Collision-induced Dissociation Spectra Research Articles

Collision-Induced Dissociations of Linear Hexose and Disaccharides with Linear Hexose at the Reducing End.

Characterization of carbohydrate structures using mass spectrometry is a challenging task. Understanding the dissociation mechanisms of carbohydrates in the gas phase is crucial for characterizing these structures through tandem mass spectrometry. In this study, we investigated the collision-induced dissociation (CID) of glucose, galactose, and mannose in their linear forms, as well as the linear forms of hexose at the reducing end of 1-6 linked disaccharides, using quantum chemistry calculations and tandem mass spectrometry. Our results suggest that the dehydration reaction in linear structures is unlikely to occur due to the significantly high reaction barrier compared to those of C═O migration and C-C bond cleavage. We demonstrate that the different intensities of the cross-ring fragments observed in the CID spectra can be explained by the different transition state energies of C═O migration and C2-C3, C3-C4, and C4-C5 bond cleavages, and the branching ratios of the cross-ring fragments are significantly different between glucose and galactose. The application of the cross-ring fragments to oligosaccharides reveals that the stereoisomers of glucose and galactose in oligosaccharides can be differentiated based on the relative intensities of the cross-ring fragments produced by the C2-C3 bond cleavage and C3-C4 bond cleavage, a differentiation that cannot be achieved by conventional tandem mass spectrometry.

Utilizing ESI-MS/MS and ion mobility spectrometry for structural characterization and isomer differentiation of 1, 2-unsaturated (1-4) linked disaccharide derivatives and their 2-C-functionalized analogues.

The 1, 2-unsaturated derivatives of (1-4) linked disaccharides serve as versatile building blocks for synthesizing biologically active compounds. Distinguishing between four pairs of stereoisomers in mixtures presents a challenging task. In this study, disaccharide derivatives are analyzed as alkali metal adducts using electrospray ionization tandem mass spectrometry (ESI-MS/MS), both as individual compounds and in mixtures by ion mobility mass spectrometry (IMS). Electrospray ionization (ESI) in combination with tandem mass spectrometry (MS/MS) in positive mode was employed to differentiate lithium adduct ions of hexa-acetyl/hexa-benzyl-D-lactals, and hexa-acetyl/hexa-benzyl-D-maltals, along with their corresponding 2-C-branched malonates. The high resolving power of trapped ion mobility spectrometry (TIMS) with the imeX™ functionality rapidly identified different metal adducts (Li, Na, Cs) as individual isomers and separated mixtures of stereoisomers. The measured collisional cross section (CCS) values were analyzed in relation to predicted CCS values. MS/MS spectra of the [M + Li]+ ions of glycal disaccharide analogues exhibited typical cross-ring and glycosidic bond cleavages. Collision-induced dissociation (CID) spectra provided insights into their fragmentation behavior, allowing differentiation of (1-4) linked disaccharides. TIMS technology delivered adjustable ion mobility resolution for suitable separation of the four sets of stereo isomeric compounds. However, accurately predicting CCS values to differentiate between respective isomeric pairs using the SigmaCCS program for sodium adducts is only partially achievable. ESI CID spectra of [M + Li]+ adduct ions for individual glycal disaccharide analogues facilitate the discrimination between alpha and beta (1-4) linked unsaturated disaccharides and their 2-C-branched analogues. Through optimized experimental conditions, complete baseline ion mobility separation of stereoisomer pairs of the Cs adducts is achieved.

Analysis of Volatile Esters and Alkanoic Acids by an Atmospheric Pressure Corona Discharge Ionization Collision-Induced Dissociation Mass Spectrometry in Positive-Ion Mode: Ionization, Fragmentation Patterns, and Discrimination between Isomeric Compounds.

The ionization and fragmentation patterns of 24 compounds with elemental composition of CmH2mO2, including isomeric esters and alkanoic acids, were investigated by atmospheric pressure corona discharge ionization collision-induced dissociation (CID) mass spectrometry in the positive-ion mode. All compounds were ionized as protonated molecules and NH4+ adducts. In addition, fragment ions were observed in mass spectra of esters other than methyl esters, which are owing to the dissociation of the alkyl group in the alcohol side from the protonated molecules. In CID spectra, protonated alkanoic acids/methyl esters split off H2O/CH3OH and CO or the alkyl group in the acid side, depending on the carbon chain length. Protonated esters (other than methyl esters) mainly fragmented the alkyl group in the alcohol side. These general rules on ionization and fragmentation patterns can provide relevant information on the discrimination of isomers.

