Higher-energy collision-activated dissociation without a dedicated collision cell

Beam-type collisional activation dissociation (HCD) offers many advantages over resonant excitation collision-activated dissociation, including improved identification of phosphorylated peptides and compatibility with isobaric tag-based quantitation (e.g. tandem mass tag (TMT) and iTRAQ). However, HCD typically requires specially designed and dedicated collision cells. Here we demonstrate that HCD can be performed in the ion injection pathway of a mass spectrometer with a standard atmospheric inlet (iHCD). Testing this method on complex peptide mixtures revealed similar identification rates to collision-activated dissociation (2883 versus 2730 IDs for iHCD/CAD, respectively) and precursor-product-conversion efficiency comparable to that achieved within a dedicated collision cell. Compared with pulsed-q dissociation, a quadrupole ion trap-based method that retains low-mass isobaric tag reporter ions, iHCD yielded isobaric tag for relative and absolute quantification reporter ions 10-fold more intense. This method involves no additional hardware and can theoretically be implemented on any mass spectrometer with an atmospheric inlet.     

Effective novel dissociation methods for intact protein: heat-assisted nozzle-skimmer collisionally induced dissociation and infrared multiphoton dissociation using a Fourier transform ion cyclotron resonance mass spectrometer equipped with a micrometal electrospray ionization emitter

Heating of a nano-electrospray ionization (nanoESI) source can improve the dissociation efficiency of collisionally induced dissociation (CID) methods, such as nozzle-skimmer CID (NS-CID) and infrared multiphoton dissociation (IRMPD), for large biomolecule fragmentation. A metal nanoESI emitter was used due to its resistance to heating above 250 degrees C. This novel method for the dissociation of large biomolecular ions is termed "heat-assisted NS-CID" (HANS-CID) or "heat-assisted IRMPD" (HA-IRMPD). Multiple charged nonreduced protein ions (8.6 Da ubiquitin, 14 kDa lysozyme, and 67 kDa bovine serum albumin) were directly dissociated by HANS-CID and HA-IRMPD to effectively yield fragment ions that could be assigned. The fragment ions of ubiquitin by HANS-CID can be analyzed by tandem mass spectrometry (MS/MS) using sustained off-resonance irradiation CID (SORI-CID) and IRMPD. In addition, a native large protein, immunoglobulin G (IgG, 150 kDa), was efficiently dissociated by HA-IRMPD. The product ions that were obtained reflected the domain structure of IgG. However, these product ions of IgG and lysozyme were not dissociated by MS/MS using the same heating energetic methods such as IRMPD and SORI-CID.     

Application of different fragmentation techniques for the analysis of phosphopeptides using a hybrid linear ion trap-FTICR mass spectrometer

Electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) present complementary techniques for the fragmentation of peptides and proteins in Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) in addition to the commonly used collisionally activated dissociation (CAD). Both IRMPD and ECD have been shown to be applicable for an efficient sequencing of peptides and proteins, whereas ECD has proven especially valuable for mapping labile posttranslational modifications (PTMs), such as phosphorylations. In this work, we compare the different fragmentation techniques and MS detection in a linear ion trap and the ICR cell with respect to their abilities to efficiently identify and characterize phosphorylated peptides. For optimizing fragmentation parameters, sets of synthetic peptides with molecular weights ranging from approximately 1 to 4 kDa and different levels of phosphorylation were analyzed. The influence of spectrum averaging for obtaining high-quality spectra was investigated. Our results show that the fragmentation methods CAD and ECD allow for a facilitated analysis of phosphopeptides; however, their general applicability for analyzing phosphopeptides has to be evaluated in each specific case with respect to the given analytical task. The major advantage of complementary peptide cleavages by combining different fragmentation methods is the increased amount of information that is obtained during MS/MS analysis of modified peptides. On the basis of the obtained results, we are planning to design LC time-scale compatible, data-dependent MS/MS methods using the different fragmentation techniques in order to improve the identification and characterization of phosphopeptides.     

