Collision-induced Dissociation Mass Spectra Research Articles

Collision-Induced Dissociation of Fucose and Identification of Anomericity.

Structural determination of carbohydrates using mass spectrometry remains challenging, particularly, the differentiation of anomeric configurations. In this work, we studied the collision-induced dissociation (CID) mechanisms of sodiated α- and β-l-fucose using an experimental method and quantum chemistry calculations. The calculations show that α-l-fucose is more likely to undergo dehydration due to the fact that O1 and O2 are on the same side of the sugar ring. In contrast, β-l-fucose is more prone to the ring-opening reaction because more OH groups are on the same side of the sugar ring as O1. These differences suggest a higher preference for the dehydration reaction in sodiated α-l-fucose but a lower preference for ring-opening compared to that of β-l-fucose. The calculation results, which are used to assign the CID mass spectra of α- and β-l-fucose separated by high-performance liquid chromatography, are supported by the fucose produced from the CID of disaccharides Fuc-β-(1 → 3)-GlcNAc and Fuc-α-(1 → 4)-GlcNAc. This study demonstrates that the correlation of cis- and trans-configurations of O1 and O2 to the relative branching ratios of dehydration and cross-ring dissociation in CID, observed in aldohexose and ketohexose in the pyranose form, can be extended to deoxyhexoses for anomericity determination.

Does deprotonated benzoic acid lose carbon monoxide in collision-induced dissociation?

Decarboxylation is known to be the major fragmentation pathway for the deprotonated carboxylic acids in collision-induced dissociation (CID). However, in the CID mass spectrum of deprotonated benzoic acid (m/z 121) recorded on a Q-orbitrap mass spectrometer, the dominant peak was found to be m/z 93 instead of the anticipated m/z 77. Based on theoretical calculations, 18 O-isotope labeling and MS3 experiments, we demonstrated that the fragmentation of benzoate anion begins with decarboxylation, but the initial phenide anion (m/z 77) can react with trace O2 in the mass analyzer to produce phenolate anion (m/z 93) and other oxygen-containing ions. Thus oxygen adducts should be considered when annotating the MS/MS spectra of benzoic acids.

Characterization of phytochemicals from twisted-leaf garlic (Allium obliquum L.) using liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry

IntroductionTwisted-leaf garlic (Allium obliquum L.) is a wild Allium species, which is traditionally used as aroma plant for culinary purposes due to its unique, garlic-like flavor. It represents an interesting candidate for domestication, breeding and cultivation.ObjectivesThe objective of this work was to explore and comprehensively characterize polar and semi-polar phytochemicals accumulating in leaves and bulbs of A. obliquum.MethodPlant material obtained from a multiyear field trial was analyzed using a metabolite profiling workflow based on ultra-high performance liquid chromatography-coupled electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC/ESI-QTOFMS) and two chromatographic methods. For annotation of metabolites, tandem mass spectrometry experiments were carried out and the resulting accurate-mass collision-induced dissociation (CID) mass spectra interpreted. Onion and garlic bulb extracts were used as reference samples.ResultsImportant metabolite classes influencing nutritional, sensory and technological properties were detected and structurally characterized including fructooligosaccharides with a degree of polymerization of 3–5, S-alk(en)ylcysteine sulfoxides and other S-substituted cysteine conjugates, flavonoids including O- and C-glycosylated flavones as well as O-glycosylated flavonols, steroidal saponins, hydroxycinnamic acid conjugates, phenylethanoids and free sphingoid bases. In addition, quantitative data for non-structural carbohydrates, S-alk(en)ylcysteine sulfoxides and flavonoids are provided.ConclusionThe compiled analytical data including CID mass spectra of more than 160 annotated metabolites provide for the first time a phytochemical inventory of A. obliquum and lay the foundation for its further use as aroma plant in food industry.

The collision-induced dissociation mechanism of sodiated Hex-HexNAc disaccharides.

