Sequencing of sulfonic acid derivatized peptides by electrospray mass spectrometry

We report the application of nanoelectrospray ionization tandem mass spectrometry (nES-MS/MS) and capillary LC/microelectrospray MS/MS (cLC/&mgr;ES-MS/MS) for sequencing sulfonic acid derivatized tryptic peptides. These derivatives were specifically prepared to facilitate low-energy charge-site-initiated fragmentation of C-terminal arginine-containing peptides, and to enhance the selective detection of a single series of y-type fragment ions. Both singly and doubly protonated peptides were analyzed by MS/MS and the results were compared with those from their derivatized counterparts. Model peptides and peptides from tryptic digests of gel-isolated proteins were analyzed. Derivatized singly protonated peptides fragment in the same way by nES-MS/MS as they do by post-source decay matrix-assisted laser desorption/ionization mass spectrometry (PSD-MALDI-MS). They produce fragment ion spectra dominated by y-ions, and the simplified spectra are readily interpreted de novo. Doubly protonated peptides fragment in much the same way as their non-derivatized doubly protonated counterparts. The fragmentation of doubly protonated derivatives is especially useful for sequencing peptides that possess a proline residue near the N-terminus of the molecule. The singly protonated forms of these proline-containing derivatives often show enhanced fragmentation on the N-terminal side of the proline and considerably reduced fragmentation on the C-terminal side. In addition, sulfonic acid derivatization increases the in-source fragmentation of arginine-containing peptides. This could be useful for sequence verification and sequence tagging for use in single stage mass spectrometry. Copyright 2000 John Wiley & Sons, Ltd.     

Substituent effects on the gas-phase fragmentation reactions of sulfonium ion containing peptides

The multistage mass spectrometric (MS/MS and MS3) gas-phase fragmentation reactions of methionine side-chain sulfonium ion containing peptides formed by reaction with a series of para-substituted phenacyl bromide (XBr where X=CH2COC6H4R, and R=--COOH, --COOCH3, --H, --CH3 and --CH2CH3) alkylating reagents have been examined in a linear quadrupole ion trap mass spectrometer. MS/MS of the singly (M+) and multiply ([M++nH](n+1)+) charged precursor ions results in exclusive dissociation at the fixed charge containing side chain, independently of the amino acid composition and precursor ion charge state (i.e., proton mobility). However, loss of the methylphenacyl sulfide side-chain fragment as a neutral versus charged (protonated) species was observed to be highly dependent on the proton mobility of the precursor ion, and the identity of the phenacyl group para-substituent. Molecular orbital calculations were performed at the B3LYP/6-31+G** level of theory to calculate the theoretical proton affinities of the neutral side-chain fragments. The log of the ratio of neutral versus protonated side-chain fragment losses from the derivatized side chain were found to exhibit a linear dependence on the proton affinity of the side-chain fragmentation product, as well as the proton affinities of the peptide product ions. Finally, MS3 dissociation of the nominally identical neutral and protonated loss product ions formed by MS/MS of the [M++H]2+ and [M++2H]3+ precursor ions, respectively, from the peptide GAILM(X)GAILK revealed significant differences in the abundances of the resultant product ions. These results suggest that the protonated peptide product ions formed by gas-phase fragmentation of sulfonium ion containing precursors in an ion trap mass spectrometer do not necessarily undergo intramolecular proton 'scrambling' prior to their further dissociation, in contrast to that previously demonstrated for peptide ions introduced by external ionization sources.     

Scrambling of sequence information in collision-induced dissociation of peptides

Collision-induced dissociation (CID) of protonated YAGFL-NH2 leads to nondirect sequence fragment ions that cannot directly be derived from the primary peptide structure. Experimental and theoretical evidence indicate that primary fragmentation of the intact peptide leads to the linear YAGFLoxa b5 ion with a C-terminal oxazolone ring that is attacked by the N-terminal amino group to induce formation of a cyclic peptide b5 isomer. The latter can undergo various proton transfer reactions and opens up to form something other than the YAGFLoxa linear b5 isomer, leading to scrambling of sequence information in the CID of protonated YAGFL-NH2.     

