Charge state separation for protein applications using a quadrupole time-of-flight mass spectrometer

A novel method for separating ions according to their charge state using a quadrupole time-of-flight mass spectrometer is presented. The benefits of charge state separation are particularly apparent in protein identification applications at low femtomole concentration levels, where in conventional TOF MS spectra peptide ions are often lost in a sea of chemical noise. When doubly and triply charged tryptic peptide ions need to be filtered from singly charged background ions, the latter are suppressed by two to three orders of magnitude, while from 10-50% of multiply charged ions remain. The suppression of chemical noise reduces the need for chromatography and can make this experimental approach the electrospray equivalent of conventional MALDI peptide maps. If unambiguous identification cannot be achieved, MS/MS experiments are performed on the precursor ions identified through charge separation, while the previously described Q2-trapping duty cycle enhancement is tuned for approximately 1.4 of the precursor m/z to enhance intensities of ions with m/z values above that of the precursor. The resulting product ion spectra contain few fragments of impurities and provide quick and unambiguous identification through database search. The multiple charge separation technique requires minimal tuning and may become a useful tool for analysis of complex mixtures.     

Structural determination of the novel fragmentation routes of zwitteronic morphine opiate antagonists naloxonazine and naloxone hydrochlorides using electrospray ionization tandem mass spectrometry

Electrospray ionization quadrupole time-of-flight (ESI-QqToF) mass spectra of the zwitteronic salts naloxonazine dihydrochloride 1 and naloxone hydrochloride 2, a common series of morphine opiate receptor antagonists, were recorded using different declustering potentials. The singly charged ion [M+H-2HCl](+) at m/z 651.3170 and the doubly charged ion [M+2H-2HCl](2+) at m/z 326.1700 were noted for naloxonazine dihydrochloride 1; and the singly charged ion [M+H-HCl](+) at m/z 328.1541 was observed for naloxone hydrochloride 2. Low-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS) experiments established the fragmentation routes of these compounds. In addition to the characteristic diagnostic product ions obtained, we noticed the formation of a series of radical product ions for the zwitteronic compounds 1 and 2, and also the formation of a distonic ion product formed from the singly charged ion [M+H-HCl](+) of naloxone hydrochloride 2. Confirmation of the various established fragmentation routes was effected by conducting a series of ESI-CID-QqTof-MS/MS product ion scans, which were initiated by CID in the atmospheric pressure/vacuum interface using a higher declustering potential. Deuterium labeling was also performed on the zwitteronic salts 1 and 2, in which the hydrogen atoms of the OH and NH groups were exchanged with deuterium atoms. Low-energy CID-QqTof-MS/MS product ion scans of the singly charged and doubly charged deuteriated molecules confirmed the initial fragmentation patterns proposed for the protonated molecules. Precursor ion scan analyses were also performed with a conventional quadrupole-hexapole-quadrupole tandem mass spectrometer and allowed the confirmation of the genesis of some diagnostic ions.     

The nature of collision-induced dissociation processes of doubly protonated peptides: comparative study for the future use of matrix-assisted laser desorption/ionization on a hybrid quadrupole time-of-flight mass spectrometer in proteomics.

Comparative MS/MS studies of singly and doubly charged electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) precursor peptide ions are described. The spectra from these experiments have been evaluated with particular emphasis on the data quality for subsequent data processing and protein/amino acid sequence identification. It is shown that, once peptide ions are formed by ESI or MALDI, their charge state, as well as the collision energy, is the main parameter determining the quality of collision-induced dissociation (CID) MS/MS fragmentation spectra of a given peptide. CID-MS/MS spectra of singly charged peptides obtained on a hybrid quadrupole orthogonal time-of-flight mass spectrometer resemble very closely spectra obtained by matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometry (MALDI-PSD-TOFMS). On the other hand, comparison of CID-MS/MS spectra of either singly or doubly charged ion species shows no dependence on whether ions have been formed by ESI or MALDI. This observation confirms that, at the time of precursor ion selection, further mass analysis is effectively decoupled from the desorption/ionization event. Since MALDI ions are predominantly formed as singly charged species and ESI ions as doubly charged, the associated difference in the spectral quality of MS/MS spectra as described here imposes direct consequences on data processing, database searching using ion fragmentation data, and de novo sequencing when ionization techniques are changed.     