Determining carbohydrate structures, such as their compositions, linkage positions, and in particular the anomers and stereoisomers, is a great challenge. Isomers of different anomers or stereoisomers have the same sequences of chemical bonds, but have different orientations of some chemical bonds which are difficult to be distinguished by mass spectrometry. Collision-induced dissociation (CID) tandem mass spectroscopy (MS/MS) is a widely used technique for characterizing carbohydrate structures. Understanding the carbohydrate dissociation mechanism is important for obtaining the structural information from MS/MS. In this work, we studied the CID mechanism of galactose-N-acetylgalactosamine (Gal-GalNAc) and glucose-N-acetylglucosamine (Glc-GlcNAc) disaccharides with 1→3 and 1→4 linkages. For Gal-GalNAc disaccharides, the CID mass spectra of sodium ion adducts show significant difference between the α- and β-anomers of GalNAc at the reducing end, while no difference in the CID mass spectra between two anomers of Glc-GlcNAc disaccharides was found. Quantum chemistry calculations show that for Gal-GalNAc disaccharides, the difference of the dissociation barriers between dehydration and glycosidic bond cleavage is significantly small in the β-anomer compared to that in the α-anomer; while these differences are similar between the α- and β-anomers of Glc-GlcNAc disaccharides. These differences can be attributed to the different orientations of hydroxyl and N-acetyl groups located at GalNAc and GlcNAc. The calculation results are consistent with the CID spectra of isotope labelled disaccharides. Our study provides an insight into the CID of 1→3 and 1→4 linked Gal-GalNAc and Glc-GlcNAc disaccharides. This information is useful for determining the anomeric configurations of GalNAc in oligosaccharides.

Calculation of Mass Spectra with the QCxMS Method for Negatively and Multiply Charged Molecules.

Analysis and validation of a mass spectrometry (MS) experiment are usually performed by comparison to reference spectra. However, if references are missing, measured spectra cannot be properly matched. To close this gap, the Quantum Chemical Mass Spectrometry (QCxMS) program has been developed. It enables fully automatic calculations of electron ionization (EI) and positive ion collision-induced dissociation (CID) mass spectra of singly charged molecular ions. In this work, the extension to negative and multiple ion charge for the CID run mode is presented. QCxMS is now capable of calculating structures carrying any charge, without the need for pretabulated fragmentation pathways or machine learning of database spectra. Mass spectra of four single negatively charged and two multiple positively charged organic ions with molecular sizes from 12 to 92 atoms were computed and compared to reference spectra. The underlying Born-Oppenheimer molecular dynamics (MD) calculations were conducted using the semiempirical quantum mechanical GFN2-xTB method, while for some small molecules, ab initio DFT-based MD simulations were performed. Detailed insights into the fragmentation pathways were gained, and the effects of the computed charge assignments on the resulting spectrum are discussed. Especially for the negative ion mode, the influence of the deprotonation site to create the anion was found to be substantial. Doubly charged fragments could successfully be calculated fully automatically for the first time, while higher charged structures introduced severe assignment problems. Overall, this extension of the QCxMS program further enhances its applicability and underlines its value as a sophisticated toolkit for CID-based tandem MS structure elucidation.

Incorporating measurement variability when comparing sets of high-resolution mass spectra

Mass spectra are an important signature by which compounds can be identified. We recently formulated a mathematical approach for incorporating measurement variability when comparing sets of high-resolution mass spectra. Leveraging replicate mass spectra, we construct high-dimensional consensus mass spectra—representing each of the compared analytes—and compute the similarity between these data structures. In this paper, we present this approach and discuss its applications and limitations when trying to discriminate methamphetamine and phentermine using in-source collision induced dissociation mass spectra collected with direct analysis in real time mass spectrometry.

The ion-neutral complex-mediated fragmentation reaction in electrospray ionization tandem mass spectrometric analysis of N-phenyl-3-(phenylthio)propanamides.

