Collision-induced Dissociation Tandem Mass Spectra Research Articles

Application of Secondary Electrospray Ionization Coupled with High-Resolution Mass Spectrometry in Chemical Characterization of Thermally Generated Aerosols.

Inhalation as a route for administering drugs and dietary supplements has garnered significant attention over the past decade. We performed real-time analyses of aerosols using secondary electrospray ionization (SESI) technology interfaced with high-resolution mass spectrometry (HRMS), primarily developed for exhaled breath analysis with the goal to detect the main aerosol constituents. Several commercially available inhalation devices containing caffeine, melatonin, cannabidiol, and vitamin B12 were tested. Chemical characterization of the aerosols produced by these devices enabled detection of the main constituents and screening for potential contaminants, byproducts, and impurities in the aerosol. In addition, a programmable syringe pump was connected to the SESI-HRMS system to monitor aerosolized active pharmaceutical ingredients (APIs) such as chloroquine, hydroxychloroquine, and azithromycin. This setup allowed us to detect caffeine, melatonin, hydroxychloroquine, chloroquine, and cannabidiol in the produced aerosols. Azithromycin and vitamin B12 in the aerosols could not be detected; however, our instrument setup enabled the detection of vitamin B12 breakdown products that were generated during the aerosolization process. Positive control was realized by liquid chromatography-HRMS analyses. The compounds detected in the aerosol were confirmed by exact mass measurements of the protonated and/or deprotonated species, as well as their respective collision-induced dissociation tandem mass spectra. These results reveal the potential wide application of this technology for the real-time monitoring of aerosolized active pharmaceutical ingredients that can be administered through the inhalation route.

HPLC-MS identification of acid degradation products of dolutegravir

Dolutegravir is an integrase strand transfer inhibitor used for the treatment of human immuno-deficiency virus infections. The present study was conducted in order to identify degradation products formed in acidic solution upon heating. The structures were assigned based on low resolution collision-induced dissociation tandem mass spectra as well as high resolution higher-energy collisional dissociation tandem mass spectra. The major degradation products resulted from hydrolytic opening of the oxepine ring leading to bis-hydroxy diastereomers (DP2 and DP3) as well as a mono-hydroxy derivative (DP1) as the result of dehydration of the diastereomers. Furthermore, two carboxylic acid derivatives (DP4 and DP5) could be identified, which can be explained as the result of the hydrolysis of the exocyclic amide bond of dolutegravir and DP1, respectively. During the fragmentation process of dolutegravir and its degradation products DP1 to DP3 a formal addition of oxygen resulting in the respective carboxylic acid fragments was detected. This could be evidenced based on high resolution masses of the fragments as well as the comparison of the MS/MS spectra of the fragments with the spectra of the carboxylic acids DP4 and DP5.

FPTMS: Frequency-based approach to identify the peptide from the low-energy collision-induced dissociation tandem mass spectra

The database search method is a widely accepted method to assign a peptide to the tandem mass spectra. In this study, a new flexible method- FPTMS is introduced to interpret the tandem mass spectra with the known peptide sequences in a protein database. Here the frequency of occurrence of fragment ion peaks extracted from the extensive spectral library is used to predict the theoretical tandem mass spectra of the peptides. The dot product scoring and windowed-xcorr scoring methods were implemented to score the experimental spectrum against the theoretical peptide spectra. Windowed-xcorr is introduced to tackle the mass errors and the cleavage position of the fragmentation process. The new method with windowed-xcorr shows an improved identification rate compared to the existing search engines Crux-Tide and X!Tandem at 1% False Discovery Rate (FDR) for the dataset considered in this study. SignificanceIdentifying or sequencing of the peptide from tandem mass spectra is an important application in mass spectrometry-based proteomics. Collision-induced dissociation (CID) fragmentation spectra have been widely used to develop a peptide identification algorithm using database search strategy. CID fragmentation behavior is a complex process and found to have dependency on the sequences of peptide, charge state, and residue content. The inclusion of more features of peptide fragmentation behavior and adaptable scoring algorithm improves the efficiency of the peptide identification algorithm.

Quantification of the Effect of Citrulline and Homocitrulline Residues on the Collision-Induced Fragmentation of Peptides.