Tandem mobility mass spectrometry study of electrosprayed tetraheptyl ammonium bromide clusters

Multiply charged electrospray ions from concentrated solutions of Heptyl4N+Br- (designated A+B- hereafter) in formamide are analyzed mass spectrometrically (MS) following mobility selection in ambient air in a differential mobility analyzer (DMA). Most of the sharp mobility peaks seen are identified as (AB)(n)A+ clusters, with 0 < or = n < ot = 5. One anomalously abundant and mobile ion is identified as NH4+(AB)4. Six ions in the (AB)n(A+)2 series are also identified, completing and correcting earlier mobility data for singly and doubly charged ions up to masses of almost 9000 Da. The more mobile of two broad humps seen in the mobility spectrum includes m/z values approximately from 2500 up to 12,000 Da. It is formed primarily by multiply charged (AB)n(A+)z clusters with multiple ammonium bromide adducts. Because of overlapping of many peaks of different m/z and charge state z, only a few individual species can be identified by MS alone in this highly congested region. However, the spectral simplification brought about by mobility selection upstream of the MS reveals a series of broad modulations in m/z space, with all ions resolved in the second, third, ...sixth modulation being in charge states z = 2, 3, ...6, respectively. Extrapolation of this trend beyond the sixth wave fixes the ion charge state (in some cases up to z = 15) and mass (beyond m = 175,000 u). This wavy structure had been previously observed and explained in terms of ion evaporation kinetics from volatile drops, though without mass identification. All observations indicate that the clusters are formed as charged residues, but their charge state is fixed by the Iribarne-Thomson ion evaporation mechanism. Consequently, the measured curve of cluster diameter versus z yields the two parameters governing ion evaporation kinetics. Clusters with z > 1 and electrical mobility Z > 0.495 cm2/V/s are metastable and evaporate a singly charged cluster, probably (AB)2A+, between the DMA and the MS. Plotting the electrical mobilities Z of the clusters in the form (z/Z)1/2 versus m(1/3) (both proportional to cluster diameter) collapse the data for all cluster sizes and charge states into one single straight line for Z below 0.495 cm2/V/s. This linear relation reveals a uniform apparent cluster density of 0.935 g/cm3 and an effective hard-sphere diameter of the air molecules of 0.44 nm. An anomalous mobility increase is observed at diameters below 3 nm.     

Use of doubly protonated molecules in the analysis of cathedulins in crude extracts of khat (Catha edulis) by liquid chromatography/serial mass spectrometry

Analysis of crude methanolic extracts of fresh khat (Catha edulis) by liquid chromatography/mass spectrometry (LC/MS) revealed the presence of 62 cathedulin alkaloids (compared with 15 published structures). Many cathedulins generated doubly protonated molecules following electrospray ionisation and the ratio of doubly to singly protonated species could be manipulated by adjusting the electrospray capillary position and source conditions. By selecting the doubly protonated species for serial mass spectrometric analysis (MS/MS), it was possible to use an ion trap mass spectrometer to observe singly charged product ions at lower m/z values than ion trap MS/MS analysis of [M+H](+) would have allowed. These spectra were particularly valuable in elucidating the acylation patterns of cathedulins where MS/MS analysis of [M+H](+) resulted in loss of a large neutral species to yield a small singly charged fragment below the lower limit for ion trapping. Acylation patterns for most of the 62 cathedulins are proposed from mass spectrometric analysis, and the data obtained for a major unreported cathedulin of mass 1001 Da suggest that it belongs to a new group of cathedulins having a cathate dilactone bridge but not an evoninate bridge.     

Doubly charged porphyrin ion tandem mass spectrometry: Implications for structure elucidation

Under electron ionization (EI) conditions, porphyrins yield unusually high intensities of doubly charged molecular and fragment ions. These doubly charged ions offer unique opportunities for the structure elucidation of porphyrins by tandem mass spectrometry (MS/MS). First, they fragment to a greater extent than the corresponding singly charged ions under both EI/MS and EI/MS/MS conditions. Second, doubly and singly charged porphyrin ions often fragment via different pathways, and can therefore yield different structural information. This paper describes several ways in which analyses of doubly charged porphyrin ions with a triple quadrupole tandem mass spectrometer can be useful in structure elucidation of porphyrins. The effect of the metal atom on the fragmentation of metalloporphyrins in an EI source is demonstrated by correlating the extent of doubly charged fragment ion formation to a stability index. Doubly charged porphyrin ions are shown to yield predominantly doubly charged daughter ions upon collisionally activated dissociation (CAD), and are also shown to fragment to a greater extent than corresponding singly charged porphyrin ions. Advantages and disadvantages of doubly charged porphyrin ion MS/MS for structure elucidation are discussed.     