Amides are the fundamental units of both peptides and proteins, and also important functional groups of medical chemicals. Investigation of the fragmentation mechanism of amides in the gas phase is scientifically important for structural analysis. However, understanding of this problem is still elusive. Protonated N-phenyl-3-(phenylthio)propanamide and its derivatives were investigated using positive ion tandem mass spectrometry (ESI-MS/MS) with an LCQ mass spectrometer. Accurate mass analysis was conducted with a micrOTOF-QII mass spectrometer. Density functional theory (DFT) calculations using the Gaussian 03 program and deuterium-labelling (D-labelling) experiments were performed to verify the proposed fragmentation mechanism. Interpretation of the fragment ions in the collision-induced dissociation mass spectra showed that the ionizing proton in the protonated ion transferred from the most thermodynamically favorable carbonyl oxygen to the dissociative protonation site at amide nitrogen or sulfur atom upon collisional activation. The dissociation of the amide or the C-S bond was induced by such proton transfer. An ion-neutral complex (INC) was generated via the dissociation of the amide bond. In the INC, it was observed that the carbocation of the ionic part attacked the ortho phenyl carbon atom adjacent to the sulfur atom, and proton transfer from the carbon atom to the nitrogen atom led to the formation of protonated aniline. The fragmentation mechanism of protonated N-phenyl-3-(phenylthio)propanamide and its derivatives was proposed and elucidated. All the compounds studied showed similar fragmentation pathways, and the competitive formation of two ions, RC9 H9 OS+ and C6 H8 N+ , was observed. The generation of protonated aniline is mediated by INC in ESI-MS/MS.

Solvent-Mediated Proton-Transfer Catalysis of the Gas-Phase Isomerization of Ciprofloxacin Protomers.

Understanding how neutral molecules become protonated during positive-ion electrospray ionization (ESI) mass spectrometry is critically important to ensure analytes can be efficiently ionized, detected, and unambiguously identified. The ESI solvent is one of several parameters that can alter the dominant site of protonation in polyfunctional molecules and thus, in turn, can significantly change the collision-induced dissociation (CID) mass spectra relied upon for compound identification. Ciprofloxacin─a common fluoroquinolone antibiotic─is one such example whereby positive-ion ESI can result in gas-phase [M + H]+ ions protonated at either the keto-oxygen or the piperazine-nitrogen. Here, we demonstrate that these protonation isomers (or protomers) of ciprofloxacin can be resolved by differential ion mobility spectrometry and give rise to distinctive CID mass spectra following both charge-directed and charge-remote mechanisms. Interaction of mobility-selected protomers with methanol vapor (added via the throttle gas supply) was found to irreversibly convert the piperazine N-protomer to the keto-O-protomer. This methanol-mediated proton-transport catalysis is driven by the overall exothermicity of the reaction, which is computed to favor the O-protomer by 93 kJ mol-1 (in the gas phase). Conversely, gas phase interactions of mobility-selected ions with acetonitrile vapor selectively depletes the N-protomer ion signal as formation of stable [M + H + CH3CN]+ cluster ions skews the apparent protomer population ratio, as the O-protomer is unaffected. These findings provide a mechanistic basis for tuning protomer populations to ensure faithful characterization of multifunctional molecules by tandem mass spectrometry.

A New Library-Search Algorithm for Mixture Analysis Using DART-MS.

Forensic analysis of seized drug evidence often involves determining whether the components of an unknown mixture are illicit compounds. One approach to this task is to screen the evidence using direct analysis in real time mass spectrometry (DART-MS) to make presumptive identifications. This manuscript introduces a new library-search algorithm that enhances presumptive identifications of mixture components using a series of in-source collision-induced dissociation mass spectra collected through DART-MS. The multistage search, titled the Inverted Library-Search Algorithm (ILSA), identifies potential components in a mixture by first searching the lowest fragmentation mass spectrum for target peaks, assuming these peaks are protonated molecules, and then scoring each target peak with possible library matches. As a proof of concept, the ILSA is demonstrated through several example searches of model seized drug mixtures of acetyl fentanyl, benzyl fentanyl, amphetamine, and methamphetamine searched against a small library of select compounds and the freely available NIST DART-MS Forensics Database. Discussion of the search results and several open areas of research to further extend the method are provided. This new approach for presumptive identification provides analysts with refined information about mixture components and will be of immediate importance in forensic analysis using DART-MS. A prototype implementation of the ILSA is available at https://github.com/asm3-nist/DART-MS-DST.

Predicting the Mass Spectra of Environmental Pollutants Using Computational Chemistry: A Case Study and Critical Evaluation.