The fate of b-ions in the two worlds of collision-induced dissociation

Fragment analysis of proteins and peptides by mass spectrometry using collision-induced dissociation (CID) revealed that the pairwise generated N-terminal b- and C-terminal y-ions have different stabilities resulting in underrepresentation of b-ions. Detailed analyses of large-scale spectra databases and synthetic peptides underlined these observations and additionally showed that the fragmentation pattern depends on utilized CID regime. To investigate this underrepresentation further we systematically compared resonant excitation energy and beam-type CID facilitated on different mass spectrometer platforms: (i) quadrupole time-of-flight, (ii) linear ion trap and (iii) three-dimensional ion trap. Detailed analysis of MS/MS data from a standard tryptic protein digest revealed that b-ions are significantly underrepresented on all investigated mass spectrometers. By N-terminal acetylation of tryptic peptides we show for the first time that b-ion cyclization reaction significantly contributes to b-ion underrepresentation even on ion trap instruments and accounts for at most 16% of b-ion loss.     

Performance characteristics of electron transfer dissociation mass spectrometry

We performed a large scale study of electron transfer dissociation (ETD) performance, as compared with ion trap collision-activated dissociation (CAD), for peptides ranging from approximately 1000 to 5000 Da (n approximately 4000). These data indicate relatively little overlap in peptide identifications between the two methods ( approximately 12%). ETD outperformed CAD for all charge states greater than 2; however, regardless of precursor charge a linear decrease in percent fragmentation, as a function of increasing precursor m/z, was observed with ETD fragmentation. We postulate that several precursor cation attributes, including peptide length, charge distribution, and total mass, could be relevant players. To examine these parameters unique ETD-identified peptides were sorted by length, and the ratio of amino acid residues per precursor charge (residues/charge) was calculated. We observed excellent correlation between the ratio of residues/charge and percent fragmentation. For peptides of a given residue/charge ratio, there is no correlation between peptide mass and percent fragmentation; instead we conclude that the ratio of residues/charge is the main factor in determining a successful ETD outcome. As charge density decreases so does the probability of non-covalent interactions that can bind a newly formed c/z-type ion pair. Recently we have described a supplemental activation approach (ETcaD) to convert these non-dissociative electron transfer product ions to useful c- and z-type ions. Automated implementation of such methods should remove this apparent precursor m/z ceiling. Finally, we evaluated the role of ion density (both anionic and cationic) and reaction duration for an ETD experiment. These data indicate that the best performance is achieved when the ion trap is filled to its space charge limit with anionic reagents. In this largest scale study of ETD to date, ETD continues to show great promise to propel the field of proteomics and, for small- to medium-sized peptides, is highly complementary to ion trap CAD.     

Ion trap collisional activation of c and z* ions formed via gas-phase ion/ion electron-transfer dissociation

A series of c- and z*-type product ions formed via gas-phase electron-transfer ion/ion reactions between protonated polypeptides with azobenzene radical anions are subjected to ion trap collision activation in a linear ion trap. Fragment ions including a-, b-, y-type and ammonia-loss ions are typically observed in collision induced dissociation (CID) of c ions, showing almost identical CID patterns as those of the C-terminal amidated peptides consisting of the same sequences. Collisional activation of z* species mainly gives rise to side-chain losses and peptide backbone cleavages resulting in a-, b-, c-, x-, y-, and z-type ions. Most of the fragmentation pathways of z* species upon ion trap CID can be accounted for by radical driven processes. The side-chain losses from z* species are different from the small losses observed from the charge-reduced peptide molecular species in electron-transfer dissociation (ETD), which indicates rearrangement of the radical species. Characteristic side-chain losses are observed for several amino acid residues, which are useful to predict their presence in peptide/protein ions. Furthermore, the unique side-chain losses from leucine and isoleucine residues allow facile distinction of these two isomeric residues.     