Charge-State-Dependent Collision-Induced Dissociation Behaviors of RNA Oligonucleotides via High-Resolution Mass Spectrometry.

Mass spectrometry (MS)-based analysis of RNA oligonucleotides (oligos) plays an increasingly important role in the development of RNA therapeutics and epitranscriptomics research. However, MS fragmentation behaviors of RNA oligomers are understood insufficiently. Herein, we characterized the negative-ion-mode fragmentation behaviors of 26 synthetic RNA oligos containing four to eight nucleotides using collision-induced dissociation (CID) on a high-resolution, accurate-mass instrument. We found that in CID spectra acquired under the normalized collision energy (NCE) of 35%, approximately 70% of the total peak intensity was attributed to sequencing ions (a-B, a, b, c, d, w, x, y, z), around 25% of the peak intensity came from precursor ions that experienced complete or partial loss of a nucleobase in the form of either a neutral or an anion, and the remainder were internal ions and anionic nucleobases. The top five sequencing ions were the y, c, w, a-B, and a ions. Furthermore, we observed that CID fragmentation behaviors of RNA oligos were significantly impacted by their precursor charge. Specifically, when the precursors had a charge from 1- to 5-, the fractional intensity of sequencing ions decreased, while that of precursors that underwent either neutral or charged losses of a nucleobase increased. Additionally, we found that RNA oligos containing 3'-U tended to produce precursors with HNCO and/or NCO- losses, which presumably corresponded to isocyanic acid and cyanate anion, respectively. These findings provide valuable insights for better comprehending the mechanism behind RNA fragmentation by MS/MS, thereby facilitating the future automated identification of RNA oligos based on their CID spectra in a more efficient manner.

RDD-HCD Provides Variable Fragmentation Routes Dictated by Radical Stability.

Radical-directed dissociation (RDD) is a fragmentation technique in which a radical created by selective 213/266 nm photodissociation of a carbon-iodine bond is reisolated and collisionally activated. In previous RDD experiments, collisional activation was effected by ion-trap collision-induced dissociation (CID). Higher-energy collisional dissociation (HCD) differs from CID both in terms of how ions are excited and in the number, type, or abundance of fragments that are observed. In this paper, we explore the use of HCD for activation in RDD experiments. While RDD-CID favors fragments produced from radical-directed pathways such as a/z-ions and side chain losses regardless of the activation energy employed, RDD-HCD spectra vary considerably as a function of activation energy, with lower energies favoring RDD while higher energies favor products resulting from cleavage directed by mobile protons (b/y-ions). RDD-HCD therefore affords more tunable fragmentation based on the HCD energy provided. Importantly, the abundance of radical products decreases as a function of increasing HCD energy, confirming that RDD generally proceeds via lower-energy barriers relative to mobile-proton-driven dissociation. The dominance of b/y-ions at higher energies for RDD-HCD can therefore be explained by the higher survivability of fragments not containing the radical after the initial or subsequent dissociation events. Furthermore, these results confirm previous suspicions that HCD spectra differ from CID spectra due to multiple dissociation events.

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.

Combining trapped ion mobility spectrometry with liquid chromatography and tandem mass spectrometry for analysis of isomeric PDE-5 inhibitor analogs

