Surface-induced dissociation of peptides and protein complexes in a quadrupole/time-of-flight mass spectrometer

A novel in-line surface-induced dissociation (SID) device was designed and implemented in a commercial QTOF instrument (Waters/Micromass QTOF II). This new setup allows efficient SID for a broad range of molecules. It also allows direct comparison with conventional collision-induced dissociation (CID) on the same instrument, taking advantage of the characteristics of QTOF instrumentation, including extended mass range, improved sensitivity, and better resolution compared with quadrupole analyzers and ion traps. Various peptides and a noncovalent protein complex have been electrosprayed and analyzed with the new SID setup. Here we present SID of leucine enkephalin, fibrinopeptide A, melittin, insulin chain-B, and a noncovalent protein complex from wheat, heat shock protein 16.9. The SID spectra were also compared to CID spectra. With the SID setup installed, ion transmission proved to be efficient. SID fragmentation patterns of peptides are, in general, similar to CID, with differences in the relative intensities of some peaks such as immonium ions, backbone cleavage b- versus y-type ions, and y- versus y-NH3 ions, suggesting enhanced accessibility to high-energy/secondary fragmentation channels with SID. Furthermore, these results demonstrate that the in-line SID setup is a valid substitute for CID, with potential advantages for activation of singly/multiply charged peptides and larger species such as noncovalent protein complexes.     

Ion mobility-mass spectrometry reveals the influence of subunit packing and charge on the dissociation of multiprotein complexes

The composition, stoichiometry, and organization of protein complexes can be determined by collision-induced dissociation (CID) coupled to tandem mass spectrometry (MS/MS). The increased use of this approach in structural biology prompts a better understanding of the dissociation mechanism(s). Here we report a detailed investigation of the CID of two dodecameric, heat-stable and toroidally shaped complexes: heat shock protein 16.9 (HSP16.9) and stable protein 1 (SP-1). While HSP16.9 dissociates by sequential loss of unfolded monomers, SP-1 ejects not only monomers, but also its building blocks (dimers), and multiples thereof (tetramers and hexamers). Unexpectedly, the dissociation of SP-1 is strongly charge-dependent: loss of the building blocks increases with higher charge states of this complex. By combining MS/MS with ion mobility (IM-MS/MS), we have monitored the unfolding and dissociation events for these complexes in the gas phase. For HSP16.9 unfolding occurs at lower energies than the ejection of subunits, whereas for SP-1 unfolding and dissociation take place simultaneously. We consider these results in the light of the structural organization of HSP16.9 and SP-1 and hypothesize that SP-1 is unable to unfold extensively due to its particular quaternary structure and unusually high charge density. This investigation increases our understanding of the factors governing the CID of protein complexes and moves us closer to the goal of obtaining structural information on subunit interactions and packing from gas-phase experiments.     

Ion mobility mass spectrometry coupled with rapid protein threading predictor structure prediction and collision-induced dissociation for probing chemokine conformation and stability

Unique to ion mobility mass spectrometry (IM-MS) is the ability to provide collision cross section (CCS) data and the capacity to delineate any dissociation and/or unfolding of protein complexes. The strong correlation of the experimentally determined CCS with theory is indicative of the retention of native structure in the gas phase, which in turn, qualifies as a means in evaluating the IM-MS data. The assessment of IM-MS data, however, is currently impeded due to the lack of appropriate structural coordinates to use as input in the in silico calculation of theory. To address this issue, this study involves the use of rapid protein threading predictor (RAPTOR) to generate tertiary structures of closely related monomeric chemokines (MCP-1, MCP-3, MCP-4, and eotaxin) and, subsequently, utilize these models to estimate the theoretical values. Experimental CCS of both the model proteins and chemokines correlate well with theory generated by RAPTOR. All conformations for z = 5+ of chemokines fall within theoretical limits. Of the four chemokines, MCP-4 with z = 6+ appears to adopt an extended conformation, while eotaxin gradually unfolds, and the extended structures of MCP-1 and MCP-3 increase in abundance upon activation. Combining RAPTOR with IM-MS and collision-induced dissociation (CID) enables us to interrogate the conformations of homologous proteins with very similar tertiary structures.     