Mass Spectrometry Study and Infrared Spectroscopy of the Complex Between Camphor and the Two Enantiomers of Protonated Alanine: The Role of Higher‐Energy Conformers in the Enantioselectivity of the Dissociation Rate Constants

The properties of the protonated complexes built from S camphor and R or S alanine were studied in a Paul ion trap at room temperature by collision‐induced dissociation (CID) and infrared multiple‐photon dissociation spectroscopy (IRMPD), as well as molecular dynamics and ab initio calculations. While the two diastereomer complexes display very similar vibrational spectra in the fingerprint region, in line with similar structures, and almost identical calculated binding energies, their collision‐induced dissociation rates are different. Comparison of the IRMPD results to computed spectra shows that the SS and SR complexes both contain protonated alanine strongly hydrogen‐bonded to the keto group of camphor. The floppiness of this structure around the NH⁺…O = C hydrogen bond results in a complex potential energy surface showing multiple minima. Calculating the dissociation rate constant within the frame of the transition state theory shows that the fragmentation rate larger for the heterochiral SR complex than the homochiral SS complex can be explained in terms of two almost isoenergetic low‐energy conformers in the latter that are not present for the former. Chirality 25:436‐443, 2013. © 2013 Wiley Periodicals, Inc.     

Protein kinase A phosphorylation characterized by tandem Fourier transform ion cyclotron resonance mass spectrometry

A microelectrospray ionization tandem Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS(n)) approach for structural characterization of protein phosphorylation is described. Identification of proteolytic peptides is based solely upon mass measurement by high field (9.4 Tesla) FT-ICR MS. The location of the modification within any phosphopeptide is then established by FT-ICR MS(2) and MS(3) experiments. Structural information is maximized by use of electron capture dissociation (ECD) and/or infrared multiphoton dissociation (IRMPD). The analytical utility of the method is demonstrated by characterization of protein kinase A (PKA) phosphorylation. In a single FT-ICR MS experiment, 30 PKA tryptic peptides (including three phosphopeptides) were mass measured by internal calibration to within an absolute mean error of |0.7 ppm|. The location of each of the three sites of phosphorylation was then determined by MS(2) and MS(3) experiments, in which ECD and IRMPD provide complementary peptide sequence information. In two out of three cases, electron irradiation of a phosphopeptide [M + nH](n+) ion produced an abundant charge-reduced [M + nH]((n-1)+*) ion, but few sequence-specific c and z(*) fragment ions. Subsequent IRMPD (MS(3)) of the charge-reduced radical ion resulted in the detection of a large number of ECD-type ion products (c and z ions), but no b or y type ions. The utility of activated ion ECD for the characterization of tryptic phosphopeptides was then demonstrated.     

Analysis of proteins and peptides on a chromatographic timescale by electron-transfer dissociation MS

Peptide and protein sequence analysis using a combination of gas-phase ion-ion chemistry and tandem MS is described. Samples are converted to multiply charged ions by ESI and then allowed to react with fluoranthene radical anions in a quadrupole linear ion trap mass spectrometer. Electron transfer from the radical anion to the multiply charged peptide or protein promotes random fragmentation along the amide backbone that is independent of peptide or protein size, sequence, or the presence of post-translational modifications. Examples are provided that demonstrate the utility of electron-transfer dissociation for characterizing post-translational modifications and for identifying proteins in mixtures on a chromatographic timescale (500 ms/protein).     

Quantitative liquid chromatographic–tandem mass spectrometric determination of orlistat in plasma with a quadrupole ion trap

This report evaluates the use of a quadrupolar ion trap for quantitation in a bioanalytical laboratory. The evaluation was accomplished with the cross-validation of an LC–MS–MS quantitative method previously validated on a triple quadrupole mass spectrometer. The method was a multi-level determination of the anti-obesity drug, orlistat, in human plasma. The method has been refined previously on a triple quadrupole instrument to provide rapid sample throughput with robust reproducibility at sub-nanogram detection limits. Optimization of the method on the ion trap required improved chromatographic separation of orlistat from interfering plasma matrix components coextracted during the initial liquid–liquid extraction of plasma samples. The ion trap produces full-scan collision-induced dissociation mass spectra containing characteristic orlistat fragment ions that are useful for quantitation. Data collection on the ion trap required a precursor ion isolation width of 3.0 Da and optimal quantitative results were obtained when three fragment ions were monitored with a 1.8 Da window for each ion. Although a direct cross-validation between the ion trap and the tandem triple quadrupole mass spectrometer was not possible, quantitative results for orlistat comparable to those obtained from the triple quadrupole instrument were achieved by the ion trap with the modified method. The limit of quantitation for orlistat in plasma on the ion trap was 0.3 ng ml⁻¹ with a linear dynamic range of 0.3 to 10 ng ml⁻¹. Precision and accuracy varied from 4 to 15% over the quantitation range. The overall results provide an example of the utility of an ion trap in bioanalytical work.     

