Electron capture dissociation of tyrosine O-sulfated peptides complexed with divalent metal cations

We compare electron capture dissociation (ECD) of doubly protonated and divalent metal-adducted tyrosine O-sulfated peptides without basic amino acid residues. ECD of doubly protonated Tyr2-sulfated cholecystokinin (CCKS) and doubly protonated Tyr12-sulfated gastrin II (GST) resulted in complete loss of SO3 from all product ions. Thus, contrary to typical ECD behavior, localization of the sulfate groups was not possible. By contrast, ECD of Ca-, Mn-, Zn-, and Fe-adducted CCKS and ECD of deprotonated GST with two calcium adducts, i.e., [GST + 2Ca - H]3+, resulted in sulfated c'- and z.-type product ions with high sequence coverage, thereby allowing both sequencing and sulfate localization. In addition, divalent metal adduction provided improved positive mode ionization efficiency for these peptides. The drastically different fragmentation behavior observed in ECD of protonated and metal-adducted CCKS and GST, respectively, is proposed to be a consequence of the absence of basic amino acid residues, promoting a mobile proton-like fragmentation mechanism, including abundant sulfate loss, for protonated species. Retention of sulfate groups was also observed in electron detachment dissociation (EDD) of CCKS and GST. However, the EDD fragmentation efficiency was much lower than that of ECD and very limited fragmentation was observed in EDD of GST, precluding localization of the sulfate group in that peptide.     

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.     

Applications of electron-ion dissociation reactions for analysis of polycationic chitooligosaccharides in Fourier transform mass spectrometry

Singly protonated, doubly protonated, and sodiated pentaglucosamide (GlcNAc)(5), oligoglucosamines (GlcN)(m)(), and (GlcN)(3)GlcN(3OH14:0) were analyzed in an FTICR mass spectrometer by electron-ion dissociation reactions and compared to collision activation. The general fragmentation mode was found as the asymmetrical sequence fragments (B(n)() and minor C(n)() ion series) with full sequence coverage. Molecular mass information of each glucosamide or glucosamine residue can be readily obtained from the ion series. Fragmentation by electron capture dissociation revealed additional fragmentation of the N-acetyl moiety compared to sustained off-resonance irradiation collision-activated dissociation (SORI-CAD) and electron-induced dissociation (EID). Sodiated GlcNAc(5) molecular adduct ions were analyzed by EID and compared to CAD. Both techniques provided full sequence coverage. EID was more effective, but CAD resulted in the cross-ring ion products (0,2)A(n)() and (2,4)A(n)() for all relevant glucosamide residues.     

Spatially resolved protein hydrogen exchange measured by subzero-cooled chip-based nanoelectrospray ionization tandem mass spectrometry

Mass spectrometry has become a valuable method for studying structural dynamics of proteins in solution by measuring their backbone amide hydrogen/deuterium exchange (HDX) kinetics. In a typical exchange experiment one or more proteins are incubated in deuterated buffer at physiological conditions. After a given period of deuteration, the exchange reaction is quenched by acidification (pH 2.5) and cooling (0 °C) and the deuterated protein (or a digest thereof) is analyzed by mass spectrometry. The unavoidable loss of deuterium (back-exchange) that occurs under quench conditions is undesired as it leads to loss of information. Here we describe the successful application of a chip-based nanoelectrospray ionization mass spectrometry top-down fragmentation approach based on cooling to subzero temperature (-15 °C) which reduces the back-exchange at quench conditions to very low levels. For example, only 4% and 6% deuterium loss for fully deuterated ubiquitin and β(2)-microglobulin were observed after 10 min of back-exchange. The practical value of our subzero-cooled setup for top-down fragmentation HDX analyses is demonstrated by electron-transfer dissociation of ubiquitin ions under carefully optimized mass spectrometric conditions where gas-phase hydrogen scrambling is negligible. Our results show that the known dynamic behavior of ubiquitin in solution is accurately reflected in the deuterium contents of the fragment ions.     