The detection and identification of phosphodiesterase type 5 enzyme (PDE-5) inhibitors in dietary supplements poses an analytical challenge due to the large number of analogs and isomers currently available and the continued introduction of novel analogs. The use of trapped ion mobility spectrometry (TIMS) in conjunction with liquid chromatography (LC) and electrospray ionization tandem mass spectrometry (MS/MS) was explored for the analysis of two groups of isomeric PDE-5 inhibitor analogs using a 5-minute method. Of the eight compounds studied, six were resolved by a combination of LC and TIMS; the two remaining isomers were distinguished by one or more unique product ions in the MS/MS spectrum. The results revealed that separation by LC corresponded to differences in substitution on the piperazine moiety of the PDE-5 inhibitors, while separation by TIMS corresponded to the position of a nitrogen atom in the fused ring region of the molecules. Samples prepared by spiking mixtures of varying amounts of the Group 2 isomers into a representative dietary supplement matrix were analyzed and concentrations determined from the mobility-adjusted extracted ion chromatograms exhibited relative standard deviations of 6.0 % or less for 17 of 20 measurements and recoveries between 80 % and 120 % for all measurements. Quantitative measurements from a short LC gradient were possible due to the reduced chemical background associated with the TIMS separation of co-eluting matrix compounds, which enabled acquisition of rapid and qualitatively relevant broadband collision induced dissociation spectra that didn’t require precursor ion isolation; the reduced chemical background permits non-targeted detection of novel analogs and eliminates the need for a separate method for quantitative measurement.

Differentiation of tetracyclines and their 4-epimers by mass spectrometry of the alkali metal adduct ions

Tetracyclines (TCs) are a family of broad-spectrum antibiotics. During the manufacturing process or storage, epimerization of tetracyclines could occur, leading to 4-epimers which are nearly inactive. From an analytical point of view, isomers are often difficult to distinguish. Previously, four pairs of TCs (oxytetracycline, tetracycline, doxycycline, chlortetracycline and their respective 4-epimers) were differentiated by mass spectrometry (MS) through protonated ions. However, they do not follow common rules and so it is still quite difficult to differentiate between them. In order to solve this, the four pairs were differentiated in the current study by collision induced dissociation (CID) spectra of the alkali adduct ions, including lithium, sodium and potassium. In the spectra of the sodium adducts, all studied tetracyclines showed a tendency to form [M+Na–NH3]+ ions, while the 4-epimers liked to form [M+Na–NH3–H2O]+ ions. Meanwhile, energy resolved mass spectrometry (ERMS) showed that all four 4-epimers' sodium adducts had the tendency to fragment at higher energy points. In the CID spectra of lithium adducts of TCs, a similar trend was observed for three pairs, except for doxycycline. For potassium adducts, the fragmentation was found to be less discriminative. As was derived from the 3D model, the four pairs all interact with the alkali metal through the dimethyl amino group at the C-4 position. The lithium adduct species also bound through the hydroxyl group at the C-5 position. If the TCs did not have a hydroxyl group at the C-5 position, they bound with the hydroxyl group at the C-6 position. For the same TC, with an increase of the diameter of the metal ion, the loss of H2O decreased gradually. As sodium adduct ions are common during the ionization process, TCs and their 4-epimers could be differentiated rapidly by ERMS of the sodium adduct ions.

Use of group IIB metal ions as charge carriers for collision-induced dissociation of glycopeptide and glycan.

Dissociation of biomolecules by tandem mass spectrometry (MS/MS) generates a variety of fragment ions which provide useful information for the structural characterization of biomolecules. Different fragmentation strategies result in different mass spectra for the same molecule and thus provide distinct features. Charge carriers play important roles in determining the dissociation pathways of the target precursor ions. The use of various transition metals ions as charge carriers of glycopeptide and glycan might provide additional structural information and needs to be investigated. A 9.4T SolariX FTICR mass spectrometer was used for collision-induced dissociation (CID) of glycopeptide and glycan. Group IIB metal ions, including Zn2+ , Cd2+ and Hg2+ , were used as charge carriers. Glycopeptide NLTK-M5 G2 and glycan G1F were used as the model systems. For Zn2+ - and Cd2+ -adducted species, cross-ring cleavages, glycosidic cleavages and cleavages along the peptide backbone could be obtained. There is a high degree of similarity in their CID spectra with that of Mg2+ ion-adducted glycopeptide species. For Hg2+ -adducted species, only glycosidic cleavages were observed in high abundance. The formation of doubly-charged ions (M2+ ) and a series of [f-H]+ fragments indicated unique dissociation pathways for Hg2+ -adducted glycopeptide. Zn2+ and Cd2+ -adducted glycopeptide species produced similar dissociation CID spectra, whereas Hg2+ -adducted species produced significantly different CID spectra. Similar CID dissociation features were also observed for Group IIB metal ions adducted glycan species. These results demonstrated that different metal ions might be used to tune the dissociation behaviors of glycopeptides and glycans.