Mass spectrometric evidence for mechanisms of fragmentation of charge-derivatized peptides

Mass spectrometry of charged derivatives of peptides has been a growing area of interest in the past decade. Fragmentation of charged derivatives of peptides is believed to be different from than that of protonated peptides when analyzed by collisionally activated dissociation-tandem mass spectrometry (CAD-MS/MS). The charged derivatives fragment by charge-remote fragmentation mechanisms, which are usually classified as high-energy (HE)-CAD processes. Our objective in the present study is to investigate the mechanism of fragmentation of charged derivatives of peptides when analyzed by matrix-assisted laser desorption/ionization-postsource decay-mass spectrometry (MALDI-PSD-MS) and electrospray ionization (ESI)-CAD-MS/MS (ion trap), which involve low-energy processes. Three major types of hydrogens (alpha, beta, and amide) are available for migration during the formation of the *a(n) ions (the predominant ion series produced from these charged derivatives). To pinpoint which of the three hydrogens is involved in the formation of the *a(n) ions, deuterium-labeled peptide derivatives with labels at specific sites were synthesized and analyzed by MALDI-PSD-MS and ESI-CAD-MS/MS. Our results suggest that the amide hydrogen of the residue at which the cleavage occurs shifts during the formation of *a(n); this observation serves as evidence for the mechanism proposed earlier by Liao et al. for fragmentation of such charged derivatives. The results also help elucidate the structure of the *a(n) ions, *b(n) ions, and others formed during cleavage at the proline residue, as well as the ions formed during loss of the C-terminal residue from these charged derivatives.     

Studies of peptide a- and b-type fragment ions using stable isotope labeling and integrated ion mobility/tandem mass spectrometry

The structures of peptide a- and b-type fragment ions were studied using synthetic peptides including a set of isomeric peptides, differing in the sequence location of an alanine residue labeled with ¹⁵N and uniformly with ¹³C. The pattern of isotope labeling of second-generation fragment ions derived via a _n and b _n ions (where n=4 or 5) suggested that these intermediates existed in part as macrocyclic structures, where alternative sites of ring opening gave rise to different linear forms whose simple cleavage might give rise to the observed final products. Similar conclusions were derived from combined ion mobility/tandem MS analyses where different fragmentation patterns were observed for isomeric a- or b-type ions that display different ion mobilities. These analyses were facilitated by a new approach to the processing of ion mobility/tandem MS data, from which distinct and separate product ion spectra are derived from ions that are incompletely separated by ion mobility. Finally, an example is provided of evidence for a macrocyclic structure for b _n ions where n=8 or 9.     

Occurrence of C-Terminal Residue Exclusion in Peptide Fragmentation by ESI and MALDI Tandem Mass Spectrometry

By screening a data set of 392 synthetic peptides MS/MS spectra, we found that a known C-terminal rearrangement was unexpectedly frequently occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTOF and TOF/TOF mass analyzer configuration, respectively. Any residue localized at the C-terminal carboxylic acid end, even a basic one, was lost, provided that a basic amino acid such arginine and to a lesser extent histidine and lysine was present in the sequence leading to a fragment ion, usually depicted as (b_n-1 + H₂O) ion, corresponding to a shortened non-scrambled peptide chain. Far from being an epiphenomenon, such a residue exclusion from the peptide chain C-terminal extremity gave a fragment ion that was the base peak of the MS/MS spectrum in certain cases. Within the frame of the mobile proton model, the ionizing proton being sequestered onto the basic amino acid side chain, it is known that the charge directed fragmentation mechanism involved the C-terminal carboxylic acid function forming an anhydride intermediate structure. The same mechanism was also demonstrated from cationized peptides. To confirm such assessment, we have prepared some of the peptides that displayed such C-terminal residue exclusion as a C-terminal backbone amide. As expected in this peptide amide series, the production of truncated chains was completely suppressed. Besides, multiply charged molecular ions of all peptides recorded in ESI mass spectrometry did not undergo such fragmentation validating that any mobile ionizing proton will prevent such a competitive C-terminal backbone rearrangement. Among all well-known nondirect sequence fragment ions issued from non specific loss of neutral molecules (mainly H₂O and NH₃) and multiple backbone amide ruptures (b-type internal ions), the described C-terminal residue exclusion is highly identifiable giving raise to a single fragment ion in the high mass range of the MS/MS spectra. The mass difference between this signal and the protonated molecular ion corresponds to the mass of the C-terminal residue. It allowed a straightforward identification of the amino acid positioned at this extremity. It must be emphasized that a neutral residue loss can be misattributed to the formation of a y_m-1 ion, i.e., to the loss of the N-terminal residue following the a₁-y_m–1 fragmentation channel. Extreme caution must be adopted when reading the direct sequence ion on the positive ion MS/MS spectra of singly charged peptides not to mix up the attribution of the N- and C-terminal amino acids. Although such peculiar fragmentation behavior is of obvious interest for de novo peptide sequencing, it can also be exploited in proteomics, especially for studies involving digestion protocols carried out with proteolytic enzymes other than trypsin (Lys-N, Glu-C, and Asp-N) that produce arginine-containing peptides.     