MS/MS simplification by 355 nm ultraviolet photodissociation of chromophore-derivatized peptides in a quadrupole ion trap

Ultraviolet photodissociation (UVPD) of chromophore-modified peptides enhances the capabilities for de novo sequencing in a quadrupole ion trap mass spectrometer. Attachment of UV chromophores allows efficient photoactivation of not only the precursor ions but also any fragments that retain the chromophore functionality. For doubly protonated peptides, UVPD leads to a vast reduction in MS/MS complexity. The array of b and y ions typically seen upon collisionally activated dissociation is reduced to a single series of either y or b ions by UVPD depending on the location of the chromophore (i.e., N- or C-terminus). The sulfonation reagent Alexa Fluor 350 (AF350) provided the best overall results for the singly and doubly charged peptides by UVPD. The nonsulfonated analogue of AF350, 7-amino-4-methylcoumarin-3-acetic acid, also led to simplified spectra for doubly charged, but not singly charged, peptides by UVPD. Dinitrophenyl-peptides also yielded simplified spectra by UVPD albeit with a small amount of internal fragments accompanying the series of diagnostic y ions. The success of this MS/MS simplification process stems from extensive secondary fragmentation of any chromophore-containing fragments upon exposure to subsequent laser pulses. Energy-variable UVPD reveals that the abundances of non-chromophore-containing y fragment ions increase linearly with laser pulse energy, suggesting secondary dissociation of these species is insignificant. The abundances of chromophore-containing a/b fragment ions follow a quadratic trend due to the extensive secondary fragmentation at higher laser energies or multiple pulses.     

Factors that influence fragmentation behavior of N-linked glycopeptide ions

The investigation of site-specific glycosylation is essential for further understanding the many biological roles that glycoproteins play; however, existing methods for characterizing site-specific glycosylation either are slow or yield incomplete information. Mass spectrometry (MS) is being applied to investigate site-specific glycosylation with bottom-up proteomic type strategies. When using these approaches, tandem mass spectrometry techniques are often essential to verify glycopeptide composition, minimize false positives, and investigate structure. The fragmentation behavior of glycopeptide ions has previously been investigated with multiple techniques including collision induced dissociation (CID), infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD); however, due to the almost exclusive analysis of multiply protonated tryptic glycopeptide ions, some dissociation behaviors of N-linked glycopeptide ions have not been fully elucidated. In this study, IRMPD of N-linked glycopeptides has been investigated with a focus on the effects of charge state, charge carrier, glycan composition, and peptide composition. Each of these parameters was shown to influence the fragmentation behavior of N-linked glycopeptide ions. For example, in contrast to previously reported accounts that IRMPD results only in glycosidic bond cleavage, the fragmentation of singly protonated glycopeptide ions containing a basic amino acid residue almost exclusively resulted in peptide backbone cleavage. The fragmentation of the doubly protonated glycopeptide ion exhibited fragmentation similar to that previously reported; however, when the same glycopeptide was sodium coordinated, a previously inaccessible series of glycan fragments were observed. Molecular modeling calculations suggest that differences in the site of protonation and metal ion coordination may direct glycopeptide ion fragmentation.     

Activation of intact electron-transfer products of polypeptides and proteins in cation transmission mode ion/ion reactions

Cationic peptide electron-transfer products that do not fragment spontaneously are exposed to ion trap collisional activation immediately upon formation while they pass through a high-pressure collision cell (Q2), where the electron-transfer reagent anions are stored. Radial ion acceleration, which is normal to the ion flow, is implemented by applying an auxiliary dipolar alternating current to a pair of opposing rods of the Q2 quadrupole array at a frequency in resonance with the surviving electron-transfer products. Collisional cooling of cations in the pressurized Q2 ensures efficient overlap of the positive and negative ions for ion/ion reactions and also gives rise to relatively long residence times (milliseconds) for ions in Q2, making it possible to fragment ions via radial excitation during their axial transmission. The radial activation for transmission mode electron-transfer ion/ion reactions has been demonstrated with a doubly protonated tryptic peptide, a triply protonated phosphopeptide, and [M + 7H]7+ ions of ubiquitin. In all cases, significant increases in fragment ion yields and structural information from electron-transfer dissociation (ETD) were observed, suggesting the utility of this method for improving transmission mode ETD performance for relatively low charge states of peptides and proteins.     