Posttranslational modifications of proteins like citrullination and carbamylation are associated with several diseases. Detailed analytical characterization of citrullinated and carbamylated proteins or peptides could be difficult due to the low concentration of the analytes in complex biological samples. High structural similarity and chemical behavior of citrullinated and carbamylated residues also pose a challenge. We previously reported the “citrulline effect” phenomenon that is manifested in the generation of intense y type ions originating from Cit-Zzz amide bond scissions in collision-induced dissociation tandem mass spectra of citrullinated tryptic peptides. In this study, we created a rigorous tryptic-like model system of both citrulline and homocitrulline-containing peptides that included appropriate and well-defined controls and fragment analogues to quantify the citrulline effect and investigate whether there is an effect for homocitrulline residues as well. Our results show that citrulline residues significantly increased fragmentation at their C-terminus relatively independent of the identity of the following amino acid. In comparison, homocitrulline residues displayed inconclusive results at the same energies. However, the strength of effects was dependent on collision energy and the position of citrulline and homocitrulline in the sequences. As newer software algorithms tend to observe structure–intensity relationships during annotation, this finding increases reliable identification of modified proteins/peptides.

Photodissociative Cross-Linking of Non-covalent Peptide-Peptide Ion Complexes in the Gas Phase.

We report a gas-phase UV photodissociation study investigating non-covalent interactions between neutral hydrophobic pentapeptides and peptide ions incorporating a diazirine-tagged photoleucine residue. Phenylalanine (Phe) and proline (Pro) were chosen as the conformation-affecting residues that were incorporated into a small library of neutral pentapeptides. Gas-phase ion-molecule complexes of these peptides with photo-labeled pentapeptides were subjected to photodissociation. Selective photocleavage of the diazirine ring at 355nm formed short-lived carbene intermediates that underwent cross-linking by insertion into H-X bonds of the target peptide. The cross-link positions were established from collision-induced dissociation tandem mass spectra (CID-MS3) providing sequence information on the covalent adducts. Effects of the amino acid residue (Pro or Phe) and its position in the target peptide sequence were evaluated. For proline-containing peptides, interactions resulting in covalent cross-links in these complexes became more prominent as proline was moved towards the C-terminus of the target peptide sequence. The photocross-linking yields of phenylalanine-containing peptides depended on the position of both phenylalanine and photoleucine. Density functional theory calculations were used to assign structures of low-energy conformers of the (GLPMG + GLL*LK + H)+ complex. Born-Oppenheimer molecular dynamics trajectory calculations were used to capture the thermal motion in the complexes within 100ps and determine close contacts between the incipient carbene and the H-X bonds in the target peptide. This provided atomic-level resolution of potential cross-links that aided spectra interpretation and was in agreement with experimental data. Graphical Abstract ᅟ.

Determination of oxidized phosphatidylcholines by hydrophilic interaction liquid chromatography coupled to Fourier transform mass spectrometry.

A novel liquid chromatography-mass spectrometry (LC-MS) approach for analysis of oxidized phosphatidylcholines by an Orbitrap Fourier Transform mass spectrometer in positive electrospray ionization (ESI) coupled to hydrophilic interaction liquid chromatography (HILIC) was developed. This method depends on three selectivity criteria for separation and identification: retention time, exact mass at a resolution of 100,000 and collision induced dissociation (CID) fragment spectra in a linear ion trap. The process of chromatography development showed the best separation properties with a silica-based Kinetex column. This type of chromatography was able to separate all major lipid classes expected in mammalian samples, yielding increased sensitivity of oxidized phosphatidylcholines over reversed phase chromatography. Identification of molecular species was achieved by exact mass on intact molecular ions and CID tandem mass spectra containing characteristic fragments. Due to a lack of commercially available standards, method development was performed with copper induced oxidation products of palmitoyl-arachidonoyl-phosphatidylcholine, which resulted in a plethora of lipid species oxidized at the arachidonoyl moiety. Validation of the method was done with copper oxidized human low-density lipoprotein (LDL) prepared by ultracentrifugation. In these LDL samples we could identify 46 oxidized molecular phosphatidylcholine species out of 99 possible candidates.

Tandem time-of-flight mass spectrometry with a curved field reflectron.

The unique focusing properties of the curved-field reflectron provide a simple solution to the problem of compensating for the broad range of energies of product ions produced postsource in a time-of-flight mass spectrometer. This has been shown previously for the technique known as postsource decay, but in this report we demonstrate its use for tandem time-of-flight mass spectrometry using a high-performance MALDI time-of-flight instrument modified by the addition of a collision chamber to enable the recording of mass-selected product ions formed by collision-induced dissociation (CID). In particular, the curved-field reflectron enables the use of the full 20-keV kinetic energy provided by the ion source extraction voltage as the collision energy in the laboratory frame and obviates the need to reaccelerate the product ions, using a second "source" or "lift" cell. Results are presented for the collision-induced dissociation of fullerenes over a range of collision gas pressures and precursor ion attenuation. In addition, CID tandem mass spectra are obtained for several peptides.

Electrospray ionization mass spectrometry of 1-phenyl-3-methyl-5-pyrazolone derivatives of neutral and N-acetylated oligosaccharides.