Gas-phase separations of protein and peptide ion fragments generated by collision-induced dissociation in an ion trap

Ion mobility/time-of-flight mass spectrometry techniques have been used to examine distributions of fragment ions generated by collision-induced dissociation (CID) in a quadrupole ion trap. The mobility-based separation step prior to mass-to-charge (m/z) analysis reduces spectral congestion and provides information that complements m/z-based assignments of peaks. The approach is demonstrated by examining fragmentation patterns of insulin chain B (a 30-residue peptide), and ubiquitin (a protein containing 76 amino acids). Some fragments of ubiquitin show evidence for multiple stable conformations.     

Tandem fourier transform mass spectrometry studies of surface-induced dissociation of benzene monomer and dimer ions on a self-assembled fluorinated alkanethiolate monolayer surface

A new instrument configuration based on a Finnigan FTMS-2000 platform has been applied to the study of surface-induced dissociation (SID) in this research. Benzene monomer ions C(6)H(6)(+) and dimer ions (C(6)H(6))(2)(+) were impacted on a fluorinated self-assembled monolayer surface at collision energies ranging from 1 to 70 eV. Benzene cations were chosen for this study because the fragmentation characteristics of the molecular cation are well known and its SID has been thoroughly investigated. SID spectra obtained by FTMS-SID are very similar to those reported in the literature for the same surface but exhibit much higher mass resolution. A comparison study of collision-induced dissociation (CID) and SID of benzene molecular cations was performed utilizing the same ICR cell and ion detection protocol. It is demonstrated that SID provides both much higher energy deposition and a narrower internal energy distribution than CID. The present instrument geometry and experimental protocol demonstrate much higher efficiencies than previous SID studies by FTMS and much higher mass resolution than previous SID studies using other types of mass analyzers.     

Ion mobility-mass spectrometry (IM-MS) for top-down proteomics: increased dynamic range affords increased sequence coverage

A general approach that combines mass spectrometry (MS), collision-induced dissociation (CID), ion mobility (IM), and MS for top-down proteomics is described, denoted as MS-CID-IM-MS. Using this approach, CID product ions are dispersed in two dimensions, specifically size-to-charge (IM) and mass-to-charge (MS), and the resulting 2D data display greatly facilitates peptide/protein mass mapping, amino acid sequence analysis, and determination of site-specific protein modifications. Also, this approach alleviates some of the inherent limitations of top-down proteomics, viz. the limitations in dynamic range for fragment ion abundances owing to the number of fragmentation channels available to large ionic systems as well as the resulting spectral congestion. For large peptides such as melittin (2845 Da), CID of the [M + 3H](3+), [M + 4H](4+), and [M + 5H](5+) ions yields amino acid sequence coverage of 42.3%, 38.5%, and 7.7%, respectively, whereas the hybrid MS-CID-IM-MS approach yields amino acid sequence coverages of 84.6%, 65.4%, and 69.2%, respectively. For large biomolecules such as ubiquitin (8565 Da), the amino acid sequence coverage increases from 39% to 76%. The MS-CID-IM-MS top-down approach allows for greater depth of information by allowing the assignment and study of internal fragment ions. Lastly, analysis of the methyl esterification of ubiquitin and single point mutation of human iron sulfur cluster U (HISCU, 14.3 kDa) demonstrates the ability of MS-CID-IM-MS to rapidly identify the presence and sites of modifications.     