Enabling MALDI-FTICR-MS/MS for high-performance proteomics through combination of infrared and collisional activation

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is a central tool for proteomic analysis, yet the singly protonated tryptic peptide ions produced by MALDI are significantly more difficult to dissociate for tandem mass spectrometry (MS/MS) than the corresponding multiply protonated ions. In order to overcome this limitation, current proteomic approaches using MALDI-MS/MS involve high-energy collision-induced dissociation (CID). Unfortunately, the use of high-energy CID complicates product ion spectra with a significant proportion of irrelevant fragments while also reducing mass accuracy and mass resolution. In order to address the lack of a high-resolution, high mass accuracy MALDI-MS/MS platform for proteomics, Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and a recently developed MS/MS technique termed CIRCA (for combination of infrared and collisional activation) have been applied to proteomic analysis. Here, CIRCA is shown to be suitable for dissociating singly protonated tryptic peptides, providing greater sequence coverage than either CID or infrared multiphoton dissociation (IRMPD) alone. Furthermore, the CIRCA fragmentation spectra are of sufficient quality to allow protein identification based on the MS/MS spectra alone or in concert with the peptide mass fingerprint (PMF). This is accomplished without compromising mass accuracy or mass resolution. As a result, CIRCA serves to enable MALDI-FTICR-MS/MS for high-performance proteomics experiments.     

Tandem mass spectrometry of very large molecules: serum albumin sequence information from multiply charged ions formed by electrospray ionization.

Serum albumin proteins, Mr approximately 66 kDa, from 10 different species (bovine, human, rat, horse, sheep, goat, rabbit, dog, porcine, and guinea pig) have been studied by electrospray ionization mass spectrometry (ESI-MS) and tandem MS using a triple-quadrupole instrument. The effectiveness of collisional activation for the multiply charged albumin ions greatly exceeds that for singly charged ions, allowing an extension by a factor of at least 20 to the molecular mass range for obtaining sequence-specific product ions by tandem MS. Efficient dissociation is largely attributed to "preheating" in the interface Coulombic instability and the large number of collisions. Increasing the electric field in the intermediate pressure region, between the nozzle-skimmer elements of the atmospheric pressure/vacuum interface, allows fragmentation of the multiply protonated (to 96+) molecules produced by ESI. The most abundant dissociation product ions assigned have a low charge state (2+ to 5+) and are attributed to "bn" mode species from cleavage of the -CO-N- peptide backbone bonds. Particularly abundant dissociation products originate from regions near residues n = 20-25 from the NH2 terminus for parent ions of moderate charge (approximately 50+). Collisionally activated dissociation (CAD) mass spectra from porcine serum albumin, in contrast to the other albumins, also gave prominent singly charged "yn" fragments formed from cleavages near the COOH terminus. Tandem mass spectrometry (MS/MS) of the multiply charged molecular ions, and of fragment species produced by dissociation in the interface (i.e., effective MS/MS/MS), produced similar "bn" species and served to confirm spectral assignments. We also show that ESI mass spectra allow a qualitative assessment of protein microheterogeneity and, in some cases, resolution of major contributions. The physical and analytical implications of the results are discussed, including the identification of possible errors in previously published sequences.     

Supplemental activation method for high-efficiency electron-transfer dissociation of doubly protonated peptide precursors