Top down mass spectrometry of < 60-kDa proteins from Methanosarcina acetivorans using quadrupole FRMS with automated octopole collisionally activated dissociation

A fragmentation geometry based upon axial acceleration of m/z-selected protein ions into a linear octopole ion trap allowed simultaneous production and external accumulation of fragment ions prior to m/z measurement in a FT mass spectrometer. Improved dynamic range resulting from this octopole collisionally activated dissociation resulted in a 2.5x increase in experimental throughput and a 2x increase in fragment ion matches to gene products identified and characterized in the top down fashion. The acceleration voltage for optimal fragmentation has a m/z and mass dependence, knowledge of which facilitated an automated platform for top down MS/MS on a quadrupole FT hybrid mass spectrometer. Controlled by improved software for data acquisition (e.g. using dynamic exclusion of previously identified species), automated octopole collisionally activated dissociation of samples fractionated using chromatofocusing and reversed-phase liquid chromatography achieved a significant increase in protein identification rate versus previous benchmarks. Also a batch analysis version of ProSight PTM facilitated probability-based identification of intact proteins obtained in a higher throughput fashion. In total, 101 unique proteins (5-59 kDa) were identified from whole cell lysates of Methanosarcina acetivorans grown anaerobically, including the characterization of several mispredicted start sites and biologically relevant mass discrepancies.     

Infrared multiphoton dissociation and electron capture dissociation of high-mannose type glycopeptides

The combination of electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) for the structural characterization of high-mannose type glycopeptides is explored in depth for the first time. Contrary to previous applications to other glycan types, our analyses reveal that IRMPD does not necessarily selectively induce glycan cleavage in high-mannose type glycopeptides; rather peptide backbone cleavage can effectively compete with glycosidic cleavage. Poor glycan cleavage with IRMPD is due to a higher gas-phase stability of mannose-linking glycosidic bonds. This reasoning also explains mannose cleavage patterns observed for a xylose type glycopeptide with IRMPD. In addition, extensive peptide backbone cleavage is observed for a >6 kDa glycopeptide with ECD, to our knowledge the largest glycopeptide examined with this technique to date.     

Collisionally induced dissociation of epoxyeicosatrienoic acids and epoxyeicosatrienoic acid-phospholipid molecular species.

Four isomers of epoxyeicosatrienoic acid (EET) can be formed by cytochrome P-450 oxidation of arachidonic acid: 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid. The collision-induced dissociation of the [M-H]- anion at m/z 319 from each of these isomers, using negative-ion fast atom bombardment ionization and a triple quadrupole mass spectrometer, resulted in a series of common ions as well as ions characteristic of each isomer. The common ions were m/z 301 [M-H2O]- and 257 [M-(H2O + CO2)]-. Unique ions resulted from cleavages alpha to the epoxide moiety to form either conjugated carbanions or aldehydes. Mechanisms involving charge site transfer are suggested for the origin of these ions. A distonic ion series that may involve a charge-remote fragmentation mechanism was also observed. The epoxyeicosatrienoic acids were also incorporated into cellular phospholipids following incubation of the free acid with murine mast cells in culture. Negative fast atom bombardment mass spectrometry of purified glycerophosphoethanolamine-EET species and glycerophosphocholine-EET species yielded abundant [M-H]- and [M-CH3]- ions, respectively. The collision-induced dissociation of these specific high-mass ions revealed fragment ions characteristic of the epoxyeicosatrienoic acids incorporated (m/z 319, 301, and 257) and the same unique ions as those seen with each isomeric epoxyeicosatrienoic acid. With this direct method of analysis, phospholipids containing the four positional isomers of EET, including the highly labile (5,6-EET), could be identified as unique molecular species in mast cells incubated with EET.     