Surface-induced dissociation in a Fourier transform ion cyclotron resonance mass spectrometer: instrument design and evaluation

A new Fourier transform ion cyclotron resonance mass spectrometer (FTICR MS) has been constructed in our laboratory. The instrument employs surface-induced dissociation (SID) as an activation method for obtaining structural information on biomolecules in the gas phase. Tandem SID mass spectra can be acquired using either a continuous or a pulsed mode of operation. Collision energy of precursor ion is controlled by a dc offset of the ICR cell. This approach eliminates defocusing of the ion beam by the ion-transfer optics as a function of ion kinetic energy and constitutes a significant improvement over our previous experimental setup. Furthermore, it can be easily implemented on any FTICR mass spectrometer. Very high signal-to-noise ratios of 200-500 were obtained in single-scan SID mass spectra of model peptides with acquisition time less than 1.1 s. Reasonable SID signal was detected in single-scan spectra with total acquisition time of only 0.3 s. The high signal-to-noise ratio and the fast acquisition time point on a potential application of SID for high-throughput studies in FTICR MS.     

Prediction of collision-induced dissociation spectra of common N-glycopeptides for glycoform identification

Confident identification of the glycan moieties in glycopeptides by collision-induced dissociation (CID) requires accurate prediction of the CID spectrum of the glycopeptides. In this Article, the kinetic model for the prediction of peptide CID spectra is extended to predict the CID spectra of N-glycopeptides. The model was trained with 1831 ion-trap CID spectra of N-glycopeptides and is able to predict ion-trap CID spectra with excellent accuracy in ion intensities for N-glycopeptides up to 8000 u in mass. A total of 524 common glycoforms including complex N-glycans with 2-4 antennas, plus high-mannose type and hybrid type, can be predicted.     

Enhanced analyte detection using in-source fragmentation of field asymmetric waveform ion mobility spectrometry-selected ions in combination with time-of-flight mass spectrometry

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry (FAIMS) is used for the selective transmission of differential mobility-selected ions prior to in-source collision-induced dissociation (CID) and time-of-flight mass spectrometry (TOFMS) analysis. The FAIMS-in-source collision induced dissociation-TOFMS (FISCID-MS) method requires only minor modification of the ion source region of the mass spectrometer and is shown to significantly enhance analyte detection in complex mixtures. Improved mass measurement accuracy and simplified product ion mass spectra were observed following FAIMS preselection and subsequent in-source CID of ions derived from pharmaceutical excipients, sufficiently close in m/z (17.7 ppm mass difference) that they could not be resolved by TOFMS alone. The FISCID-MS approach is also demonstrated for the qualitative and quantitative analysis of mixtures of peptides with FAIMS used to filter out unrelated precursor ions thereby simplifying the resulting product ion mass spectra. Liquid chromatography combined with FISCID-MS was applied to the analysis of coeluting model peptides and tryptic peptides derived from human plasma proteins, allowing precursor ion selection and CID to yield product ion data suitable for peptide identification via database searching. The potential of FISCID-MS for the quantitative determination of a model peptide spiked into human plasma in the range of 0.45-9.0 μg/mL is demonstrated, showing good reproducibility (%RSD < 14.6%) and linearity (R(2) > 0.99).     

Prediction of collision-induced-dissociation spectra of peptides with post-translational or process-induced modifications

Mass spectrometry, combined with collision-induced dissociation (CID), has become the method of choice for analyzing protein post-translational and process-induced modifications. However, confident and automated identification of modifications and modification sites is often challenged by the diversity of modifications and their labile nature under typical CID conditions. An accurate prediction of the CID spectra of modified peptides will improve the reliability of automated determination of modifications and modification sites. In this article, the kinetic model for the prediction of peptide CID spectra is extended to the prediction of the CID spectra of modified peptides. The mathematical model for predicting CID spectra of peptides with enzymatic and chemical modifications such as (1) phosphorylation of serine, threonine, and tyrosine, (2) S-carboxymethylation and carbamidomethylation of cysteine, (3) different stages of oxidation of methionine, tryptophan, and cysteine, (4) glycation of lysine, (5) O-mannosylation of serine, (6) hydroxylation of lysine, and (7) N-monomethylation and N-dimethylation of lysine is described. The mathematical model, once established with CID spectra of peptides with known modifications and modification sites, is able to predict CID spectra with excellent accuracy in ion intensities, facilitating more reliable identification of modification and modification sites.     

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.     

Comparative study of photodissociation and surface-induced dissociation by laser desorption Fourier transform mass spectrometry.