Organic pollutants can be identified by comparing their electron ionization (EI) mass spectra with those in libraries or obtained from authentic standards. Nevertheless, libraries are incomplete; standards may be unavailable or too costly, or their synthesis may be too time-consuming. This study evaluates the performance of quantum chemical electron ionization mass spectrometry (QCEIMS) vis-à-vis competitive fragmentation modeling (CFM) for suspect screening and unknown identification. EI mass spectra of 35 compounds, including halogenated organics, organophosphorus flame retardants (OPFRs), and disinfection byproducts were computed. Computational results were compared with EI mass spectra compiled in the NIST Library as well as collision-induced dissociation (CID) mass spectra obtained from radical cations M•+ generated by charge-exchange atmospheric pressure chemical ionization (APCI). The results indicate that QCEIMS performs equivalently or better than CFM. Average match factors between computed and experimental (NIST) EI mass spectra were 656 vs 503 for the halogenated organics, and on average, QCEIMS predicted 55% of the products generated by CID vs 17% predicted by CFM. QCEIMS predicted 37% of the OPFR CID products whereas CFM predicted 29%. QCEIMS performed comparably to a commercial combinatorial fragmentation method for suspect screening of a dust sample, identifying 19/20 targets. Examples of unknown pollutants, whose reference spectra were unavailable at the time of discovery, are also presented. The computational results suggest that QCEIMS can help guide the analyst in obtaining authentic standards and raise the possibility that, with advances in computing, an unknown may eventually be confirmed in hours as opposed to the days or months required to obtain authentic standards.

In Silico Tandem Mass Spectrometer: an Analytical and Fundamental Tool

AbstractIn this article, we summarize some aspects of the recently developed computational approach to model and predict collision induced dissociation mass spectra. In particular, we describe how initial conditions can be set to model fragmentation conditions and then obtain different results from the analysis of an ensemble of reactive trajectories. This approach was studied and validated on different systems, from small organic molecules to large biomolecules. Recently an iterative procedure was proposed to increase the fragmentation sampling, reducing computational time and providing a more comprehensive fragmentation pattern. All these fundamental developments are at the basis of the application of this approach to analytical problems. One important and possible outcome will be in creating an in silico data base which will be an useful complement to the experimental ones. This is discussed within other possible future outlooks.

Comprehensive profiling of semi\u2010polar phytochemicals in whole wheat grains (Triticum aestivum) using liquid chromatography coupled with electrospray ionization quadrupole time\u2010of\u2010flight mass spectrometry

IntroductionWheat (Triticum aestivum) it is one of the most important staple food crops worldwide and represents an important resource for human nutrition. Besides starch, proteins and micronutrients wheat grains accumulate a highly diverse set of phytochemicals.ObjectivesThis work aimed at the development and validation of an analytical workflow for comprehensive profiling of semi-polar phytochemicals in whole wheat grains.MethodReversed-phase ultra-high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC/ESI-QTOFMS) was used as analytical platform. For annotation of metabolites accurate mass collision-induced dissociation mass spectra were acquired and interpreted in conjunction with literature data, database queries and analyses of reference compounds.ResultsBased on reversed-phase UHPLC/ESI-QTOFMS an analytical workflow for comprehensive profiling of semi-polar phytochemicals in whole wheat grains was developed. For method development the extraction procedure and the chromatographic separation were optimized. Using whole grains of eight wheat cultivars a total of 248 metabolites were annotated and characterized by chromatographic and tandem mass spectral data. Annotated metabolites comprise hydroquinones, hydroxycinnamic acid amides, flavonoids, benzoxazinoids, lignans and other phenolics as well as numerous primary metabolites such as nucleosides, amino acids and derivatives, organic acids, saccharides and B vitamin derivatives. For method validation, recovery rates and matrix effects were determined for ten exogenous model compounds. Repeatability and linearity were assessed for 39 representative endogenous metabolites. In addition, the accuracy of relative quantification was evaluated for six exogenous model compounds.ConclusionsIn conjunction with non-targeted and targeted data analysis strategies the developed analytical workflow was successfully applied to discern differences in the profiles of semi-polar phytochemicals accumulating in whole grains of eight wheat cultivars.