Analysis of tryptic peptides using desorption electrospray ionisation combined with ion mobility spectrometry/mass spectrometry

A novel method is reported for rapid protein identification by the analysis of tryptic peptides using desorption electrospray ionisation (DESI) coupled with hyphenated ion mobility spectrometry and quadrupole time-of-flight mass spectrometry (IMS/Q-ToF-MS). Confident protein identification is demonstrated for the analysis of tryptically digested bovine serum albumin (BSA), with no sample pre-treatment or clean-up. Electrophoretic ion mobility separation of ions generated by DESI allowed examination of charge-state and mobility distributions for tryptic peptide mixtures. Selective interrogation of singly charged ions allowed isobaric peptide responses to be distinguished, along with a reduction in spectral noise. The mobility-selected singly charged peptide responses were presented as a pseudo-peptide mass fingerprint (p-PMF) for protein database searching. Comparative data are shown for electrospray ionisation (ESI) of the BSA digest, without sample clean-up, from which confident protein identification could not be made. Implications for the robustness of the DESI method, together with potential insights into mechanisms for DESI of proteolytic digests, are discussed.     

The structural significance of doubly charged ion spectra. Phenylenediamine derivatives

The distribution of doubly charged ions found in the mass spectra of various N, N′-alkyl substituted phenylenediamines is compared to that of their singly charged counterparts. Structural and electronic requirements for the resonance stabilization of doubly charged organic ions are suggested and it is shown that doubly charged ions could be used to differentiate between certain structural isomers which give practically identical singly charged ions. The doubly charged ions were distinguished from singly charged ions of the same integral m/e value by high resolution techniques.     

Electron transfer dissociation with supplemental activation to differentiate aspartic and isoaspartic residues in doubly charged peptide cations

Electron-transfer dissociation (ETD) with supplemental activation of the doubly charged deamidated tryptic digested peptide ions allows differentiation of isoaspartic acid and aspartic acid residues using the c + 57 or z ^• − 57 peaks. The diagnostic peak clearly localizes and characterizes the isoaspartic acid residue. Supplemental activation in ETD of the doubly charged peptide ions involves resonant excitation of the charge reduced precursor radical cations and leads to further dissociation, including extra backbone cleavages and secondary fragmentation. Supplemental activation is essential to obtain a high quality ETD spectrum (especially for doubly charged peptide ions) with sequence information. Unfortunately, the low-resolution of the ion trap mass spectrometer makes detection of the diagnostic peak, [M-60], for the aspartic acid residue difficult due to interference with side-chain loss from arginine and glutamic acid residues.     

Site-specific N-glycosylation analysis: matrix-assisted laser desorption/ionization quadrupole-quadrupole time-of-flight tandem mass spectral signatures for recognition and identification of glycopeptides

The identification of glycosylation sites in proteins is often possible through a combination of proteolytic digestion, separation, mass spectrometry (MS) and tandem MS (MS/MS). Liquid chromatography (LC) in combination with MS/MS has been a reliable method for detecting glycopeptides in digestion mixtures, and for assigning glycosylation sites and glycopeptide sequences. Direct interfacing of LC with MS relies on electrospray ionization, which produces ions with two, three or four charges for most proteolytic peptides and glycopeptides. MS/MS spectra of such glycopeptide ions often lead to ambiguous interpretation if deconvolution to the singly charged level is not used. In contrast, the matrix-assisted laser desorption/ionization (MALDI) technique usually produces singly charged peptide and glycopeptide ions. These ions require an extended m/z range, as provided by the quadrupole-quadrupole time-of-flight (QqTOF) instrument used in these experiments, but the main advantages of studying singly charged ions are the simplicity and consistency of the MS/MS spectra. A first aim of the present study is to develop methods to recognize and use glycopeptide [M+H]+ ions as precursors for MS/MS, and thus for glycopeptide/glycoprotein identification as part of wider proteomics studies. Secondly, this article aims at demonstrating the usefulness of MALDI-MS/MS spectra of N-glycopeptides. Mixtures of diverse types of proteins, obtained commercially, were prepared and subjected to reduction, alkylation and tryptic digestion. Micro-column reversed-phase separation allowed deposition of several fractions on MALDI plates, followed by MS and MS/MS analysis of all peptides. Glycopeptide fractions were identified after MS by their specific m/z spacing patterns (162, 203, 291 u) between glycoforms, and then analyzed by MS/MS. In most cases, MS/MS spectra of [M+H]+ ions of glycopeptides featured peaks useful for determining sugar composition, peptide sequence, and thus probable glycosylation site. Peptide-related product ions could be used in database search procedures and allowed the identification of the glycoproteins.     