Collision-Induced Dissociation of α-Isomaltose and α-Maltose.

Determination of carbohydrate structures remains a considerable challenge. Collision-induced dissociation (CID) tandem mass spectroscopy (MS/MS) is widely used for carbohydrate structure determination. Structural information derived from MS/MS relies on an understanding of the carbohydrate dissociation mechanism. Among various hexose disaccharides, the major dissociation channels (dehydration, glycosidic bond cleavage, and cross-ring dissociation) of 1→2-, 1→3-, and 1→4-linked disaccharide sodium ion adducts can be explained by the dissociation mechanism derived from hexose monosaccharides. However, 1→6-linked disaccharides, which have low branching ratios for dehydration and glycosidic bond cleavage, cannot be explained by the same dissociation mechanism. In this study, we performed high-level quantum chemistry calculations to examine the CID mechanism of the α-isomaltose sodium ion adduct, a 1→6-linked glucose disaccharide. For comparison, we examined the CID dissociation mechanism of the α-maltose sodium ion adduct, a 1→4-linked glucose-disaccharide. Calculations revealed that although α-isomaltose and α-maltose had similar dissociation mechanisms, energy differences between the lowest transition states of various dissociation channels led to different CID fragmentation patterns. The dissociation barriers of dehydration and glycosidic bond cleavage were similar for the two disaccharides, but the cross-ring dissociation, which has the lowest dissociation barrier, exhibited differences in barriers between the disaccharides. The cross-ring dissociation barrier for α-maltose was only slightly lower than those of dehydration and glycosidic bond cleavage. However, the cross-ring dissociation barrier for α-isomaltose was substantially lower than those of dehydration and glycosidic bond cleavage. In addition, most of the α-isomaltose conformers that led to dehydration also led to cross-ring dissociation, resulting in suppression of dehydration by cross-ring dissociation. The findings can explain the low branching ratios for dehydration and glycosidic bond cleavage observed in α-isomaltose CID spectra.

Method optimization of the simultaneous detection of B12 congeners leading to the detection of a novel isomer of hydroxycobalamin in seawater.

More than half of surveyed microalgae and over 90% of harmful algae have an obligate requirement for vitamin B12 , but methods for directly measuring dissolved B12 in seawater are scarce due to low concentrations and rapid light-induced hydrolysis. We present a method to detect and measure the four main congeners of vitamin B12 dissolved in seawater. The method includes solid-phase extraction, separation by ultrahigh-performance liquid chromatography and detection by triple-quadrupole tandem mass spectrometry utilizing an electrospray ion source. This method was applied to coastal field samples collected in the German Bay, Baltic Sea and the Danish Limfjord system. The total dissolved B12 pool ranged between 0.5 and 2.1pM. Under ambient conditions methyl-B12 and adenosyl-B12 were nearly fully hydrolyzed to hydroxy-B12 in less than 1 h. Hydroxy-B12 and a novel, corresponding isomer were the main forms of B12 found at all field sites. This isomer eluted well after the OH-B12 peak and was also detected in commercially available OH-B12 . Both compounds showed very high similarity in their collision-induced dissociation spectra. The high instability of the biologically active forms of Me-B12 and Ado-B12 towards hydrolysis was shown, highlighting the importance of reducing the duration of the extraction protocol. In addition, the vitamin B12 pool in the study area was mostly comprised of a previously undescribed isomer of OH-B12 . Further studies into the structure of this isomer and its bioavailability are needed.

The Good, the Bad, and the Ugly Memories of Carbohydrate Fragments in Collision-Induced Dissociation.

