Formation of protein-protein complexes in vacuo.
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Gases↗
Biomedical subjects
S A McLuckey
Publications and source records attributed to S A McLuckey.
Charge-state-dependent sequence analysis of protonated ubiquitin ions via ion trap tandem mass spectrometry.
One of the major factors governing the "top-down" sequence analysis of intact multiply protonated proteins by tandem mass spectrometry is the effect of the precursor ion charge state on the formation of product ions. To more fully understand this effect, electrospray ionization coupled to a quadrupole ion trap mass spectrometer, collision-induced dissociation, and gas-phase ion/ion reactions have been employed to examine the fragmentation of the [M + 12H]12+ to [M + H]+ ions of bovine ubiquitin. At low charge states (+1 to +6), loss of NH3 or H2O from the protonated precursor and directed cleavage at aspartic acid residues was observed. At intermediate charge states, (+7, +8, and +9), extensive nonspecific fragmentation of the protein backbone was observed, with 50% sequence coverage obtained from the [M + 8H]8+ ion alone. At high charge states, (+10, +11, +12), the single dominant channel that was observed was the preferential fragmentation of a single proline residue. These data can be readily explained in terms of the current model for intramolecular proton mobilization, that is, the "mobile proton model", the mechanisms for amide bond dissociation developed for protonated peptides, as well as the structures of the multiply charged ions of ubiquitin in the gas phase, examined by ion mobility and hydrogen/deuterium exchange measurements.
Amino Acid Sequence↗
Identification of bacteriophage MS2 coat protein from E. coli lysates via ion trap collisional activation of intact protein ions.
Collisional activation of the intact MS2 viral capsid protein with subsequent ion/ion reactions has been used to identify the presence of this virus in E. coli lysates. Tandem ion trap mass spectrometry experiments on the +7, +8, and +9 charge states, followed by ion/ion reactions, provided the necessary sequence tag information (and molecular weight data) needed for protein identification via database searching. The most directly informative structural information is obtained from those charge states that produce a series of product ions arising from fragmentation at adjacent residues. The formation of these product ions via dissociation at adjacent amino acid residues depends greatly on the charge state of the parent ion. Database searching of the charge-state-specific sequence tags was performed by two different search engines: the ProteinInfo program from the Protein information Retrieval On-line World Wide Web Lab or PROWL and the TagIdent program from the ExPASy molecular biology server. These search engines were used in conjunction with the sequence tag information generated via collisional activation of the intact viral coat protein. These programs were used to evaluate the feasibility of generating sequence tags from collisional activation of intact multiply charged protein ions in a quadrupole ion trap.
Capsid↗
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.
Apoproteins↗
Dissociation reactions of gaseous ferro-, ferri-, and apo-cytochrome c ions.
Electrochemical reduction of the iron bound in the heme group of cytochrome c is shown to occur in the nano-electrospray capillary if the protein is sprayed from neutral water using a steel wire as the electrical contact. Quadrupole ion trap collisional activation is used to study the dissociation reactions of cytochrome c as a function of the oxidation state of the iron. Oxidized (Fe(III)) cytochrome c dissociates via sequence-specific amide bond cleavage, while the reduced (Fe(II)) form of the protein dissociates almost exclusively by loss of protonated heme. Apo-cytochrome c, from which the heme has been removed either via gas-phase dissociation of the reduced holo-protein or via solution chemistry, dissociates via amide bond cleavage in similar fashion to the oxidized holo-protein.
Apoproteins↗
Mass analysis at the advent of the 21st century.