Radical <Emphasis Type="BoldItalic">a</Emphasis>-Ions in Electron Capture Dissociation: On the Origin of Species

Radical a* ions appear in electron capture dissociation mass spectra sporadically, but sometimes with high intensity. Mechanistically, radical a ions are hypothesized to arise due to thermodynamically disadvantaged charge solvation on the backbone nitrogen (instead of carbonyl), which upon neutralization produces a hypervalent group instantly fragmenting into a radical b* and conventional y' ion. The former species is unstable and, after releasing a CO molecule, decays to an a* ion. Here we validate this scenario by direct observation of the complementarity of a* and y' ions by interrogation of an ECD MS/MS database of >10,000 doubly and >5,000 triply charged tryptic peptides. Intriguingly, the most abundant a*/y' pairs are found to come from the cleavage of the same backbone link as the most abundant c' and z* complementary ions. This result gives strong support to the “local” N-Cα bond cleavage mechanism, in which the dissociation occurs at the site of charge solvation. However, a second strong peak is observed in the c'/z* fragment distribution four residues away from the a*/y' cleavage, which supports the indirect N-Cα bond cleavage mechanism. The size distribution of a ions from doubly (but not triply!) charged precursors shows deficit of a3 ions, and possibly a6 ions.     

Automated deconvolution and deisotoping of electrospray mass spectra

Electrospray ionization (ESI) of peptides and proteins produces a series of multiply charged ions with a mass/charge (m/z) ratio between 500 and 2000. The resulting mass spectra are crowded by these multiple charge values for each molecular mass and an isotopic cluster for each nominal m/z value. Here, we report a new algorithm simultaneously to deconvolute and deisotope ESI mass spectra from complex peptide samples based on their mass-dependent isotopic mean pattern. All signals corresponding to one peptide in the sample were reduced to one singly charged monoisotopic peak, thereby significantly reducing the number of signals, increasing the signal intensity and improving the signal-to-noise ratio. The mass list produced could be used directly for database searching. The developed algorithm also simplified interpretation of fragment ion spectra of multiply charged parent ions.     

On-line capillary liquid chromatography tandem mass spectrometry on an ion trap/ reflectron time-of-flight mass spectrometer using the sequence tag database search approach for peptide sequencing and protein identification

Capillary high-performance liquid chromatography has been coupled on-line with an ion trap storage/reflectron time-of-flight mass spectrometer to perform tandem mass spectrometry for tryptic peptides. Selection and fragmentation of the precursor ions were performed in a three-dimensional ion trap, and the resulting fragment ions were pulsed out of the trap into a reflectron time-of-flight mass spectrometer for mass analysis. The stored waveform inverse Fourier transform waveform was applied to perform ion selection and an improved tickle voltage optimization scheme was used to generate collision-induced dissociation. Tandem mass spectra of various doubly charged tryptic peptides were investigated where a conspicuous y ion series over a certain mass range defined a partial amino acid sequence. The partial sequence was used to determine the identity of the peptide or even the protein by database search using the sequence tag approach. Several peptides from tryptic digests of horse heart myoglobin and bovine cytochrome c were selected for tandem mass spectrometry (MS/MS) where it was demonstrated that the proteins could be identified based on sequence tags derived from MS/MS spectra. This approach was also utilized to identify protein spots from a two-dimensional gel separation of a human esophageal adenocarcinoma cell line.     

Electrospray Ionization Tandem Mass Spectrometry of Ammonium Cationized Polyethers

Quaternary ammonium salts (Quats) and amines are known to facilitate the MS analysis of high molar mass polyethers by forming low charge state adduct ions. The formation, stability, and behavior upon collision-induced dissociation (CID) of adduct ions of polyethers with a variety of Quats and amines were studied by electrospray ionization quadrupole time-of-flight, quadrupole ion trap, and linear ion trap tandem mass spectrometry (MS/MS). The linear ion trap instrument was part of an Orbitrap hybrid mass spectrometer that allowed accurate mass MS/MS measurements. The Quats and amines studied were of different degree of substitution, structure, and size. The stability of the adduct ions was related to the structure of the cation, especially the amine’s degree of substitution. CID of singly/doubly charged primary and tertiary ammonium cationized polymers resulted in the neutral loss of the amine followed by fragmentation of the protonated product ions. The latter reveals information about the monomer unit, polymer sequence, and endgroup structure. In addition, the detection of product ions retaining the ammonium ion was observed. The predominant process in the CID of singly charged quaternary ammonium cationized polymers was cation detachment, whereas their doubly charged adduct ions provided the same information as the primary and tertiary ammonium cationized adduct ions. This study shows the potential of specific amines as tools for the structural elucidation of high molar mass polyethers.     