Data-Independent Identification of Suspected Organic Pollutants Using Gas Chromatography-Atmospheric Pressure Chemical Ionization-Mass Spectrometry.

The identity of an unknown environmental pollutant is reflected by the mass and dissociation chemistry of its (quasi)molecular ion. Gas chromatography-atmospheric pressure chemical ionization-mass spectrometry (GC-APCI-MS) increases the yield of molecular ions (compared to conventional electron ionization) by collisional cooling. Scanning quadrupole data-independent acquisition (SQDIA) permits unbiased, unattended selection of (quasi)molecular ions and acquisition of structure-diagnostic collision-induced dissociation mass spectra, while minimizing interferences, by sequentially cycling a quadrupole isolation window through the m/z range. This study reports on the development of a suspect screening method based on industrial compounds with bioaccumulation potential. A comparison of false and correct identifications in a mixed standard containing 30 analytes suggests that SQDIA results in a markedly lower false-positive rate than standard DIA: 5 for SQDIA and 82 for DIA. Electronic waste dust was analyzed using GC and quadrupole time-of-flight MS with APCI and SQDIA acquisition. A total of 52 brominated, chlorinated, and organophosphorus compounds were identified by suspect screening; 15 unique elemental compositions were identified using nontargeted screening; 17 compounds were confirmed using standards and others identified to confidence levels 2, 3, or 4. SQDIA reduced false-positive identifications, compared to experiments without quadrupole isolation. False positives also varied by class: 20% for Br, 37% for Cl, 75% for P, and >99% for all other classes. The structure proposal of a previously reported halogenated compound was revisited. The results underline the utility of GC-SQDIA experiments that provide information on both the (quasi)molecular ions and its dissociation products for a more confident structural assignment.

Ground and Excited States of Gas-Phase DNA Nucleobase Cation-Radicals. A UV-vis Photodisociation Action Spectroscopy and Computational Study of Adenine and 9-Methyladenine.

Cation radicals of adenine (A•+) and 9-methyladenine (MA•+) were generated in the gas phase by collision-induced intramolecular electron transfer in copper-terpyridine-nucleobase ternary complexes and characterized by collision-induced dissociation (CID) mass spectra and UV-vis photodissociation action spectroscopy in the 210-700 nm wavelength region. The action spectra of both A•+ and MA•+ displayed characteristic absorption bands in the near-UV and visible regions. Another tautomer of A•+ was generated as a minor product by multistep CID of protonated 9-(2-bromoethyl)adenine. Structure analysis by density functional theory and coupled-clusters ab initio calculations pointed to the canonical 9-H-tautomer Ad1•+ as the global energy minimum of adenine cation radicals. The canonical tautomer MA1•+ was also calculated to be a low-energy structure among methyladenine cation radicals. However, two new noncanonical tautomers were found to be energetically comparable to MA1•+. Vibronic absorption spectra were calculated for several tautomers of A•+ and MA•+ and benchmarked on equation-of-motion coupled-clusters excited-state calculations. Analysis of the vibronic absorption spectra of A•+ tautomers pointed to the canonical tautomer Ad1•+ as providing the best match with the action spectrum. Likewise, the canonical tautomer MA1•+ was the unequivocal best match for the MA•+ ion generated in the gas phase. According to potential-energy mapping, MA1•+ was separated from energetically favorable noncanonical cation radicals by a high-energy barrier that was calculated to be above the dissociation threshold for loss of a methyl hydrogen atom, thus preventing isomerization. Structures and energetics of all four DNA nucleobase cation radicals are compared and discussed.

Variation in the Relative Isomer Abundance of Synthetic and Biologically Derived Phosphatidylethanols and Its Consequences for Reliable Quantification.