There have been many new and exciting developments in mass spectrometer systems in recent years. Many of these developments are being driven by challenges presented by molecular biology. The activity is fueled by resources being devoted to drug development, for example, and other medically and biologically related activities. Progress in these applications will be accelerated by improved sensitivity, specificity, and speed. In mass spectrometry, this translates to greater mass resolving power, mass accuracy, mass-to-charge range, efficiency, and speed. It is safe to say that the demands resulting from current analytical needs are likely to be met to varying degrees but probably not by a single analyzer technology or hybrid instrument. On-line and/or off-line separations and manipulations combined with mass spectrometry will also play increasingly important roles. For any analyzer, or combination of analyzers, to become widely used it must have an important application for which its figures of merit are best suited, relative to competing approaches. The relative cost of competing technologies is also an important factor. The mass filter has seen so much use in the past 30 years because its characteristics best fit a wide range of applications. As an example, biological applications, which are currently driving many instrument development activities in mass spectrometry, demand more information, of higher quality, from less material, faster, and at lower cost. Which technologies will dominate biological applications in the coming years is open to speculation. However, in considering the relative merits of today's dominant mass analyzers, areas of opportunity for improvement are apparent. Furthermore, new and more demanding measurement needs are constantly being recognized that will continue to exercise the creativity of the mass spectrometry community.
Journal Article↗
Ion trap collisional activation of the (M + 2H)2+ - (M + 17H)17+ ions of human hemoglobin beta-chain.
The parent ions of human hemoglobin beta-chain ranging in charge from 2+ to 17+ have been subjected to ion trap collisional activation. The highest charge-state ions (17+ to 13+) yielded series of products arising from dissociation of adjacent residues. The intermediate charge-state ions (12+ to 5+) tended to fragment preferentially at the N-terminal sides of proline residues and the C-terminal sides of acidic residues. Many, but not all, of the possible cleavages at proline, aspartic acid, and glutamic acid residues were represented in the spectra. The lowest charge-state ions were difficult to dissociate with high efficiency and yielded spectra with poorly defined product ion signals. This observation is attributed to sequential fragmentations arising from losses of small molecules such as water and/or ammonia. The poor fragmentation efficiency observed for the low charge states is due at least in part to the low trapping wells used to store the ions. Higher ion stabilities due to lower Coulombic repulsion and charges being sequestered at highly basic sites may also play an important role. Ion/ion proton-transfer reactions involving protein parent ions allows for the formation of a wide range of parent ion charge states. In addition, the ion/ion proton-transfer reactions involving protein dissociation products simplify interpretation of the product ion spectra.
Amino Acid Sequence↗
Measurement of collision-induced dissociation rates for tantalum oxide ions in a quadrupole ion trap.
A study of factors influencing the collision-induced dissociation (CID) rate of strongly bound diatomic ions effected via resonance excitation in a quadrupole ion trap is presented. From these studies, an approach to measuring the CID rates is described wherein product ion recovery is optimized and the effect of competitive processes (e.g., parent ion ejection and product ion reactions) on rate measurements are prevented from influencing rate measurements. Tantalum oxide ions (dissociation energy = 8.2 eV), used as a model system, were formed via reactions of glow discharge generated Ta+ ions with residual gases in the ion trap. Neon (0.5 mtorr) was found to be a more favorable target gas for the dissociation of TaO+ than He and Ar, but collisional activation of TaO+ ions in neon during ion isolation by mass selective instability necessitated ion cooling prior to dissociation. A 25 ms delay time at qz = 0.2 allowed for kinetic cooling of stored TaO+ ions and enabled precise dissociation rate measurements to be made. CID of TaO+ was determined to be most efficient at qz = 0.67 (226 kHz for m/z 197). Suitable resonance excitation voltages and times ranged from 0.56 to 1.2 V(p-p) and 1 to 68 ms, respectively. Under these conditions, measurement of rates approaching 80 s(-1) for the dissociation of TaO+ could be made without significant complications associated with competing processes, such as ion ejection.
Journal Article↗
Gas phase H/D exchange kinetics: DI versus D2O.