Photodissociation (PD) and surface-induced dissociation (SID) are compared for structural analysis of several nonvolatile compounds analyzed by laser desorption Fourier transform mass spectrometry (LD/FTMS). SID and PD of a porphyrin and two metalloporphyrins were investigated using a variety of experimental conditions. Optimum structural information is obtained from PD when parent ions are irradiated for relatively long times (10-30 s) using 575-nm radiation and short times (0.5-1 s) using 308- or 388-nm radiation. Shorter irradiation times in the visible region resulted in less efficient production of structurally significant product ions, while longer times in the ultraviolet region produced more nonspecific fragment ions, apparently at the expense of more structurally significant fragment ions. SID conversion efficiencies for the porphyrins are estimated for collision energies from 25 to 360 eV, with maximum conversion efficiency found using 62- and 115-eV collision energies for the two porphyrins studied. Results from a concurrent study on the combined use of PD and SID for MS/MS/MS are discussed in the context of these results. The MS3 ion spectra generated by the two dissociation techniques differ more significantly than MS2 product ion spectra. These data suggest some general guidelines for MSn studies of nonvolatile compounds analyzed by LD/FTMS, employing PD and SID for ion activation.     

Determinants of gas-phase disassembly behavior in homodimeric protein complexes with related yet divergent structures

The overall structure of a protein-protein complex reflects an intricate arrangement of noncovalent interactions. Whereas intramolecular interactions confer secondary and tertiary structure to individual subunits, intermolecular interactions lead to quaternary structure--the ordered aggregation of separate polypeptide chains into multisubunit assemblies. The specific ensemble of noncovalent contacts dictates the stability of subunit folds, enforces protein-protein binding specificity, and determines multimer stability. Consequently, noncovalent architecture is likely to play a role in the gas-phase dissociation of these assemblies during tandem mass spectrometry (MS/MS). To further advance the applicability of MS/MS to analytical problems in structural biology, a better understanding of the interplay between the structures and fragmentation behaviors of noncovalent protein complexes is essential. The present work constitutes a systematic study of model protein homodimers (bacteriophage N15 Cro, bacteriophage λ Cro, and bacteriophage P22 Arc) with related but divergent structures, both in terms of subunit folds and protein-protein interfaces. Because each of these dimers has a well-characterized structure (solution and/or crystal structure), specific noncovalent features could be correlated with gas-phase disassembly patterns as studied by collision-induced dissociation, surface-induced dissociation, and ion mobility. Of the several respects in which the dimers differed in structure, the presence or absence of intermolecular electrostatic contacts exerted the most significant influence on the gas-phase dissociation behavior. This is attributed to the well-known enhancement of ionic interactions in the absence of bulk solvent. Because salt bridges are general contributors to both intermolecular and intramolecular stability in protein complexes, these observations are broadly applicable to aid in the interpretation or prediction of dissociation spectra for noncovalent protein assemblies.     

Thermally assisted collision-induced dissociation in a quadrupole ion trap mass spectrometer

Thermally assisted collision-induced dissociation (TA-CID) provides increased dissociation in comparison with CID performed at ambient temperature in a quadrupole ion trap mass spectrometer. Heating the bath/collision gas during CID increases the initial internal energy of the ions and reduces the collisional cooling rate. Thus, using the same CID parameters, the parent ion can be activated to higher levels of internal energy, increasing the efficiency of dissociation and the number of dissociation pathways. The increase in the number of dissociation pathways can provide additional structural information. A consequence of the increase in initial internal energy is the ability to use less power to effect collisional activation. This allows lower q(z) values to be used and, thus, a greater mass range of product ions to be observed. TA-CID alleviates the problems associated with traditional CID and results in more available information than traditional CID.     