Derivatization using 1-phenyl-3-methyl-5-pyrazolone (PMP) was selected among a number of reported methods for labeling carbohydrates, since it gives a quantitative yield, proceeds through a rapid reaction and involves a simple clean-up procedure. Moreover, PMP derivatives provide an increase in sensitivity with ultraviolet and mass spectrometric detection relative to native neutral sugars. Sensitivity studies were carried out using a standard oligosaccharide, tetraglucose. One of the aims of these studies was to determine the minimum amounts of PMP-tetraglucose necessary to generate informative full-scan electrospray ionization (ESI) mass spectra and collision-induced dissociation tandem mass spectra. Another aim was to characterize the fragmentation pattern of PMP derivatives. Quantitative and qualitative studies were also carried out with a typical N-linked oligosaccharide obtained commercially. The PMP-labeled compound underwent directed cleavages which produced fragments containing the reducing end. The native N-linked sugar yielded fragments corresponding to cleavages from both ends of the molecule. Under the same ESI conditions, the N-linked oligosaccharide exhibited more lability, or tendency to fragment, than neutral tetraglucose, in both the derivatized and native forms. Also, PMP labeling was shown to enhance sensitivity in the case of a neutral oligosaccharide, i.e. tetraglucose, whereas the labeling of an N-acetylated oligosaccharide, NGA3, did not yield a noticeable improvement in sensitivity.

Mass spectrometric analysis of oxidized tryptophan

Oxindolylalanine and oxindolylalanine-containing peptides were prepared by treatment of tryptophan and tryptophan-containing peptides with mixtures of dimethyl sulfoxide and hydrochloric acid in acetic acid (DMSO–HCl–HAc). The reaction between tryptophan and DMSO–HCl–HAc was monitored by fast atom bombardment mass spectrometry (FAB-MS) and the proposed chlorotryptophan intermediate in the reaction was observed. Almost complete conversion of tryptophan to oxindolylalanine was obtained in reaction mixtures containing 3.75 M HCl when the reaction was performed in an open tube. A higher HCl concentration (5.5 M) and a closed reaction tube promoted the formation of by-products, such as dioxindolylalanine and 3-chlorooxindolylalanine. Extensive hydrolysis C-terminal of tryptophan was observed when tryptophan-containing peptides were treated with DMSO–HCl–HAc containing 5.5 M HCl, during which the tryptophan residue was modified to dioxindolylalanine lactone. Hydrolysis was not observed in mixtures containing 3.75 M HCl. The presence of oxindolylalanine in peptides could be demonstrated by characteristic peaks in FAB collision-induced dissociation tandem mass spectra. © 1998 John Wiley & Sons, Ltd.

Investigation of beam-induced reactions occurring under fast-atom bombardment conditions between triethanolamine and various phospholipids

The generation of adduct ions when using a triethanolamine (TEA) matrix under conditions of fast-atom bombardment has been investigated. In particular, phospholipids containing a free amino group react in the TEA matrix to form abundant adduct ions 26 and 42 mass units higher than the analyte pseudomolecular ions. The identity of these adduct ions was investigated using specific phosphatidylethanolamines and phosphatidylserine as well as 2-aminoethyldihydrogenphosphate and 2-aminoethylmonomethylphosphate as model compounds. Evidence obtained from positive- and negative-ion high-energy collision-induced dissociation tandem mass spectra, high resolution mass spectra, deuterium labelling and experiments using diethanolamine/TEA mixtures revealed that the intensities of the adduct ions are dependent on the percentage of TEA in the matrix and on the polarity of the analyte. The nature of the adduct ions is best reflected by a structure in which a hydrogen atom of the free amino group is substituted by a vinyl (+26) or by an acetyl (+42) group. A mechanism for the adduct ion formation is proposed. © 1997 John Wiley & Sons, Ltd.

Microheterogeneity characterization of a paracelsin mixture from Trichoderma reesei using high-energy collision-induced dissociation tandem mass spectrometry.

The microheterogeneity of the paracelsin mixture broth of Trichoderma reesei was analysed using mass spectrometric methods, in particular high-energy collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). Based on the liquid secondary ion mass spectrum of the mixture, there are three main components, with molecular masses M and (M +/- 14), together with two minor components of molecular weight (M +/- 28). The high-energy CID tandem mass spectra of both the protonated and sodiated molecules yielded abundant and characteristic fragment ions, but of very different types. It was found that a paracelsin peptaibol in a mixture could be successfully sequenced based on the tandem mass spectra of its protonated and sodiated molecules or, alternatively, on the tandem mass spectra of its y7 and b13 fragment ions. A terminology for indicating these sequential peptide fragments is proposed. To determine the sequence of new analogues, tandem mass spectra of the y7, (y7 +/- 14), b13, (b13 +/- 14) and (MH +/- 14) positive ions were also taken. Based on these experiments, four new paracelsin components (PA-F, PA-G, PA-H and PA-I) were sequenced successfully. The microheterogeneity of the mixture was found to be more pronounced than had been assumed previously. In these new analogues, besides positions 6 and 9, position 17 is also involved in the exchange. MS/MS studies on minor fragment ions, such as (b13-28) and (b8-14) show further microheterogeneity at positions 3, 5, 10 and 12, which increase the number of possible minor components.