The gas phase H/D exchange reactions of bradykinin (M + 3H)3+ ions with D2O and DI were monitored in a quadrupole ion trap mass spectrometer. The H/D exchange kinetics of both chemical probes (D2O and DI) indicate the presence of two noninterconverting reactive gas phase ion populations of bradykinin (M + 3H)3+ at room temperature. The H/D exchange involving DI, however, generally proceeds faster than that involving D2O. The rate observations described here can be rationalized on the basis of the "relay mechanism" (see Campbell et al. J. Am. Chem. Soc. 1995, 117, 12840-12854) recently proposed to account for H/D exchange between D2O and gaseous protonated polypeptides. The higher exchange rate with DI is believed to arise primarily as a result of its lower gas-phase acidity relative to that of D2O and, secondarily, as a result of the longer bond length of DI relative to that of OD in D2O.
Bradykinin↗
Ion trap collisional activation of disulfide linkage intact and reduced multiply protonated polypeptides.
The presence of disulfide linkages in multiply charged polypeptide ions tends to inhibit the formation of structurally informative product ions under conventional quadrupole ion trap collisional activation conditions. In particular, fragmentation that requires two cleavages (i.e., cleavage of a disulfide linkage and a peptide linkage) is strongly suppressed. Reduction of the disulfide linkage(s) by use of dithiothreitol yields parent ions upon electrospray without this complication. Far richer structural information is revealed by ion trap collisional activation of the disulfide-reduced species than from the native species. These observations are illustrated with doubly protonated native and reduced somatosin, the [M + 5H](5+) ion of native bovine insulin and the [M + 4H](4+) and [M + 3H](3+) ions of the B-chain of bovine insulin produced by reduction of the disulfide linkages in insulin, and the [M + 11H](11+) ion of native chicken lysozyme and the [M + 11H](11+) and [M + 14H](14+) ions of reduced lysozyme. In each case, the product ions produced by ion trap collisional activation were subjected to ion/ion proton transfer reactions to facilitate interpretation of the product ion spectra. These studies clearly suggest that the identification of polypeptides with one or more disulfide linkages via application of ion trap collisional activation to the multiply charged parent ions formed directly by electrospray could be problematic. Means for cleaving the disulfide linkage, such as reduction by dithiothreitol prior to electrospray, are therefore desirable in these cases.
Amino Acid Sequence↗
Hydroiodic acid attachment kinetics as a chemical probe of gaseous protein ion structure: bovine pancreatic trypsin inhibitor.
The kinetics of attachment of hydroiodic acid (HI) to the (M + 6H)6+ ions of native and reduced forms of bovine pancreatic trypsin inhibitor (BPTI) in the quadrupole ion trap environment are reported. Distinctly nonlinear (pseudo first-order) reaction kinetics are observed for reaction of the native ions, indicating two or more noninterconverting structures in the parent ion population. The reduced form, on the other hand, shows very nearly linear reaction kinetics. Both forms of the parent ion attach a maximum of five molecules of hydroiodic acid. This number is expected based on the amino acid composition of the protein. There is a total of 11 strongly basic sites in the protein (i.e., six arginines, four lysines, and one N-terminus). An ion with protons occupying six of the basic sites has five available for hydroiodic acid attachment. The kinetics of successive attachment of HI to the native and reduced forms of BPTI also differ, particularly for the addition of the fourth and fifth HI molecules. A very simple kinetic model describes the behavior of the reduced form reasonably well, suggesting that all of the neutral basic sites in the reduced BPTI ions have roughly equal reactivity. However, the behavior of the native ion is not well-described by this simple model. The results are discussed within the context of differences in the three-dimensional structures of the ions that result from the presence or absence of the three disulfide linkages found in native BPTI. The HI reaction kinetics appears to have potential as a chemical probe of protein ion three-dimensional structure in the gas phase. Hydroiodic acid attachment chemistry is significantly different from other chemistries used to probe three-dimensional structure and hence, promises to yield complementary information.
Acids↗
Simplification of product ion spectra derived from multiply charged parent ions via ion/ion chemistry.