Fragmentation studies on monensin A and B by accurate-mass electrospray tandem mass spectrometry

Monensin A and B were studied by electrospray ionisation tandem mass spectrometry (ESI-MS/MS) and the fragment ions were confirmed by accurate-mass measurements. Analyses were performed on both a quadrupole time-of-flight (QTOF) and a Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer. The analysis revealed that fragment ions were produced by Grob-Wharton fragmentations and pericyclic rearrangements in addition to various simple neutral losses. A study of the protonated and sodiated sodium salt revealed different fragmentation pathways for these species, thus complementary structural information could be gained. A complete fragmentation pathway of monensin A and B protonated sodium salt [(M-H+Na)+H])+) and sodiated sodium salt [(M-H+Na)+Na](+) is proposed. MS(3) analysis confirmed the separate fragmentation pathways.     

三唑仑苯二氮(艹卓)类药物的电喷雾质谱裂解特征

采用电喷雾/四极杆飞行时间质谱(ESI-QqTOF)联用技术,对3种三唑仑苯二氮(艹卓)类药物进行CID研究,并以质子化准分子离子[M+H]+作为内标物,对碎片离子进行了准确质量测定,确认了这些碎片离子的元素组成,探讨了该类化合物的质谱裂解规律.研究发现,它们的ESI-MS2(源内)和ESI-MS3质谱分别生成脱去N2分子、HCN或CH3CN分子和Cl原子的碎片离子,其中m/z 205为3种药物共有的碎片离子,这些特征可用于三唑仑苯二氮(艹卓)类药物的体内代谢转化和定量研究.     

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.     

Rapid analysis of lubricants by atmospheric solid analysis probe–ion mobility mass spectrometry

Formulated lubricants are complex mixtures composed of base oil(s) and additives with various functions (detergents, corrosion inhibiter, antioxidant, viscosity modifiers, etc.). Because of the aliphatic nature of base oil and the chemical diversity of additives, the characterization of lubricant is currently a long and complex process. The comprehensive analysis of lubricant samples involves several techniques such as nuclear magnetic resonance, mass spectrometry, chromatography and infrared spectroscopy. The coupling of atmospheric solid analysis probe (ASAP) with ion mobility‐mass spectrometry (IM‐MS) has been shown to be an efficient tool for the characterization of complex mixture containing vaporizable polar to non‐polar compounds. This approach affords the coupling of a direct ionization technique that does not require sample preparation, with a bi‐dimensional separation method with high peak capacity. In this work, we show that ASAP‐IM‐MS is a suitable method for rapid and direct characterization of lubricant samples. Indeed, base oil and additives yielded, by ASAP, ions series which could be separated by IM‐MS. Molecular additives such as Zn‐dithiocarbamate, phosphite, thiophosphate and Alkyl diphenylamine were ionized as molecular ions [M]^+? or protonated molecules [M + H]⁺, depending of their polarity. In some cases, fragment ions were observed, confirming the additive identification. In addition, high molecular weight polymeric additives such as poly(alkyl methacrylate) (PAM) were pyrolized in the ASAP source leading to characteristic fragment ions. ASAP‐IM‐MS is shown to be a powerful tool for studying complex mixtures, allowing the first comprehensive analysis of lubricants in just a few minutes. Copyright © 2014 John Wiley & Sons, Ltd.     