Phosphatidylethanol (PEth) in human blood samples is a marker for alcohol usage. Typically, PEth is detected by reversed-phase liquid chromatography coupled with negative ion tandem mass spectrometry, investigating the fatty acyl anions released from the precursor ion upon collision-induced dissociation (CID). It has been established that in other classes of asymmetric glycerophospholipids, the unimolecular fragmentation upon CID is biased depending on the relative position (known as sn-position) of each fatty acyl chain on the glycerol backbone. As such, the use of product ions in selected-reaction-monitoring (SRM) transitions could be prone to variability if more than one regioisomer is present in either the reference materials or the sample. Here, we have investigated the regioisomeric purity of three reference materials supplied by different vendors, labeled as PEth 16:0/18:1. Using CID coupled with ozone-induced dissociation, the regioisomeric purity (% 16:0 at sn-1) was determined to be 76, 80 and 99%. The parallel investigation of the negative ion CID mass spectra of standards revealed differences in product ion ratios for both fatty acyl chain product ions and ketene neutral loss product ions. Furthermore, investigation of the product ion abundances in CID spectra of PEth within authentic blood samples appears to indicate a limited natural variation in isomer populations between samples, with the cannonical, PEth 16:0/18:1 (16:0 at sn-1) predominant in all cases. Different reference material isomer distributions led to variation in fully automated quantification of PEth in 56 authentic dried blood spot (DBS) samples when a single quantifier ion was used. Our results suggest caution in ensuring that the regioisomeric compositions of reference materials are well-matched with those of the authentic blood samples.

Ion trap MS using high trapping gas pressure enables unequivocal structural analysis of three isobaric compounds in a mixture by using energy-resolved mass spectrometry and the survival yield technique.

Recently, it has been shown that energy-resolved mass spectrometry (MS) can provide quantitative information from two isomeric or isobaric compounds in mixtures by using the survival yield (SY) technique together with "gas-phase collisional purification" (GPCP) strategy (Anal. Chem., 2016, 88, p.10821). Herein, we present an improvement and an extension of this concept to the structural analysis of a model mixture of three isobaric compounds (two peptides and a polyether). By using default collision-induced dissociation (CID) tandem MS parameters on an ion trap instrument, the previous approach did not show any signs of isobaric contamination. However, by modifying CID conditions and using a threefold increase of the He trapping gas pressure (to reach 3.00·10-5 mbar), the SY curve was unexpectedly and strongly shifted to higher excitation voltages with two plateaus appearing. Those plateaus, indicating clearly the presence of three isobaric compounds, were taken as reliable indicators to perform GPCP at carefully selected excitation voltages in order to selectively fragment one compound after the other. In this way, CID mass spectra of each compound were correctly recovered, both in terms of fragment ion peaks and in terms of relative intensities, from energy-resolved MSn spectra of the three compounds mixture. This feature enables their unequivocal structural analysis as if samples were free from isobaric interferences. In this paper, we also discuss the possibility for recovering SY curves for pure compounds directly from the mixture. Clearly, in this case, the higher He trapping gas pressure made it possible to use the SY technique, for the first time, for the structural analysis in the case of mixtures of three isobaric compounds. This observation, quite unexpected, demonstrates that the trapping gas pressure is of paramount importance although it is usually not considered in energy-resolved MS for structural and/or quantitative analysis.

De novo structural determination of mannose oligosaccharides by using a logically derived sequence for tandem mass spectrometry

Carbohydrates play important roles in biological recognition processes. However, determining the structures of carbohydrates remains challenging because of their complexity. A simple tandem mass spectrometry-based method for determining the structure of underivatized mannose tetrasaccharides was demonstrated. This method employed the multistage low-energy collision-induced dissociation (CID) of sodium adducts in an ion trap, a logically derived sequence (LODES) from the dissociation mechanism for deciding the sequence of CID, and a specially prepared disaccharide spectrum database. Through this method, the linkages, anomeric configurations, and branch locations of carbohydrates could be determined without the prior assumption of possible structures. We validated this method by blind test of all the commercial available mannose tetrasaccharides. We showed that the structure of a given tetrasaccharide can be determined from 928 isomers by using only three to six appropriately selected CID mass spectra according to the proposed procedure. This method is simple and rapid and has the potential to be applied to other hexoses and oligosaccharides larger than tetrasaccharides. The CID procedures can be built in a computer-controlled mass spectrometer for automatic structural determination of underivatized oligosaccharides. Graphical abstract.

Differentiation of isomeric heptylamines by in-source collision-induced dissociation of [M + H]+ ions.