Electron-transfer dissociation (ETD) delivers the unique attributes of electron capture dissociation to mass spectrometers that utilize radio frequency trapping-type devices (e.g., quadrupole ion traps). The method has generated significant interest because of its compatibility with chromatography and its ability to: (1) preserve traditionally labile post-translational modifications (PTMs) and (2) randomly cleave the backbone bonds of highly charged peptide and protein precursor ions. ETD, however, has shown limited applicability to doubly protonated peptide precursors, [M + 2H]2+, the charge and type of peptide most frequently encountered in "bottom-up" proteomics. Here we describe a supplemental collisional activation (CAD) method that targets the nondissociated (intact) electron-transfer (ET) product species ([M + 2H]+*) to improve ETD efficiency for doubly protonated peptides (ETcaD). A systematic study of supplementary activation conditions revealed that low-energy CAD of the ET product population leads to the near-exclusive generation of c- and z-type fragment ions with relatively high efficiency (77 +/- 8%). Compared to those formed directly via ETD, the fragment ions were found to comprise increased relative amounts of the odd-electron c-type ions (c+*) and the even-electron z-type ions (z+). A large-scale analysis of 755 doubly charged tryptic peptides was conducted to compare the method (ETcaD) to ion trap CAD and ETD. ETcaD produced a median sequence coverage of 89%-a significant improvement over ETD (63%) and ion trap CAD (77%).     

Electron capture dissociation for structural characterization of multiply charged protein cations

For proteins of < 20 kDa, this new radical site dissociation method cleaves different and many more backbone bonds than the conventional MS/MS methods (e.g., collisionally activated dissociation, CAD) that add energy directly to the even-electron ions. A minimum kinetic energy difference between the electron and ion maximizes capture; a 1 eV difference reduces capture by 10(3). Thus, in an FTMS ion cell with added electron trapping electrodes, capture appears to be achieved best at the boundary between the potential wells that trap the electrons and ions, now providing 80 +/- 15% precursor ion conversion efficiency. Capture cross section is dependent on the ionic charge squared (z2), minimizing the secondary dissociation of lower charge fragment ions. Electron capture is postulated to occur initially at a protonated site to release an energetic (approximately 6 eV) H. atom that is captured at a high-affinity site such as -S-S- or backbone amide to cause nonergodic (before energy randomization) dissociation. Cleavages between every pair of amino acids in mellitin (2.8 kDa) and ubiquitin (8.6 kDa) are represented in their ECD and CAD spectra, providing complete data for their de novo sequencing. Because posttranslational modifications such as carboxylation, glycosylation, and sulfation are less easily lost in ECD than in CAD, ECD assignments of their sequence positions are far more specific.     

A mechanistic investigation of the enhanced cleavage at histidine in the gas-phase dissociation of protonated peptides

Enhanced gas-phase cleavage of peptides adjacent to histidine was investigated. The peptides examined were angiotensins III (RVYIHPF) and IV (VYIHPF) as well as synthetic peptide analogues with altered key residues ((R)VYI-X-Z-F; X = F or H and Z = A, P, or Sar) or a fixed charge M3P(+)CH(2)C(O)-VYIHPF. While all singly protonated peptide ions containing both histidine and arginine fragment nonselectively, the doubly protonated peptide ions with arginine and histidine, and the singly protonated peptides containing histidine but not arginine, cleave in a selective manner. In particular, dominant complementary b+/y+ product ions resulting from cleavage between the HP amide bond are observed. For the fixed-charge derivative, selective cleavage occurs only if a proton is added to produce a doubly charged precursor. The results are consistent with involvement of a protonated histidine in the selective cleavage. The ratio of b+/y+ is determined by the identity of the residue C-terminal to histidine and by the ability of protonated histidine to transfer a proton to the C-terminal leaving fragment. This was probed further by systematically changing the residue C-terminal to histidine and by alkylating histidine. The results indicate that while b+/y+ complementary ion pairs dominate in doubly protonated RVYIHPF, b5(2+) and b6(2+) product ions dominate the spectra of doubly protonated RVYIHAF. Also, dominant b5(2+) product ions are observed when the histidine side chain is alkylated (H) in doubly protonated RVYIHPF. Based on all of the results, a selective fragmentation mechanism for enhanced cleavage at histidine involving an atypical b ion structure is proposed.     