Electron capture dissociation mass spectrometry of peptide cations containing a lysine homologue: a mobile proton model for explaining the observation of b-type product ions

Eleven doubly protonated peptides with a residue homologous to lysine were investigated by electron capture dissociation mass spectrometry (ECD-MS). Lysine homologues provide the unique opportunity to examine the ECD fragmentation behavior by allowing us to vary the length of the lysine side chain, with minimal structural change. The lysine homologue has a primary amine side chain with a length that successively decreases by one methylene (CH(2)) unit from the --CH(2)CH(2)CH(2)CH(2)NH(2) of lysine and the accompanying decrease of its proton affinities: lysine (K), 1006.5(+/-7.2) kJ/mol; ornithine (K(*)), 1001.1(+/-6.6) kJ/mol; 2,4-diaminobutanoic acid (K(**)), 975.8(+/-7.4) kJ/mol; 2,3-diaminopropanoic acid (K(***)), 950.2(+/-7.2) kJ/mol. In general, the lysine-homologous peptides exhibited overall ECD fragmentation patterns similar to that of the lysine-containing peptides in terms of the locations, abundances, and ion types of products, such as yielding c(+) and z(+.) ions as the dominant product ions. However, a close inspection of product ion mass spectra showed that ECD-MS for the alanine-rich peptides with an ornithinyl or 2,4-diaminobutanoyl residue gave rise to b ions, while the lysinyl-residue-containing peptides did not, in most cases, produce any b ions. The peptide selectivity in the generation of b(+) ions could be understood from within the framework of the mobile proton model in ECD-MS, previously proposed by Cooper (Ref. 29). The exact mass analysis of the resultant b ions reveals that these b ions are not radical species but rather the cationic species with R-CO(+) structure (or protonated oxozalone ion), that is, b(+) ions. The absence of [M+2H](+.) species in the ECD mass spectra and the selective b(+)-ion formation are evidence that the peptides underwent H-atom loss upon electron capture, and then the resulting reduced species dissociated following typical MS/MS fragmentation pathways. This explanation was further supported by extensive b(+) ions generated in the ECD of alanine-based peptides with extended conformations.     

Travelling‐wave ion mobility and negative ion fragmentation of high‐mannose N‐glycans

The isomeric structure of high‐mannose N‐glycans can significantly impact biological recognition events. Here, the utility of travelling‐wave ion mobility mass spectrometry for isomer separation of high‐mannose N‐glycans is investigated. Negative ion fragmentation using collision‐induced dissociation gave more informative spectra than positive ion spectra with mass‐different fragment ions characterizing many of the isomers. Isomer separation by ion mobility in both ionization modes was generally limited, with the arrival time distributions (ATD) often showing little sign of isomers. However, isomers could be partially resolved by plotting extracted fragment ATDs of the diagnostic fragment ions from the negative ion spectra, and the fragmentation spectra of the isomers could be extracted by using ions from limited areas of the ATD peak. In some cases, asymmetric ATDs were observed, but no isomers could be detected by fragmentation. In these cases, it was assumed that conformers or anomers were being separated. Collision cross sections of the isomers in positive and negative fragmentation mode were estimated from travelling‐wave ion mobility mass spectrometry data using dextran glycans as calibrant. More complete collision cross section data were achieved in negative ion mode by utilizing the diagnostic fragment ions. Examples of isomer separations are shown for N‐glycans released from the well‐characterized glycoproteins chicken ovalbumin, porcine thyroglobulin and gp120 from the human immunodeficiency virus. In addition to the cross‐sectional data, details of the negative ion collision‐induced dissociation spectra of all resolved isomers are discussed. Copyright © 2016 John Wiley & Sons, Ltd.     