Mass spectrometry of peptides and proteins

This tutorial article introduces mass spectrometry (MS) for peptide fragmentation and protein identification. The current approaches being used for protein identification include top-down and bottom-up sequencing. Top-down sequencing, a relatively new approach that involves fragmenting intact proteins directly, is briefly introduced. Bottom-up sequencing, a traditional approach that fragments peptides in the gas phase after protein digestion, is discussed in more detail. The most widely used ion activation and dissociation process, gas-phase collision-activated dissociation (CAD), is discussed from a practical point of view. Infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD) are introduced as two alternative dissociation methods. For spectral interpretation, the common fragment ion types in peptide fragmentation and their structures are introduced; the influence of instrumental methods on the fragmentation pathways and final spectra are discussed. A discussion is also provided on the complications in sample preparation for MS analysis. The final section of this article provides a brief review of recent research efforts on different algorithmic approaches being developed to improve protein identification searches.     

Low-energy collision-activated dissociation electrospray ionization tandem mass spectrometric analysis of Sinorhizobial succinoglycan monomers

Succinoglycan monomers (M1, M2, and M3) are octasaccharides with acetyl, pyruvyl, and/or succinyl groups as substituents derived from Sinorhizobium meliloti 1021. The dissociation patterns of the octasaccharides caused by low-energy collision-activated dissociation (CAD) were investigated using triple quadrupole tandem mass spectrometry (MS) equipped with an electrospray ionization (ESI) source with increasing collision energy (CE) in negative ion mode. None of the succinoglycan monomers were fragmented at a CE of −25 eV. When the CE was applied to −50 or −70 eV, the loss of the terminal Gal residue and/or the succinyl group of the monomers was observed in the product ion scan mode. Interestingly, the acetyl and the pyruvyl groups in the succinoglycan monomers were not lost even when a CE of −70 eV was applied, indicating that the substituents are more stable than the succinyl group in the octasaccharides.     

Mass spectrometry-based proteomics using Q Exactive, a high-performance benchtop quadrupole Orbitrap mass spectrometer

Mass spectrometry-based proteomics has greatly benefitted from enormous advances in high resolution instrumentation in recent years. In particular, the combination of a linear ion trap with the Orbitrap analyzer has proven to be a popular instrument configuration. Complementing this hybrid trap-trap instrument, as well as the standalone Orbitrap analyzer termed Exactive, we here present coupling of a quadrupole mass filter to an Orbitrap analyzer. This "Q Exactive" instrument features high ion currents because of an S-lens, and fast high-energy collision-induced dissociation peptide fragmentation because of parallel filling and detection modes. The image current from the detector is processed by an "enhanced Fourier Transformation" algorithm, doubling mass spectrometric resolution. Together with almost instantaneous isolation and fragmentation, the instrument achieves overall cycle times of 1 s for a top 10 higher energy collisional dissociation method. More than 2500 proteins can be identified in standard 90-min gradients of tryptic digests of mammalian cell lysate- a significant improvement over previous Orbitrap mass spectrometers. Furthermore, the quadrupole Orbitrap analyzer combination enables multiplexed operation at the MS and tandem MS levels. This is demonstrated in a multiplexed single ion monitoring mode, in which the quadrupole rapidly switches among different narrow mass ranges that are analyzed in a single composite MS spectrum. Similarly, the quadrupole allows fragmentation of different precursor masses in rapid succession, followed by joint analysis of the higher energy collisional dissociation fragment ions in the Orbitrap analyzer. High performance in a robust benchtop format together with the ability to perform complex multiplexed scan modes make the Q Exactive an exciting new instrument for the proteomics and general analytical communities.     