Localization of the O-glycosylated sites in peptides by fixed-charge derivatization with a phosphonium group

The present study demonstrates that matrix-assisted laser desorption ionization/postsource decay (MALDI/PSD) analysis of the molecular cation of glycopeptides derivatized at their amino terminus with a phosphonium group cleaves peptide backbone without removing the glycan. The predictable a-type fragment ions retain the glycan moiety, enabling unambiguous localization of O-glycans on the peptide chain. In contrast, collision-activated dissociation tandem mass spectrometry analysis carried out on the doubly charged protonated phosphonium cation results in the predominant loss of the sugar moiety from the peptide. This result supports the previously proposed charge-induced fragmentation mechanism of the sugar-peptide bond. MALDI/PSD analysis of glycopeptides converted to their acetyl phosphonium derivatives is an effective alternative to electron capture dissociation, as illustrated by the positioning of up to three GalNac residues along the full tandem repeat peptide sequence derived from the MUC 5AC mucin.     

Statistical characterization of ion trap tandem mass spectra from doubly charged tryptic peptides

Collision-induced dissociation (CID) is a common ion activation technique used to energize mass-selected peptide ions during tandem mass spectrometry. Characteristic fragment ions form from the cleavage of amide bonds within a peptide undergoing CID, allowing the inference of its amino acid sequence. The statistical characterization of these fragment ions is essential for improving peptide identification algorithms and for understanding the complex reactions taking place during CID. An examination of 1465 ion trap spectra from doubly charged tryptic peptides reveals several trends important to understanding this fragmentation process. While less abundant than y ions, b ions are present in sufficient numbers to aid sequencing algorithms. Fragment ions exhibit a characteristic series-specific relationship between their masses and intensities. Each residue influences fragmentation at adjacent amide bonds, with Pro quantifiably enhancing cleavage at its N-terminal amide bond and His increasing the formation of b ions at its C-terminal amide bond. Fragment ions corresponding to a formal loss of ammonia appear preferentially in peptides containing Gln and Asn. These trends are partially responsible for the complexity of peptide tandem mass spectra.     

Fragmentation of singly protonated peptides via a combination of infrared and collisional activation

The coupling of matrix-assisted laser desorption/ionization (MALDI) to Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) provides an exceptionally capable platform for peptide analysis, but an important limitation of this approach is the difficulty in obtaining informative tandem mass spectra (MS/MS) of singly protonated peptides. This difficulty is especially pronounced with peptide ions containing basic amino acid residues (for example, tryptic peptides). While such ions can be fragmented in some instrument configurations, most FTICR instruments have comparatively little facility for high-energy fragmentation. Here, a novel MS/MS approach implemented with MALDI-FTICR-MS and specifically intended for enhanced fragmentation of singly protonated peptides is described. The method involves infrared irradiation in concert with the simultaneous application of sustained off-resonance irradiation collision-induced dissociation (SORI-CID). This form of MS/MS, described as a combination of infrared and collisional activation (CIRCA), is shown to provide a greater capacity for dissociation of singly charged model peptide ions as compared to infrared multiphoton dissociation (IRMPD) or SORI-CID alone. Overall, the CIRCA approach is demonstrated to be a feasible technique for accessing useful fragmentation pathways of singly charged peptides, including those harboring basic amino acid residues--a crucial feature in the context of proteomics.     

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.     

Surface-induced dissociation of ions produced by matrix-assisted laser desorption/ionization in a fourier transform ion cyclotron resonance mass spectrometer

Intermediate pressure matrix-assisted laser desorption/ionization (MALDI) source was constructed and interfaced with a 6-T Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially configured for surface-induced dissociation (SID) studies. First MALDI-SID results in FT-ICR are presented, demonstrating unique advantages of SID over conventional FT-ICR MS ion activation techniques for structural characterization of singly protonated peptide ions. Specifically, we demonstrate that SID on a diamond surface results in a significantly better sequence coverage for singly protonated peptides than SORI-CID. A combination of two effects contributes to the improved sequence coverage: shattering of peptide ions on surfaces opens up a variety of dissociation channels at collision energies above 40 eV, and second, wide internal energy distribution deposited by collision with a stiff diamond surface provides an efficient mixing between the primary reaction channels that are dominant at low internal energies and extensive fragmentation at high internal excitation that results from shattering. Activation of MALDI-generated ions by collisions with surfaces in FT-ICR MS is a new powerful method for characterization and identification of biomolecules     

Ion trap versus low-energy beam-type collision-induced dissociation of protonated ubiquitin ions

