purified charge state, followed by activation of each charge state, is introduced as a means to obtain more protein structural information than is available from dissociation of a single charge state alone. This approach is illustrated using off-resonance collisional activation of the [M + 8H]8+ to [M + 6H]6+ precursor ions of the bacteriophage MS2 viral coat protein following concentration and purification of the [M + 8H]8+ charge state. This range of charge states was selected on the basis of an ion trap collisional activation study of the effects of precursor ion charge state on the dissociation of the [M + 12H]12+ to [M + 5H]5+ ions. Gas-phase ion/ion proton-transfer reactions and the ion parking technique were applied to purify and concentrate selected precursor ion charge states as well as to simplify the product ion spectra. The high-charge-state ions fragment preferentially at the N-terminal side of proline residues while the product ion spectra of the lowest charge states investigated are dominated by C-terminal aspartic acid cleavages. Maximum structural information is obtained by fragmentation of the intermediate-charge states.

'Top down' protein characterization via tandem mass spectrometry.

Technological and scientific advances over the past decade have enabled protein identification and characterization strategies to be developed that are based on subjecting intact protein ions and large protein fragments directly to tandem mass spectrometry. These approaches are referred to collectively as 'top down' to contrast them with 'bottom up' approaches whereby protein identification is based on mass spectrometric analysis of peptides derived from proteolytic digestion, usually with trypsin. A key step in enabling top down approaches has been the ability to assign tandem mass spectrometer product ion identities, which can be done either via high resolving power or through product ion charge state manipulation. The ability to determine product ion charge states has permitted studies of the reactions, including dissociation, ion-molecule reactions, ion-electron reactions and ion-ion reactions of high-mass, multiply charged protein ions. Electrospray ionization combined with high magnetic field strength Fourier transform ion cyclotron resonance has proven to be particularly powerful for detailed protein characterization owing to its high mass resolution and mass accuracy and its ability to effect electron capture-induced dissociation. Other types of tandem mass spectrometers are also beginning to find increasing use in top down protein identification/characterization studies. Charge state manipulation via ion-ion reactions in electrodynamic ion traps, for example, enables top down strategies to be considered using instruments with relatively modest mass resolution capabilities. Precursor ion charge state manipulation techniques have also recently been demonstrated to be capable of concentrating and charge-state purifying proteins in the gas phase. Advances in technologies applied to the structural analysis of whole protein ions and in understanding their reactions, such as those described here, are providing new options for the study of complex protein mixtures.

Charge state dependent fragmentation of gaseous protein ions in a quadrupole ion trap: bovine ferri-, ferro-, and apo-cytochrome c

The dissociation behavior of bovine ferri-, ferro-, and apo-cytochrome c ions formed by electrospray ionization has been studied as a function of precursor ion charge state under quadrupole ion trap collisional activation conditions. Tandem mass spectrometry data collected under solution conditions in which the oxidation state of the heme-iron is known show that cytochrome c ions dissociate by oxidation state dependent channels. Dissociation of precursor ions in the oxidized form [Fe(III)heme] yields predominantly b- and y-type ion backbone cleavages with preferential cleavages observed C-terminal to basic residues including lysine, arginine and histidine, C-terminal to aspartic and glutamic acids as well as N-terminal to proline. At the highest charge states studied, an abundant Leu–Met cleavage is apparent. The relative contribution of each type of fragmentation was found to be a function of precursor ion charge state. Dissociation of cytochrome c ions in the reduced form [Fe(II)heme] show, in addition to backbone cleavages, loss of protonated heme, which dominates the product ion spectrum for the [M+7H] 7+ and [M+8H] 8+ precursor ion charge states. Apo-cytochrome c ions dissociate via similar fragmentation channels to those observed in the product ion spectra for the oxidized form of cytochrome c. The results presented in this study are expected to reflect predominantly first generation cleavage products from cytochrome c precursor ions in the temperature range of 500–650 K.

Tandem mass spectrometry of ribonuclease A and B: N-linked glycosylation site analysis of whole protein ions.

Recently, an approach for the "top down" sequence analysis of whole protein ions has been developed, employing electrospray ionization, collision-induced dissociation, and ion/ion proton-transfer reactions in a quadrupole ion trap mass spectrometer. This approach has now been extended to an analysis of the [M + 12H]12+ to [M + 5H]5+ ions of ribonuclease A and its N-linked glycosylated analogue, ribonuclease B, to determine the influence of the posttranslational modification on protein fragmentation. In agreement with previous studies on the fragmentation of a range of protein ions, facile gas-phase fragmentation was observed to occur along the protein backbone at the C-terminal of aspartic acid residues, and at the N-terminal of proline, depending on the precursor ion charge state. Interestingly, no evidence was found for gas-phase deglycosylation of the N-linked sugar in ribonuclease B, presumably due to effective competition from the facile amide bond cleavage channels that "protect" the N-linked glycosidic bond from cleavage. Thus, localization of the posttranslational modification site may be determined by analysis of the "protein fragment ion mass fingerprint".

Loss of charged versus neutral heme from gaseous holomyoglobin ions.

The dissociation of holomyoglobin ions ranging in charge state from +10 to +2 has been studied using collisional activation in a quadrupole ion trap. Collisional activation times and amplitudes were varied to investigate the effects of these variables on dissociation of the heme group from the holoprotein. The onset of neutral heme loss occurs at a lower activation amplitude than loss of charged heme. For solutions of ferri-myoglobin, charged heme loss was prominent for +10 to +4 holomyoglobin ions, while neutral heme loss product was found to be dominant for charge states +3 and +2. For any given charge state, activation of holomyoglobin ions from a solution containing primarily ferro-myoglobin yielded significantly more abundant neutral heme loss products than was observed for activation of ions from solutions containing primarily ferri-myoglobin. The relative concentrations of the two oxidation states were shown to be affected by redox chemistry within the nano-electrospray emitter used in this work. Results from a double activation experiment revealed that the precursor ions of a given charge state contained a mixture of two populations, with ferro-myoglobin giving rise to neutral heme loss upon dissociation and ferri-myoglobin yielding charged heme. No evidence for electron transfer upon collisional activation of ferri-myoglobin ions was observed. Furthermore, little or no evidence for electron transfer associated with ion/ion reactions with anions derived from perfluoro-1,3-dimethylcyclohexane was observed. Definitive results could not be drawn for the lowest precursor ion charge states (+3 and +2) due to low dissociation efficiencies.