The role of proton mobility in determining the energy-resolved vibrational activation/dissociation channels of N-glycopeptide ions

Site-specific glycoproteomic analysis largely hinges on the use of tandem mass spectrometry (MS/MS) to identify glycopeptides. Experiments of this type are usually aimed at drawing connections between individual oligosaccharide structures and their specific sites of attachment to the polypeptide chain. These determinations inherently require ion dissociation methods capable of interrogating both the monosaccharide and amino acid connectivity of the glycopeptide. Collision-induced dissociation (CID) shows potential to satisfy this requirement, as the vibrational activation/dissociation of protonated N-glycopeptides has been observed to access cleavage of either glycosidic bonds of the glycan or amide bonds of the peptide in an energy-resolved manner. Nevertheless, the relative energy requirement for these fragmentation pathways varies considerably among analytes. This research addresses the influence of proton mobility on the vibrational energy necessary to achieve either glycan or peptide cleavage in a collection of protonated N-glycopeptide ions. While greater proton mobility of the precursor ion was found to correlate with lower energy requirements for precursor ion depletion and appearance of glycosidic fragments, the vibrational energy deposition necessary for appearance of peptide backbone fragments showed no relation to the precursor ion proton mobility. These results are consistent with observations suggesting that peptide fragments arise from an intermediate fragment which is generally of lower proton mobility than the precursor ion. Such findings have potential to facilitate the rational selection of CID conditions which are best suited to provide either glycan or peptide cleavage products in MS/MS based N-glycoproteomic analysis.     

离子淌度质谱技术在中药化学成分分析中的研究进展

离子淌度质谱(IM-MS)是一种将离子淌度分离与质谱分析相结合的新型分析技术。IM-MS的主要优势不仅是在质谱检测前提供了基于气相离子形状、大小、电荷数等因素的多一维分离,而且能够提供碰撞截面积、漂移时间等质谱信息进而辅助化合物鉴定。近年来,随着IM-MS技术的不断发展,该技术在中药化学成分分析中受到越来越多的关注。首先,IM-MS已成功应用于改善中药复杂成分尤其是同分异构体或等量异位素等成分的分离;其次,IM-MS可通过多重碎裂模式辅助高质量中药小分子质谱信息的获取;此外,IM-MS提供的高维质谱数据信息还可促进中药复杂体系多成分的整合分析。该文在对IM-MS分类和基本原理进行概述的基础上,从分离能力及分离策略、多重碎裂模式、多维质谱数据处理策略3个方面,重点综述了IM-MS在中药化学成分分析中的应用,以期为IM-MS在中药化学成分研究提供参考。     

Electrospray ionization tandem mass spectrometry of model peptides reveals diagnostic fragment ions for protein ubiquitination

In this work, synthetic peptides were used to determine the fragmentation behavior of ubiquitinated peptides and to find ions diagnostic for peptide ubiquitination. The ubiquitin-calmodulin peptide1 was chosen as the model peptide for naturally occurring ubiquitinated proteins cleaved with endoproteinase gluC. In addition, the fragmentation behavior of model ubiquitinated peptides produced by tryptic digestion was also of great interest since the standard protocols for proteomics-based protein identification use trypsin as the protease. Attachment of ubiquitin to a target protein results in a branched structure, but only ions from the ubiquitin side chain (and the lysine to which it is attached) can be used as diagnostic ions, since fragment ions that contain other amino acids from the parent protein will vary in mass. Characteristic b-type fragment ions from the gluC cleavage of the ubiquitin side chain (designated as b ions) were found which involve only the ubiquitin tail (b2, b3, b4, b5 and b6 ions at m/z 189.06, 302.12, 439.18, 552.30 and 651.30, respectively). Maximum production of these ions occurred at a collision energy of 45 eV in a Q-TOF instrument. Although a non-ubiquitinated peptide may produce isobaric fragment ions, it is unlikely that it can produce these ions in combination. With liquid chromatography/tandem mass spectrometry (LC/MS/MS) experiments, ubiquitinated peptides can readily be determined by surveying the reconstructed or extracted ion chromatograms of the diagnostic fragment ions for common peaks. Characteristic ions resulting from tryptic cleavage of the side chain were found in cleavage products with a missed cleavage, resulting in a LRGG- tag instead of a GG- tag. For the LRGG-tagged peptide, diagnostic MS/MS fragment ions (at m/z 270.17 and 384.21) from the ubiquitin tail (b2 and b4, respectively) were found, along with an internal fragment ion (LRGGK-28) at m/z 484.30. These ions should prove useful in precursor-ion scanning experiments for identifying peptides modified by attachment of ubiquitin, and for locating the site of ubiquitin attachment.     