Native electrospray and electron-capture dissociation FTICR mass spectrometry for top-down studies of protein assemblies

The high sensitivity, extended mass range, and fast data acquisition/processing of mass spectrometry and its coupling with native electrospray ionization (ESI) make the combination complementary to other biophysical methods of protein analysis. Protein assemblies with molecular masses up to MDa are now accessible by this approach. Most current approaches have used quadrupole/time-of-flight tandem mass spectrometry, sometimes coupled with ion mobility, to reveal stoichiometry, shape, and dissociation of protein assemblies. The amino-acid sequence of the subunits, however, still relies heavily on independent bottom-up proteomics. We describe here an approach to study protein assemblies that integrates electron-capture dissociation (ECD), native ESI, and FTICR mass spectrometry (12 T). Flexible regions of assembly subunits of yeast alcohol dehydrogenase (147 kDa), concanavalin A (103 kDa), and photosynthetic Fenna-Matthews-Olson antenna protein complex (140 kDa) can be sequenced by ECD or "activated-ion" ECD. Furthermore, noncovalent metal-binding sites can also be determined for the concanavalin A assembly. Most importantly, the regions that undergo fragmentation, either from one of the termini by ECD or from the middle of a protein, as initiated by CID, correlate well with the B-factor from X-ray crystallography of that protein. This factor is a measure of the extent an atom can move from its coordinated position as a function of temperature or crystal imperfections. The approach provides not only top-down proteomics information of the complex subunits but also structural insights complementary to those obtained by ion mobility.     

Application of atmospheric pressure ionization time-of-flight mass spectrometry coupled with liquid chromatography for the characterization of in vitro drug metabolites

Atmospheric pressure ionization time-of-flight mass spectrometry coupled with high-performance liquid chromatography was used to characterize the in vitro metabolites of glyburide. Metabolic products formed in vitro by human microsomes were separated using a C18 column with gradient elution at a flow rate of 200 microL/min without postcolumn splitting. In-source collision-induced dissociation (CID) by automated nozzle potential switching was employed to obtain both abundant protonated molecules and characteristic fragments whose accurate masses were measured simultaneously by internal mass calibration, performed by continuous postcolumn infusion of two reference standards. The mass errors were within 9 ppm for all ions measured, whose abundance was greater than 5%, relative to the most abundant isotopic "A" ion. Exact mass differences between the parent drug and metabolite(s) were determined and these values corresponded to a unique elemental composition. The elemental compositions of all metabolite fragment ions were generated based upon the known compositional elements of the protonated molecule. The structures of metabolites and their fragment ions were proposed based on the determined elemental composition and in-source CID spectra. The elemental composition and fragmentation pathways of four cyclohexyl hydroxylation metabolites and one ethylhydroxy metabolite are discussed.     

Submicrosecond surface-induced dissociation of peptide ions in a MALDI TOF MS

Surface-induced dissociation (SID) has been implemented in a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI TOF MS), allowing production of tandem mass spectrometric information for peptide ions (MALDI TOF SID TOF). The instrument retains the standard operational modes such as the reflectron monitoring of the MALDI-generated intact ions and postsource decay. We show through ion trajectory simulations and experimental results that implementing SID in a commercial MALDI TOF spectrometer is feasible and that the SID products in this instrument fall in an observation time frame that allows the specific detection of fast-fragmentation channels. The instrument design, pulse timing sequence, and high-voltage electronics together with SID spectra of MALDI-generated peptide ions are presented. Standard peptides such as YGGFLR, angiotensin III, fibrinopeptide A, and des-Arg1-bradykinin were dissociated by means of hyperthermal collisions with a gold surface coated with a self-assembled monolayer of 2-(perfluorodecyl)ethanethiol. With the extraction fields and the short observation times used, the spectra obtained show intense low-mass ion signals such as immonium, b2, b3, and y2 ions. TOF data analysis involved matching simulated and experimental flight times and indicates that the observed fragments are produced at approximately 250 ns after the precursor ion collides with the surface. This submicrosecond gas-phase fragmentation time frame is complementary to the observation time frame of existing SID spectrometers, which are on the order of 10 micros for tandem quadrupoles and are larger than a few milliseconds for SID implemented in Fourier transform ion cyclotron resonance spectrometers.     