1,3-Dimethylamylamine (1,3-DMAA), 1,4-dimethylamylamine (1,4-DMAA) and 2-aminoheptane are isomeric heptylamines commonly used as stimulants. The fragmentations of their [M + H]+ ions (m/z 116) are almost identical and consist of the formation of a fragment ion C4 H9 + at m/z 57. It is interesting to check if analysis of electrospray ionization (ESI) mass spectra of these three amines enables their differentiation. In order to better understand the fragmentation of isomeric heptylamines, 1-, 3- and 4-aminoheptane were also included in the study. ESI in-source collision-induced dissociation (CID) mass spectra were obtained with a Waters/Micromass ZQ2000 mass spectrometer (single quadrupole-type instrument) at a cone voltage of 12-34 V. The influence of three commonly used solvents on the fragmentation of [M + H]+ ions was tested, namely methanol, methanol/water (1/1) and acetonitrile/water (1/1). Only one quantitative difference was observed in the mass spectra of 1,3-DMAA, 1,4-DMAA and 2-aminoheptane, namely formation of a product ion at m/z 57 from the [M + H]+ ion of 1,3-DMAA is more efficient than the formation of this ion from [M + H]+ ions of 2-aminoheptane and 1,4-DMAA. In-source CID of 3- and 4-aminoheptane consists of (besides the formation of ion C4 H9 + ) the formation of a low-abundance, but clearly seen, fragment ion at m/z 114 (loss of H2 ). Differentiation of 1,3-DMAA from 1,4-DMAA and 2-aminoheptane seems to be possible by in-source CID of their [M + H]+ ions. However, comparison of the mass spectrum obtained for the analysed sample with that of a standard seems to be necessary. Only 3- and 4-aminoheptane may be easily differentiated from the other heptylamines by the presence of fragment ions at m/z 114.

A Tandem Mass Spectrometry Sequence Database Search Method for Identification of O-Fucosylated Proteins by Mass Spectrometry.

Thrombospondin type 1 repeats (TSRs), small adhesive protein domains with a wide range of functions, are usually modified with O-linked fucose, which may be extended to O-fucose-β1,3-glucose. Collision-induced dissociation (CID) spectra of O-fucosylated peptides cannot be sequenced by standard tandem mass spectrometry (MS/MS) sequence database search engines because O-linked glycans are highly labile in the gas phase and are effectively absent from the CID peptide fragment spectra, resulting in a large mass error. Electron transfer dissociation (ETD) preserves O-linked glycans on peptide fragments, but only a subset of tryptic peptides with low m/ z can be reliably sequenced from ETD spectra compared to CID. Accordingly, studies to date that have used MS to identify O-fucosylated TSRs have required manual interpretation of CID mass spectra even when ETD was also employed. In order to facilitate high-throughput, automatic identification of O-fucosylated peptides from CID spectra, we re-engineered the MS/MS sequence database search engine Comet and the MS data analysis suite Trans-Proteomic Pipeline to enable automated sequencing of peptides exhibiting the neutral losses characteristic of labile O-linked glycans. We used our approach to reanalyze published proteomics data from Plasmodium parasites and identified multiple glycoforms of TSR-containing proteins.

Differentiating Positional Isomers of Nucleoside Modifications by Higher-Energy Collisional Dissociation Mass Spectrometry (HCD MS).

The analytical identification of positional isomers (e.g., 3-, N4-, 5-methylcytidine) within the > 160 different post-transcriptional modifications found in RNA can be challenging. Conventional liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) approaches rely on chromatographic separation for accurate identification because the collision-induced dissociation (CID) mass spectra of these isomers nearly exclusively yield identical nucleobase ions (BH2+) from the same molecular ion (MH+). Here, we have explored higher-energy collisional dissociation (HCD) as an alternative fragmentation technique to generate more informative product ions that can be used to differentiate positional isomers. LC-MS/MS of modified nucleosides characterized using HCD led to the creation of structure- and HCD energy-specific fragmentation patterns that generated unique fingerprints, which can be used to identify individual positional isomers even when they cannot be separated chromatographically. While particularly useful for identifying positional isomers, the fingerprinting capabilities enabled by HCD also offer the potential to generate HPLC-independent spectral libraries for the rapid analysis of modified ribonucleosides. Graphical Abstract ᅟ.