Differentiation of three pairs of Boc-beta,gamma- and gamma,beta-hybrid peptides by electrospray ionization tandem mass spectrometry

A new series of Boc-N-beta(3), gamma(4)-/gamma(4), beta(3)-isomeric hybrid peptides (containing repeats of beta(3)-Caa and gamma(4)-Caa's, Caa = C-linked carbo beta(3)-/gamma(4)-amino acids derived from D-xylose) have been differentiated by both positive and negative ion electrospray ionization (ESI) ion-trap and high resolution quadrupole time-of-flight/tandem mass spectrometry (Q-TOF MS/MS). MS(n) of protonated isomeric peptides and [M + H - Boc + H](+) produce characteristic fragmentation involving the peptide backbone, the Boc-group, and the side chain. The positional isomers are differentiated from one another by the presence of y(n) (+), b(n) (+), and other fragment ions of different m/z values. It is observed that the peptides with beta-Caa at the N-terminus produce extensive fragmentation, whereas gamma-Caa gave rise to much less fragmentation. Peptides with gamma-Caa at the N-terminus lose NH(3), whereas this process is absent for the carbopeptides with beta-Caa at the N-terminus. Two pairs of dipeptide diastereomers are clearly differentiated by the collision-induced dissociation (CID) of their protonated molecules. The loss of 2-methylprop-1-ene is more pronounced for Boc-NH-(R)-beta-Caa-(R)-gamma-Caa-OCH(3) (6) and Boc-NH-(R)-gamma-Caa-(R)-beta-Caa-OCH(3) (12), whereas it is insignificant or totally absent for its protonated diastereomeric pair Boc-NH-(S)-beta-Caa-(S)-gamma-Caa-OCH(3) (1) and Boc-NH-(S)-gamma-Caa-(S)-beta-Caa-OCH(3) (7). Further, ESI negative ion tandem mass spectrometry has also been found to be useful for differentiating these isomeric peptide acids. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Fragmentation studies on monensin A by sequential electrospray mass spectrometry

Monensin A was studied by electrospray ionisation sequential mass spectrometry (ESI-MSn) and all fragments were confirmed by accurate-mass measurements. Analyses were performed on both a quadrupole time-of-flight (Q-tof) and a Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer. MSn analysis shows that depending on sample preparation the ion at m/z 671 consists of two different ions with the same accurate-mass. It is either the monensin protonated parent ion or a different ion structure derived from the loss of water from the water adduct of monensin. Both ions show different fragmentation patterns. Major fragment ions from the protonated parent ion were produced by Grob-Wharton type fragmentations in addition to various simple neutral losses. The fragmentation pathways of the two different m/z 671 ions are proposed.     

Gas-phase fragmentation study of novel synthetic 1,5-benzodiazepine derivatives using electrospray ionization tandem mass spectrometry