Collision-induced dissociation (CID) tandem mass spectrometry is commonly used for carbohydrate structural determinations. In the CID tandem mass spectrometry approach, carbohydrates are dissociated into fragments, and this is followed by the structural identification of fragments through subsequent CID. The success of the structural analysis depends on the structural correlation of fragments before and after dissociation, that is, structural memory of fragments. Fragments that completely lose the memory of their original structures cannot be used for structural analysis. By contrast, fragments with extremely strong correlations between the structures before and after fragmentation retain the information on their original structures as well as have memories of their precursors' entire structures. The CID spectra of these fragments depend on their own structures and on the remaining parts of the precursor structures, making structural analysis impractical. For effective structural analysis, the fragments produced from a precursor must have good structural memory, meaning that the structures of these fragments retain their original structure, and they must not be strongly affected by the remaining parts of the precursors. In this study, we found that most of the carbohydrate fragments produced by low-energy CID have good memory in terms of linkage position and anomericity. Fragments with ugly memory, where fragment structures change with the remaining parts of the precursors, can be attributed to C ion formation in a linear form. Fragments with ugly memory can be changed to have good memory by preventing linear C ion generation by using an alternative CID sequence, or the fragments of ugly memory can become useful in structural analysis when the contribution of linear C ions in fragmentation patterns is understood.

The impact of the complexing agent on the sensitivity of collision-induced dissociation spectra to fatty acid position for a set of XYZ-type triglycerides.

The development of an automated platform for the positional analysis of triglycerides (TAGs) based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) continues to be pursued. This work evaluates the positional sensitivities of the collision-induced dissociation (CID) spectra of a representative set of XYZ triglycerides using sodium, lithium, and ammonium salts as complexing agents. A set of triglycerides were synthesized and analyzed via ESI-MS/MS using an ion trap mass spectrometer. Using three different complexing agents, the product ion spectra of the corresponding precursor ions for twelve XYZ TAGs were collected, where X, Y, and Z represent C16:0 , C18:1(c-9) , C18:2(cc-9,12) , and C20:4(cccc-5,8,11,14) fatty acid chains. These data were then used to prepare ternary plots for four positional isomer systems to evaluate the positional sensitivity differences among the three different complexing agents. The positional sensitivities for each of the four positional isomer systems were robust for the sodium and lithium adducts. The CID data for the sodium and lithium TAGs demonstrated an unfavorable loss of the fatty acid in the center position and showed a higher sensitivity to fatty acid position, when compared with the CID data for ammonium adducts, especially for the arachidonic acid containing triglycerides. The data shows that that the relative abundances of the DAG product ions for the XYZ-type TAGs when using sodium and lithium complexing agent adducts are sensitive to fatty acid position and are consistent for the diverse array of TAGs studied in this work. This suggests that using sodium or lithium as the complexing agent may be advantageous for the development of an automated platform for the positional analysis of complex TAG mixtures based on ESI-MS/MS.

Identification of N-glycans with GalNAc-containing antennae from recombinant HIV trimers by ion mobility and negative ion fragmentation

Negative ion collision-induced dissociation (CID) of underivatized N-glycans has proved to be a simple, yet powerful method for their structural determination. Recently, we have identified a series of such structures with GalNAc rather than the more common galactose capping the antennae of hybrid and complex glycans. As part of a series of publications describing the negative ion fragmentation of different types of N-glycan, this paper describes their CID spectra and estimated nitrogen cross sections recorded by travelling wave ion mobility mass spectrometry (TWIMS). Most of the glycans were derived from the recombinant glycoproteins gp120 and gp41 from the human immunodeficiency virus (HIV), recombinantly derived from human embryonic kidney (HEK 293T) cells. Twenty-six GalNAc-capped hybrid and complex N-glycans were identified by a combination of TWIMS, negative ion CID, and exoglycosidase digestions. They were present as the neutral glycans and their sulfated and α2→3-linked sialylated analogues. Overall, negative ion fragmentation of glycans generates fingerprints that reveal their structural identity.

A unique sequential C-S or N-S inductive cleavage and retro-Diels-Alder fragmentation mechanism for alkylsulfonyl piperidine- and piperazine-containing compounds.