The influence and utility of varying field strength for the separation of tryptic peptides by ion mobility-mass spectrometry

The influence of field strength on the separation of tryptic peptides by drift tube-based ion mobility-mass spectrometry is reported. Operating the ion mobility drift tube at elevated field strengths (expressed in V cm(-1) torr(-1)) reduces separation times and increases ion transmission efficiencies. Several accounts in the literature suggest that performing ion mobility separation at elevated field strength can change the selectivity of ion separation. To evaluate the field strength dependant selectivity of ion mobility separation, we examined a data set of 65 singly charged tryptic peptide ion signals (mass range 500-2500 m/z) at six different field strengths and four different drift gas compositions (He, N2, Ar, and CH4). Our results clearly illustrate that changing the field strength from low field (15 V cm(-1) torr(-1)) to high field (66 V cm(-1) torr(-1)) does not significantly alter the selectivity or peak capacity of IM-MS. The implications of these results are discussed in the context of separation methodologies that rely on the field strength dependence of ion mobility for separation selectivity, e.g., high-field asymmetric ion mobility spectrometry (FAIMS).     

Analysis of intact proteins on a chromatographic time scale by electron transfer dissociation tandem mass spectrometry

Direct analysis of intact proteins on a chromatographic time scale is demonstrated on a modified linear ion trap mass spectrometer using sequential ion/ion reactions, electron transfer and proton transfer, to dissociate the sample and to convert the resulting peptide fragments to a mixture of singly and doubly charged species. Proteins are converted to gas-phase, multiply-charged, positive ions by electrospray ionization and then allowed to react with fluoranthene radical anions. Electron transfer to the multiply charged protein promotes random fragmentation of amide bonds along the protein backbone. Multiply charged fragment ions are then deprotonated in a second ion/ion reaction with even-electron benzoate anions. M/z values for the resulting singly and doubly charged ions are used to read a sequence of 15-40 amino acids at both the N-terminus and the C-terminus of the protein. This information, along with the measured mass of the intact protein, are employed to identify known proteins and to detect the presence of post-translational modifications. In this study, we analyze intact proteins from the Escherchia coli 70S ribosomal protein complex and identify 46 of the 55 known unique components in a single, 90 min, on-line, chromatography experiment. Truncated versions of the above proteins along with several post-translational modifications are also detected.     

Unimolecular and collision-induced reactions of doubly charged porphyrins

Results from a tandem mass spectrometry (MS/MS) study, obtained with a reverse-geometry mass spectrometer, of the unimolecular and collision-induced reactions of doubly charged free-base and metal containing alkyl-substituted porphyrins formed by electron ionization are reported. These doubly charged porphyrin ions dissociate to yield both singly and doubly charged product ions via a number of reactions. This article classifies the major reactions observed, illustrating each with the appropriate spectra. Supplementary data from the same porphyrins, acquired with a tandem quadrupole MS/MS instrument, are also presented. The potential utility of some of these reactions as new methods for porphyrin analysis is discussed.     

‘Doubly charged ion’ mass spectra of some simple aromatic compounds

Benzene, toluene, phenol, diphenyl ether and the three isomeric dihydroxy-benzenes have been examined using an MS-9 mass spectrometer under conditions that allowed only ions having twice the normal amount of kinetic energy to be detected. These ions are, in fact, singly charged ions arising from charge exchange reactions of doubly charged ions of the same mass, occuring in the first field free region of the Spectrometer. It is argued that the spectra obtained yield essentially the distribution of doubly charged ions in the source region. These ‘doubly charged ion’ mass spectra are compared with the normal singly charged ion spectra of the compounds and the implications of the significant differences that are found, are discussed.     

Primary and secondary locations of charge sites in angiotensin II (M + 2H)2+ ions formed by electrospray ionization

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 multiple charged ions. The most probable kinetic energy released (Tm.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, (b7" + 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.     

Proton transfer reactions for improved peptide characterisation

The combination of deprotonation (via ion/molecule and ion/ion reactions) and low-energy collision-induced dissociation (CID) has been explored for the enhanced characterisation of tryptic peptides via access to different precursor charge states. This approach allows instant access to fragmentation properties of singly and doubly protonated precursors (arising from the availability of mobile protons) in a single experiment. Considering both charge states extended our base of structurally informative data (in comparison with considering just a single charge state) due to generation of additional sequence ions and by obtaining supplementary structural information derived from selective cleavages. Roughly 37% of combined data sets (CID spectra of doubly and singly charged precursor) showed a greater database identification confidence than each set alone. Moreover, comparison between a number of sequence ions of the singly charged precursor and the doubly charged precursor provided a mean of distinguishing the two classes of tryptic peptides (arginine or lysine containing).