Structural studies of gangliosides by fast atom bombardment ionization, low-energy collision-activated dissociation, and tandem mass spectrometry

Negative ion fast atom bombardment, low-energy collision-activated dissociation, and tandem mass spectrometry techniques were applied for the structural elucidation of gangliosides. The mass spectra were simplified by selecting a single molecular ion or fragment ion in the analysis of mixtures, and interference by background signals from the liquid matrix could be avoided. Introduction of collision-activated dissociation produced abundant fragment ions convenient for structural analysis. In the daughter scan mode, ions were produced by cleavage of the glycosidic bonds, and not by cleavage at the sugar ring. These ions all contain ceramide moieties, except the sialic acid fragment ion. In the parent scan mode, product ions resulting from cleavage at the sugar ring were detected beside the ions resulting from cleavage at the glycosidic bonds, and ions of oligosaccharide fragments were also detected. In parent scan mode spectra of gangliosides based on the sialic acid ion, all ions contained a sialic acid residue, and the observed ions were similar to those obtained in the high-energy collision-activated dissociation daughter scan mode. These results indicate the usefulness of low-energy collision-activated dissociation tandem mass spectrometry in the daughter and parent scan modes for the analysis of ganglioside structure, in combination with fast atom bombardment mass spectrometry and high-energy collision-activated dissociation mass spectrometry.     

Tandem mass spectrometric characterization of bile acids and steroid conjugates based on low-energy collision-induced dissociation

We examined the characteristics of several bile acids and some steroid conjugates under low-energy-collision-induced dissociation conditions using a triple quadrupole tandem mass spectrometer. According to conjugation types, we observed characteristic product ions and/or neutral losses in the product ion spectra. Amino acid conjugates afforded specific product ions. For example, glycine-conjugated metabolites routinely produced a product ion at m/z 74, and taurine-conjugated metabolites produced product ions at m/z 124, 107, and 80. When a strong peak appeared at m/z 97, the molecule contained a sulfate group. In contrast to amino acid conjugates, carbohydrate conjugates required a combination of product ions and neutral losses for identification. We could discriminate a glucoside from an acyl galactoside according to the presence or absence of a product ion at m/z 161 and a neutral loss of 180 Da. Discrimination among esters, aliphatic ethers, and phenolic ether types of glucuronides was based upon differences in the intensities of a product ion at m/z 175 and a neutral loss of 176 Da. Furthermore, N-acetylglucosamine conjugates showed a characteristic product ion at m/z 202 and a neutral loss of 203 Da, and the appearance of a product ion at m/z 202 revealed the existence of N-acetylglucosamine conjugated to an aliphatic hydroxyl group without a double bond in the immediate vicinity.Together, the data presented here will help to enable the identification of unknown conjugated cholesterol metabolites by using low-energy collision-induced dissociation.     

Analysis of glycosylphosphatidylinositol membrane anchors by electrospray ionization-mass spectrometry and collision induced dissociation

The multi-component nature of glycosylphosphatidylinositol membrane anchors makes the analysis of their structure complex. Nuclear magnetic resonance spectroscopy of delipidated glycosylphosphatidylinositol-peptide fractions can supply considerable information but requires relatively large quantities of material. High-sensitivity sequencing techniques are available for the oligosaccharide portions of glycosylphosphatidylinositol anchors, but there is no simple and generally applicable technique to complement this information. In this paper we describe the application of electrospray ionization-mass spectrometry and collision induced dissociation to study intact glycosylphosphatidylinositol-peptides from aTrypanosoma brucei variant surface glycoprotein. Collision of the [M + 4H]⁴⁺ pseudomolecular ions of two glycosylphosphatidylinositol-peptide glycoforms produced easily interpretable daughter ion spectra, from which detailed information on the lipid moiety, carbohydrate sequence and site of peptide attachment could be obtained. All of the collision induced dissociation cleavage events occurred in the glycosylphosphatidylinositol portion of the glycosylphosphatidylinositol-peptide. This technique supplies complementary data to the high-sensitivity oligosaccharide sequencing procedures and should greatly assist glycosylphosphatidylinositol anchor structure-function studies, particularly when sample quantities are limiting.