BI 605906 undergoes a collision-induced dissociation (CID) fragmentation resulting in the loss of methylsulfinic acid and butadiene to produce a corresponding imine. The fragmentation is hypothesized to occur via inductive cleavage of the C-S bond, generating a six-membered cyclic ene, followed by the retro-Diels-Alder (RDA) reaction. The aim of this study was to provide mechanistic evidence for the proposed fragmentation by investigating the CID spectra of BI 605906 and other alkylsulfonyl piperidine- and piperazine-containing compounds. The positive electrospray ionization tandem mass spectrometric (ESI+ -MS/MS) fragmentations of BI 605906, D9 -BI 605906, GK02935, GK02942, ketoconazole, terazosin, and homopiperazine were investigated. Additionally, incubations of BI 605906 and GK02942 in human liver microsomes (HLM) preparations were conducted. Metabolite identification experiments were performed following these incubations to investigate corresponding in vitro metabolism. BI 605906, D9 -BI 605906, GK02935, and GK02942 demonstrated the same fragmentation pattern by generating a respective imine ion, supporting the hypothesized inductive cleavage and subsequent RDA mechanism. Ketoconazole and terazosin, which contain either an N-acetyl or tetrahydrofuranyl piperazine group, respectively, did not demonstrate this mechanism, notably because they do not have the alkylsulfonyl moiety as a good leaving group. Although homopiperazine contains an arylsulfonyl diazepane group, and the initial step produced an unsaturated diazepane ring, the subsequent RDA reaction was unable to proceed due to the absence of a six-membered cyclic ene intermediate. Additionally, we identified oxidative metabolites of BI 605906 and GK02942 in HLM incubations utilizing the proposed fragmentation pattern. In the mass spectrometer, compounds containing alkylsulfonyl piperidine or piperazine groups can undergo inductive cleavage, leading to a six-membered cyclic ene intermediate. This intermediate will then form a corresponding imine ion via the RDA reaction. A practical application of this work is to utilize this fragmentation for elucidating structures of metabolites arising from parent compounds containing alkylsulfonyl piperidine or piperazine moieties.

From QCEIMS to QCxMS: A Tool to Routinely Calculate CID Mass Spectra Using Molecular Dynamics.

Mass spectrometry (MS) is a powerful tool in chemical research and substance identification. For the computational modeling of electron ionization MS, we have developed the quantum-chemical electron ionization mass spectra (QCEIMS) program. Here, we present an extension of QCEIMS to calculate collision-induced dissociation (CID) spectra. The more general applicability is accounted for by the new name QCxMS, where "x" refers to EI or CID. To this end, fragmentation and rearrangement reactions are computed "on-the-fly" in Born-Oppenheimer molecular dynamics (MD) simulations with the semiempirical GFN2-xTB Hamiltonian, which provides an efficient quantum mechanical description of all elements up to Z = 86 (Rn). Through the explicit modeling of multicollision processes between precursor ions and neutral gas atoms as well as temperature-induced decomposition reactions, QCxMS provides detailed insight into the collision kinetics and fragmentation pathways. In combination with the CREST program to determine the preferential protonation sites, QCxMS becomes the first standalone MD-based program that can predict mass spectra based solely on molecular structures as input. We demonstrate this for six organic molecules with masses ranging from 159 to 296 Da, for which QCxMS yields CID spectra in reasonable agreement with experiments.

Statistical Characterization of the Multi-Charged Fragment Ions in the CID and HCD Spectrum

Collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) are the widely used fragmentation technique in mass spectrometry-based proteomics studies. Understanding the fragmentation pattern from the tandem mass spectra using statistical methods helps to implement efficient spectrum analysis algorithms. The study characterizes the frequency of occurrence of multi-charged fragment ions and their neutral loss events of doubly and triply charged peptides in the CID and HCD spectrum. The dependency of the length of the fragment ion on the occurrence of multi-charged fragment ion is characterized here. Study shows that the singly charged fragment ions are generally dominated in the doubly charged peptide spectrum. However, as the length of the product ion increases, the frequency of occurrence of charge 2 fragment ions increases. The y- ions have more tendencies to generate charge 2 fragment ions than b- ions, both in CID and HCD spectrum. The frequency of occurrence of charge 2 fragment ion peaks is prominent upon the dissociation of the triply charged peptides. For triply charged peptides, product ion of higher length occurred in multiple charge states in CID spectrum. The neutral loss peaks mostly exist in charge 2 states in the triply charged peptide spectrum. The b-ions peaks are observed in much less frequency than y-ions in HCD spectrum as the length of the fragment increases. Isotopic peaks are occurred in charge 2 state both in doubly and triply charged peptide’s HCD spectrum.