High-mass multiply charged ions fragment to yield a mixture of products of varying mass and charge. When the measurement of mass-to-charge ratio is used to determine products ion mass, product ion charge must first be established. To minimize charge-state ambiguity in product ion spectra derived from multiply charged parent ions, product ions have been subjected to proton-transfer reactions with oppositely charged ions to reduce product ion charge states largely to +1. This procedure greatly simplifies the interpretation of product ion spectra derived from multiply charged ions. Illustrative data are presented for the +4 and +3 parent ions derived from electrospray of melittin and the +12 to +4 parent ions of bovine ubiquitin, whereby product ions were formed in a conventional quadrupole ion trap tandem mass spectrometry experiment. Data are also shown for product ion mixtures derived from interface-induced dissociation of multiply charged ions derived from bovine ubiquitin, tuna cytochrome c, bovine cytochrome c, and equine cytochrome c. The use of ion/ion chemistry to simplify product ion spectra derived from multiply charged parent ions significantly extends the size range of macromolecules for which the quadrupole ion trap can derive structural information.
Amino Acid Sequence↗
Ion/ion proton-transfer kinetics: implications for analysis of ions derived from electrospray of protein mixtures.
Protein ions of different mass and charge but similar mass-to-charge ratios are shown to undergo significantly different rates of differential neutralization, defined as the rate of change of charge with time, upon initiation of reactions with oppositely charged ions in the quadrupole ion trap. Overlapping charge state distributions arising from mixtures of ions of dissimilar charge are separated on the mass-to-charge scale at short reactions times. It is also demonstrated that the time frame for near total neutralization, defined as charge reduction to the 1+ ion, is relatively insensitive to initial charge state. It is shown, for example, that the (M + 11H)(11+)-(M + 22H)22+ ions derived from horse skeletal muscle apomyoglobin yield the (M + H)+ ion as the major ion/ion reaction product over the same reaction period that largely converts doubly protonated bradykinin to the singly protonated species. Less than 25% of the bradykinin ions are expected to be totally neutralized when roughly 7% of the myoglobin ions are expected to be totally neutralized. The phenomenon of significantly different initial differential neutralization rates for ions of dissimilar charge, and the relative insensitivity to ion charge for total neutralization, can be used to advantage in strategies for protein ion mixture analysis.
Animals↗
Charge manipulation for improved mass determination of high-mass species and mixture components by electrospray mass spectrometry.
The manipulation of the charge states of high-mass ions can facilitate mass determination in electrospray (ES) mass spectrometry. Specifically, the reduction of charge (which leads to ions of higher mass-to-charge ratios) can significantly reduce peak overlap. Signals associated with various charge states of high-mass ions are more easily resolved at low charge states and chemical noise tends to be significantly lower at high mass-to-charge ratios than in the normal mass-to-charge window typically associated with electrospray. Algorithms that transform ES mass spectra to zero-charge spectra are most likely to yield unambiguous results when charge states are clearly resolved and when signal-to-noise ratios are relatively high. Charge manipulation can enhance the value of the transformation algorithms in cases in which compromises their utility. Such situations include ES mass spectra of high-mass species that yield charge states that are not baseline resolved, mixtures with many components and mixtures in which the signals from major components overwhelm signals from minor components. Examples of improved mass determination are illustrated for proteins using ion-ion chemistry as the means for charge state manipulation and the quadrupole ion trap as the mass analyzer.
Algorithms↗
Ion/ion reactions for oligopeptide mixture analysis: application to mixtures comprised of 0.5-100 kDa components.