Relative information content and top-down proteomics by mass spectrometry: utility of ion/ion proton-transfer reactions in electrospray-based approaches

Computer simulations of electrospray ionization (ESI) and collision-induced dissociation (CID) experiments were employed to examine the informing power associated with "top-down" proteomics implemented with some commonly used mass analyzers, i.e., the quadrupole ion trap (QIT), the Fourier transform-ion cyclotron resonance mass spectrometer (FT-ICRMS), and the time-of-flight (TOF) mass spectrometer. Using a ratio of the separated (or resolved) peaks to the total number of predicted peaks as a measure of informing power, the ESI-MS simulation of a mixture of proteins showed that the FT-ICRMS exhibited the highest informing power among the three instruments being studied, with the QIT giving the lowest informing power, which was expected from the analysis of the "component capacity" of the three approaches. Also as expected on the basis of resolving elements per component, a dramatic increase in the informing power of the approach was obtained when ion/ion proton-transfer reactions were used to reduce the number of peaks and to minimize overlap between ions of different mass and charge but similar mass-to-charge ratio. With the assumptions made in this study, the informing power of the TOF + ion/ion approach rivaled or even exceeded that of the FT-ICRMS approach, despite significantly lower mass resolution. This result stemmed from both a reduction in the number of peaks and their dispersion over a much wider range of mass-to-charge ratios. Similar results were obtained from the CID simulation, where the informing power of different approaches was evaluated on the basis of the ratio of the number of ions for which a mass could be determined unambiguously to the total number of ions in the spectra.     

Preparation, separation, and conformational analysis of differentially sulfated heparin octasaccharide isomers using ion mobility mass spectrometry

Heparin is a linear sulfated polysaccharide widely used in medicine because of its anticoagulant properties. The various sulfation and/or acetylation patterns on heparin impart different degrees of conformational change around the glycosidic bonds and subsequently alter its function as an anticoagulant, anticancer, or antiviral drug. Characterization of these structures is important for eventual elucidation of its function but presents itself as an analytical challenge due to the inherent heterogeneity of the carbohydrates. Heparin octasaccharide structural isomers of various sulfation patterns were investigated using ion mobility mass spectrometry (IMMS). In addition to distinguishing the isomers, we report the preparation and tandem mass spectrometry analysis for multiple sulfated or acetylated oligosaccharides. Herein, our data indicate that heparin octasaccharide isomers were separated on the basis of their structural conformations in the ion mobility cell. Subsequent to this separation, isomers were further distinguished using product ions resulting from tandem mass spectrometry. Overall, IMMS analysis was used to successfully characterize and separate individual isomers and subsequently measure their conformations.     

Separation and characterization of metallosupramolecular libraries by ion mobility mass spectrometry

The self-assembly of Zn(II) ions and bis(terpyridine) (tpy) ligands carrying 120° or 180° angles between their metal binding sites was utilized to prepare metallosupramolecular libraries with the connectivity. These combinatorial libraries were separated and characterized by ion mobility mass spectrometry (IM MS) and tandem mass spectrometry (MS(2)). The 180°-angle building blocks generate exclusively linear complexes, which were used as standards to determine the architectures of the assemblies resulting from the 120°-angle ligands. The latter ligand geometry promotes the formation of macrocyclic hexamers, but other n-mers with smaller (n = 5) or larger ring sizes (n = 7-9) were identified as minor products, indicating that the angles in the bis(terpyridine) ligand and within the coordinative tpy-Zn(II)-tpy bonds are not as rigid, as previously believed. Macrocyclic and linear isomers were detected in penta- and heptameric assemblies; in the larger octa- and nonameric assemblies, ring-opened conformers with compact and folded geometries were observed in addition to linear extended and cyclic architectures. IM MS(2) experiments provided strong evidence that the macrocycles present in the libraries were already formed in solution, during the self-assembly process, not by dissociation of larger complexes in the gas phase. The IM MS/MS(2) methods provide a means to analyze, based on size and shape (architecture), supramolecular libraries that are not amenable to liquid chromatography, LC-MS, NMR, and/or X-ray techniques.