Characterization of Fatty Acyl Modifications in Phosphatidylcholines and Lysophosphatidylcholines via Radical-Directed Dissociation.

Phosphatidylcholines (PCs) are the major structural components of the plasma membrane of mammalian cells, while lysophosphatidylcholines (LPCs) are critical intermediates in lipid remodeling. Conventional tandem mass spectrometric (MSn) methods via collision-induced dissociation (CID) are blind to intrachain modifications such as the location of the carbon-carbon double bond (C═C) and methyl branching point. In this study, we demonstrate that almost complete structural information can be inferred from a single MS2 CID spectrum of the bicarbonate anion adducts of PC or LPC ([M + HCO3]-), including the identity of the headgroup, composition of fatty acyl chains, their sn-positions, the location of C═C, and the point of methyl branching in fatty acyls. We have integrated this MS2 CID method onto liquid chromatography for the analysis LPCs in human plasma, revealing the existence of multiple sn-isomers, branched chain isomers, and C═C location isomers of LPC.

Comparison of in-source collision-induced dissociation and beam-type collision-induced dissociation of emerging synthetic drugs using a high-resolution quadrupole time-of-flight mass spectrometer.

In-source collision-induced dissociation (CID) is commonly used with single-stage high-resolution mass spectrometers to gather both a molecular formula and structural information through the collisional activation of analytes with residual background gas in the source region of the mass spectrometer. However, unlike tandem mass spectrometry, in-source CID does not involve an isolation step prior to collisional activation leading to a product ion spectrum composed of fragment ions from any analyte present during the activation event. This work provides the first comparison of in-source CID and beam-type CID spectra of emerging synthetic drugs on the same instrument to understand the fragmentation differences between the two techniques and to contribute to the scientific foundations of in-source CID. Electrospray ionization-quadrupole time-of-flight (ESI-Q-TOF) mass spectrometry was used to generate product ion spectra from in-source CID and beam-type CID for a series of well-characterized fentanyl analogs and synthetic cathinones. A comparison between the fragmentation patterns and relative ion abundances for each technique was performed over a range of fragmentor offset voltages for in-source CID and a range of collision energies for beam-type CID. The results indicate that large fragmentor potentials for in-source CID tend to favor higher energy fragmentation pathways that result in both kinetically favored pathways and consecutive neutral losses, both of which produce more abundant lower mass product ions relative to beam-type CID. Although conditions can be found in which in-source CID and beam-type CID provide similar overall spectra, the in-source CID spectra tend to contain elevated noise and additional chemical background peaks relative to beam-type CID.

Full-Range Profiling of tRNA Modifications Using LC-MS/MS at Single-Base Resolution through a Site-Specific Cleavage Strategy.

Transfer RNAs (tRNAs) are the most heavily modified RNA species. Liquid chromatography coupled with mass spectrometry (LC-MS/MS) is a powerful tool for characterizing tRNA modifications, which involves pretreating tRNAs with base-specific ribonucleases to produce smaller oligonucleotides amenable to MS. However, the quality and quantity of products from base-specific digestions are severely impacted by the base composition of tRNAs. This often leads to a loss of sequence information. Here, we report a method for the full-range profiling of tRNA modifications at single-base resolution by combining site-specific RNase H digestion with the LC-MS/MS and RNA-seq techniques. The key steps were designed to generate high-quality products of optimal lengths and ionization properties. A linear correlation between collision energies and the m/z of oligonucleotides significantly improved the information content of collision-induced dissociation (CID) spectra. False positives were eliminated by up to 95% using novel inclusion criteria for collecting a census of modifications. This method is illustrated by the mapping of mouse mitochondrial tRNAHis(GUG) and tRNAVal(UAC), which were hitherto not investigated. The identities and locations of the five species of modifications on these tRNAs were fully characterized. This approach is universally applicable to any tRNA species and provides an experimentally realizable pathway to the de novo sequencing of post-transcriptionally modified tRNAs with high sequence coverage.