The beam-type and ion trap collision-induced dissociation (CID) behaviors of protonated bovine ubiquitin ions were studied for charge states ranging from +6 to +12 on a modified triple quadrupole/linear ion trap tandem mass spectrometer. Both beam-type CID and ion trap CID were conducted in a high-pressure linear ion trap, followed by proton-transfer ion/ion reactions to reduce the charge states of product ions mostly to +1. The product ions observed under each activation condition were predominantly b- and y-type ions. Fragmentation patterns showed a much stronger dependence on parent ion charge state with ion trap CID than with beam-type CID using nitrogen as the collision gas, with preferential cleavages C-terminal to aspartic acid at relatively low charge states, nonspecific fragmentation at moderate charge states, and favored cleavages N-terminal to proline residues at high charge states. In the beam-type CID case, extensive cleavage along the protein backbone was noted, which yielded richer sequence information (77% of backbone amide bond cleavages) than did ion trap CID (52% of backbone amide bond cleavages). Collision gas identity and collision energy were also evaluated in terms of their effects on the beam-type CID spectrum. The use of helium as collision gas, as opposed to nitrogen, resulted in CID behavior that was sensitive to changes in collision energy. At low collision energies, the beam-type CID data resembled the ion trap CID data with preferential cleavages predominant, while at high collision energies, nonspecific fragmentation was observed with increased contributions from sequential fragmentation.     

Mass spectrometric differentiation of linear peptides composed of L-amino acids from isomers containing one D-amino acid residue

MS/MS of electrosprayed ions is shown to have the capacity to discriminate between peptides that differ by configuration about their alpha-carbons. It is not necessary for the peptides to possess tertiary structures that are affected by stereochemistry, since five epimers of the pentapeptide, H2N-Gly-Leu-Ser-Phe-Ala-OH (GLSFA) all display different collisionally activated dissociation (CAD) patterns of their protonated parent ions. The figure of merit, r, is a ratio of ratios of fragment ion abundances between stereoisomers, where r = 1 corresponds to no stereochemical effect. Values of r as high as 3.8 are seen for diastereomer pairs. Stereochemical effects are also seen for the diprotonated dodecapeptide H2N-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-OH (LVFFAEDVGSNK), a tryptic fragment from the amyloid beta-protein. Triply charged complexes of the protonated dodecapeptide with cobalt(II) ions undergo CAD at lower collision energies than do doubly protonated LVFFAEDVGSNK ions. Statistically significant (p < 0.01) differences between the all-L-dodecapeptide and the ones containing a d-serine or a D-aspartic acid are observed.     

Multipole-storage-assisted dissociation for the characterization of large proteins and simple protein mixtures by ESI-FTICR-MS

In Fourier transform ion cyclotron resonance mass spectrometry, collisionally activated dissociation (CAD) typically is accomplished within the analyzer ion cell. An alternative approach of multipole-storage-assisted dissociation (MSAD) has previously been demonstrated by inducing collisional fragmentation in the external multipole that is usually employed for ion accumulation. To explore the utility of MSAD for interrogating intact proteins and simple protein mixtures in a multiplexed manner, we have investigated the means of controlling the collisional energy and the fragmentation pattern for this experimental approach. With protein samples in the low micromolar concentration range, the two major experimental parameters affecting MSAD in the hexapole region were found to be the dc offset voltage and accumulation time. While low-energy MSAD of intact proteins yields fragment ions similar to sustained off resonance irradiation collision-activated dissociation (SORI-CAD), high-energy MSAD induces sequential fragmentation for intact proteins to yield a rich variety of singly charged ions in the m/z 600-1200 Da region. Each of the seven proteins (Mr range of 8.5-116 kDa) examined in this study exhibited their own characteristic MSAD fragmentation pattern, which could be used as a signature of the presence of a given protein, even in a mixture. In addition, any MSAD fragment can be isolated and dissociated further by SORI-CAD in an MS3-type experiment inside the FTICR analyzer cell. This presents a novel way to interrogate the identities of these fragment ions as well as obtain amino acid sequence tag information that can be used to identify proteins from mixtures.