Oligopeptide mixtures have been subjected to electrospray ionization, accumulated within a quadrupole ion trap, and subjected to ion/ion proton transfer reactions with anions derived from perfluoro-1,3-dimethylcyclohexane. Various mixtures were studied with approximate molecular weight ranges of 0.5-8.5, 12-30, 45-100, and 0.5-100 kDa. Mixtures of known composition were studied to evaluate the mixture complexity amenable to electrospray combined with ion/ion reactions to reduce spectral complexity associated with multiple charging. Mixture analysis with at least 40 components of low and medium molecular weight and roughly comparable solution concentrations appears to be straightforward. No matrix effects upon ionization were implicated in the data for the low and medium molecular weight mixtures but bovine albumin appeared to inhibit signals from bovine transferrin and chicken conalbumin in the high molecular weight mix. Furthermore, the presence of abundant low mass-to-charge ions appeared to inhibit signals from high molecular weight proteins (> 40 kDa) in the 0.5-100 kDa mix. Such an observation is consistent with dynamic range limitations that can arise from discrimination based on ion space charge effects, although an ionization matrix effect could not be precluded from the data reported here. The results reported here indicate that the limitation to mixture complexity amenable to electrospray mass spectrometry imposed by spectral congestion associated with multiple charging can be significantly reduced via ion/ion reactions. The use of ion/ion reactions can therefore facilitate the study of other factors that can impose limitations to mixture analysis, such as matrix effects upon ionization and differences in ion transmission, accumulation, storage, and detection efficiencies.
Algorithms↗
Counting basic sites in oligopeptides via gas-phase ion chemistry.
Cations derived from oligopeptides ranging from laminin fragment (5 residues) to beta-lactoglobulin (162 residues) have been subjected to gas-phase ion/molecule reactions with hydroiodic acid. The sum of the ion charge state and the maximum number of molecules of hydroiodic acid that attach to the ion is equal to the total number of lysines, arginines, histidines, and N-termini consisting of a primary amine for ions derived from all 21 oligopeptides studied. These results suggest that ion/molecule reactions can provide useful information regarding oligopeptide basic site number, which might be used as a criterion for searching protein data bases instead of, or in conjunction with, use of proteolytic digestion or gas-phase ion dissociation procedures.
Amino Acid Sequence↗
Charge reduction of oligonucleotide anions via gas-phase electron transfer to xenon cations.
Multiply-charged anions derived from electrospray (ES) ionization of the oligonucleotide 5'-d(GTCTTAGCGCTAAGAC)-3' have been subjected to electron transfer reactions with ionized xenon in a quadrupole ion trap and found to undergo minimal fragmentation. This observation stands in contrast with electron transfer to rare gases from anions of smaller oligonucleotides which have been shown to undergo extensive fragmentation. The present results are attributed to longer anion lifetimes for the larger oligonucleotide anions (following highly exothermic electron transfer) which then allow for collisional cooling by the helium bath gas. Reduction to singly charged anions with minimal fragmentation is noted, indicating that xenon cations can be considered as a reagent for simplifying ES mass spectra of moderately sized oligonucleotides. The relative merits of the use of xenon cations in the quadropole ion trap for this purpose is discussed.
Anions↗
Ion/ion proton transfer reactions for protein mixture analysis.
Ion/ion proton transfer reactions are shown to be an effective means to facilitate the resolution of ions in electrospray mass spectrometry that differ in mass and charge but are similar in mass-to-charge ratio. Examples are shown in which a minor contaminant protein in a ribonuclease B solution is clearly apparent after ion/ion proton transfer but not in the conventional electrospray mass spectrum. A further example involving a mixture of bovine serum albumin and bovine transferrin also showed the identification of previously unnoticed "contaminant" polymer. The latter mixture also illustrated important issues in the use of the quadrupole ion trap as a reaction vessel and mass analyzer for high mass-to-charge ratio ions. The results suggest that the use of ion trap operating parameters specifically tailored for storage, ejection, detection, and mass-to-charge analysis of high mass-to-charge ratio ions can have attractive analytical figures of merit for determining mixtures of relatively high-mass proteins and, by extension, other types of high-mass biopolymers.
Animals↗