The fragmentation patterns of a series of three novel synthesized 3-hydroxy-4-phenyl-tetrahydro-1,5-benzodiazepin-2-ones (1-3), possessing the same backbone structure, were investigated using electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) techniques. A simple methodology, based on the use of ESI (positive ion mode) and by increasing the declustering potential in the atmospheric pressure/vacuum interface, collision-induced dissociation (CID), was used to enhance the formation of the fragment ions. In general, the novel synthetic 1,5-benzodiazepine derivatives afforded, in the gas phase, both protonated and sodiated molecules. This led to the confirmation of the molecular masses and chemical structures of the studied compounds. Exact accurate masses were measured using a high-resolution ESI-quadrupole orthogonal time-of-flight (QqToF)-MS/MS hybrid mass spectrometer instrument.The breakdown routes of the protonated molecules were rationalized by conducting low-energy collision CID-MS/MS analyses (product ion- and precursor ion scans) using a conventional quadrupole-hexapole-quadrupole (QhQ) tandem mass spectrometer. All the observed major fragmentations for the 1,5-benzodiazepines occurred in the saturated seven-membered ring containing the nitrogen atoms. These formed a multitude of product ions by different breakdown routes. All the major fragmentations involved cleavages of the N-1-C-2 and C-3-C-4 bonds. These occurred with concomitant eliminations of glyoxal, benzene and ethyl formate, forming the product ion at m/z 119, which was observed in all the studied compounds. In addition, an unique simultaneous CID-MS/MS fragmentation was noticed for the 1,5-benzodiazepines 1 and 3, which occurred by a pathway dictated by the substituent located on the N-1-position. It was evident that the aromatic ring portion of the 1,5-benzodiazepines was resistant to CID-MS/MS fragmentation. Re-confirmation of the various geneses of the product ions was achieved by conducting a series of precursor ion scans. ESI-MS and CID-MS/MS analyses have thus proven to be a specific and very sensitive method for the structural identification of these novel 1,5-benzodiazepine derivatives.     

Mannose7 Glycan Isomer Characterization by IMS-MS/MS Analysis

The isomers of the Man₇GlcNAc₂ glycan obtained from bovine ribonuclease B have been characterized by ion mobility spectrometry-tandem mass spectrometry (IMS-MS/MS). In these experiments, [Man7 + 2Na]²⁺ precursors having different mobilities are selected by ion mobility spectrometry and analyzed by MS/MS techniques in an ion trap. The fragmentation spectra obtained for various precursor ions are specific, suggesting the isolation or enrichment of different glycan isomers. One fragment ion with a mass-to-charge ratio (m/z) of 903.8 is found to correspond to the loss of an internal mannose residue of a specific isomer. Extracted fragment ion drift time distributions (XFIDTDs) yield distinctive precursor ion drift time profiles indicating the existence of four separate isomers as proposed previously.     

On performing simultaneous electron transfer dissociation and collision-induced dissociation on multiply protonated peptides in a linear ion trap

We propose a tandem mass spectrometry method that combines electron-transfer dissociation (ETD) with simultaneous collision-induced dissociation (CID), termed ETD/CID. This technique can provide more complete sequence coverage of peptide ions, especially those at lower charge states. A selected precursor ion is isolated and subjected to ETD. At the same time, a residual precursor ion is subjected to activation via CID. The specific residual precursor ion selected for activation will depend upon the charge state and m/z of the ETD precursor ion. Residual precursor ions, which include unreacted precursor ions and charge-reduced precursor ions (either by electron-transfer or proton transfer), are often abundant remainders in ETD-only reactions. Preliminary results demonstrate that during an ETD/CID experiment, b, y, c, and z-type ions can be produced in a single experiment and displayed in a single mass spectrum. While some peptides, especially doubly protonated ones, do not fragment well by ETD, ETD/CID alleviates this problem by acting in at least one of three ways: (1) the number of ETD fragment ions are enhanced by CID of residual precursor ions, (2) both ETD and CID-derived fragments are produced, or (3) predominantly CID-derived fragments are produced with little or no improvement in ETD-derived fragment ions. Two interesting scenarios are presented that display the flexibility of the ETD/CID method. For example, smaller peptides that show little response to ETD are fragmented preferentially by CID during the ETD/CID experiment. Conversely, larger peptides with higher charge states are fragmented primarily via ETD. Hence, ETD/CID appears to rely upon the fundamental reactivity of the analyte cations to provide the best fragmentation without implementing any additional logic or MS/MS experiments. In addition to the ETD/CID experiments, we describe a novel dual source interface for providing front-end ETD capabilities on a linear ion trap mass spectrometer.