Sulphated mucin oligosaccharides from porcine small intestine analysed by four-sector tandem mass spectrometry.

The fraction of sulphated oligosaccharide alditols isolated from mucin glycopeptides of porcine small intestine 'insoluble' mucin complex was analysed by negative-ion fast atom bombardment (FAB) tandem mass spectrometry. Collision-induced dissociation (CID) tandem mass spectra of native and peracetylated species were compared with standards of sulphated monosaccharides. The tandem mass spectra revealed structural information of the carbohydrate sequence and sulphate position. Negative-ion FAB ionization of the peracetylated sulphated oligo-saccharide alditols was at least three times more sensitive than that of the native sulphated oligosaccharide alditols, as revealed by comparing the signal-to-noise ratios, and allowed the detection of eleven compared with six pseudo-molecular ions. Fourteen structures were determined from the CID tandem mass spectra obtained. The main sulphation site was C-6 of an N-acetylglucosamine 6-linked to the N-acetylgalactosaminitol. C-3 of the N-acetylgalactosaminitol could be unsubstituted or extended with a series of up to three monosaccharide residues including blood group H determinants and blood group A determinants. Also, the sulphated N-acetylglucosamine could be further extended. The most abundant structure was a monosulphated trisaccharide with the sequence Gal-->3(SO3-->6GlcNAc-->6)GalNAcol. The sulphation at C-6 of N-acetylglucosamine seems to be a common feature for O-linked oligosaccharides, and has been described both for skeletal keratan sulphates and respiratory mucin oligosaccharides. Low-abundance ions were also detected from oligosaccharides with sulphation at C-3 of an amino sugar residue. This seems to be a novel sulphation site for mucin oligosaccharides.

Comparison of charged derivatives for high energy collision-induced dissociation tandem mass spectrometry

Fixed-charge derivatives have been used to direct the fragmentation pattern of high energy collision-induced dissociation tandem mass spectra for several years. It has been noted that a fixed-charge placed at a terminus of a peptide will simplify the pattern of fragment ions that are produced in collision-induced dissociation. Trimethylammoniumacetyl, dimethyloctylammoniumacetyl, and triphenylphosphoniumethyl derivatives have been cited in the literature for this purpose and many other structures are possible. This work compares the cited derivatives as well as some new structures. The criteria used include the ease of synthesis and purification of the derivatized peptide and the effects of the derivative on the peptide sequence fragment ion yield and ionization efficiency. The trimethylammoniumacetyl derivative is concluded to be the most practical for general use, whereas the dimethyloctylammoniumacetyl derivative is found to be desirable for use with hydrophilic peptides.

Characteristics of high energy collision-induced dissociation tandem mass spectra of polycyclic aromatic hydrocarbon diolepoxide adducted peptides

Polycyclic aromatic hydrocarbon (PAH) diolepoxides are known to covalently modify serum albumin and hemoglobin. Mass spectrometric techniques have proven quite useful in the characterization of the site of adduction on these proteins. To facilitate the study of PAH diolepoxide adducted peptides, model peptide adducts of benzo[a]pyrene- rans-7,8-dihydrodiol-9,10-epoxide [ anti-B aP(9,10)DE] and benzo[ a]anthracene- trans-8,9-dihydrodiol-10,11-epoxide [anti-B aA(10,11)DE] have been synthesized for the purpose of studying their high energy collision-induced dissociation tandem mass spectra. These spectra are dominated by ions produced from cleavage of the peptide—adduct bond with charge retention by the adducting moiety. Such ions allow for the facile identification of adducted peptides in a mixture by use of neutral loss scans. The peptide sequence can still be deduced from the data in most cases, and the site of adduction can be determined. For those peptide—adducts in which this is not possible, a charged derivative placed at the N-terminus simplifies the peptide fragmentation pattern and makes the spectrum more interpretable.

Fast atom bombardment and tandem mass spectrometry at high and low collision energy for the sequence analysis of low to middle-mass peptides.

The information derived from the high and low-energy collision-induced dissociation tandem mass spectra of some low to middle-mass peptides is presented, in order to show the feasibility of peptide sequencing by fast atom bombardment and tandem mass spectrometry.