Tuning the Internal Energy of Ions Produced by Atmospheric and High-Pressure Electrospray by Modulating the Gas Throughput into the First Vacuum Stage.

In a high-pressure environment, electrospray ionization (ESI) can be achieved without discharge between the emitter and the counter electrode, thus enabling the generation of gas-phase ions from liquid with high surface tension, such as pure water, which requires a high onset voltage for stable ESI. In this study, the ion dissociation during the transferring of ions/charged droplets from a superatmospheric pressure environment to vacuum has been systematically investigated using benzyl ammonium thermometer ions. The ion source pressure did not affect the internal energy distribution of ions, whereas the gas throughput into the first vacuum stage clearly influences the internal energy distribution of the ions. The increase in the gas throughput increased the density of molecules/atoms presented in ion transfer/focusing electrodes located in the first vacuum stage. As a result, the mean free path of ions in the first vacuum stage decreases, and the energy of ions decreases by decreasing the kinetic energy involved in each collision between ions and residue gas. The gas throughput into the first vacuum stage is found to describe the internal energy distribution of ions associated with the local conditions more quantitatively instead of using the measured pressure of the vacuum stage, which is different from the effective local pressure. This study also demonstrated the controlled dissociation of ions using the ion transfer settings of the instrument in combination with ion inlet tubes of different sizes.

Predictive Value of Vancomycin AUC24/MIC Ratio for 30-day Mortality in Patients with Severe or Complicated Methicillin-Resistant Staphylococcus aureus Infections: A Multicenter Retrospective Study.

BACKGROUND: Although vancomycin is typically employed against methicillin-resistant Staphylococcus aureus (MRSA) infections, the optimal ratio of 24-h area under the concentration-time curve to minimum inhibitory concentration (AUC24/MIC) for severe or complicated infections lacks clear guideline recommendations. This study aimed to determine the target AUC24/MIC ratio associated with treatment outcomes of infections treated with vancomycin. METHODS: This retrospective multicenter cohort study included adult patients receiving ≥ 5 days of vancomycin for severe/complicated MRSA infections (e.g., osteoarticular, pulmonary, endocarditis, etc.) between January 2018 and December 2023. The primary outcome was 30-day mortality, with secondary outcomes including clinical success, microbiological eradication, and nephrotoxicity. Receiver operating characteristic (ROC) curve analysis was used to identify the AUC24/MIC cutoff for 30-day mortality. Multivariate regression analysis was used to determine association between AUC24/MIC and outcomes. RESULTS: This study included 82 patients. ROC identified a target AUC24/MIC of ≥ 505 for 30-day mortality. The overall 30-day mortality rate (22.0%) was significantly higher for below average AUC24/MIC cutoff (34.1%) than for above AUC24/MIC cutoff group (9.8%). Multivariate analysis confirmed AUC24/MIC of < 505 as an independent predictor (adjusted odds ratio, 5.001; 95% confidence interval, 1.335-18.75). The clinical success rate differed significantly between below- and above-cutoff groups, whereas microbiological eradication tended to favor the above-cutoff group. The nephrotoxicity rates were comparable between groups. CONCLUSIONS: In treating severe/complicated MRSA infections, vancomycin AUC24/MIC ratio ≥ 505 was independently associated with favorable 30-day mortality. Given the retrospective nature of this study, further prospective studies are essential to confirm the reliability of the target AUC24/MIC ratios.

Discrimination of Aspartic and Isoaspartic Acid Residues in Peptides by Tandem Mass Spectrometry with Hydrogen Attachment Dissociation.

Long-lived proteins undergo chemical modifications that can cause age-related diseases. Among these chemical modifications, isomerization is the most difficult to identify. Isomerization often occurs at the aspartic acid (Asp) residues. In this study, we used tandem mass spectrometry equipped with a newly developed ion activation method, hydrogen attachment dissociation (HAD), to analyze peptides containing Asp isomers. Although HAD preferentially produces [cn + 2H]+ and [zm + 2H]+ via N-Cα bond cleavage, [cn + 58 + 2H]+ and [zm - 58 + 2H]+ originate from the fragmentation of the isoAsp residue. Notably, [cn + 58 + 2H]+ and [zm - 58 + 2H]+ could be used as diagnostic fragment ions for the isoAsp residue because these fragment ions did not originate from the Asp residue. The detailed fragmentation mechanism was investigated by computational analysis using density functional theory. According to the results, hydrogen attachment to the carbonyl oxygen in the isoAsp residue results in the Cα-Cß bond cleavage. The experimental and theoretical joint study indicates that the present method allows us to discriminate Asp and isoAsp residues, including site identification of the isoAsp residue. Moreover, we demonstrated that the molar ratio of peptide isomers in the mixture could be estimated from their fragment ion abundance. Therefore, tandem mass spectrometry with HAD is a useful method for the rapid discrimination and semiquantitative analysis of peptides containing isoAsp residues.

Potential risk factors for early acute kidney injury in patients treated with vancomycin.

PURPOSE: The acute kidney injury (AKI) onset owing to vancomycin (VCM) is reported that depend on the area under the blood concentration-time curve (AUC) and occur comparison early phase (early AKI). This study aimed to investigate the occurrence of early AKI in patients treated with VCM and new indicators to avoid early AKI. METHODS: Adult patients who received VCM treatment for more than 4 days and whose trough values measured at least once on or after day 4 and serum creatinine before day 7 from the initiation of VCM administration between August 2021 and September 2022 at the Yamanashi Prefectural Central Hospital were enrolled. Early AKI (defined as AKI occurring within day 7 from VCM administration) and the association between each AUC (0-24, 24-48, 48-72, 0-48, 24-72, 0-72) were investigated. Furthermore, each AUC cut-off value for early AKI was calculated. RESULT: In total, 164 patients were enrolled; early AKI developed in 21 patients and most frequently occurred on day 4. All stratified AUC were associated with early AKI development. The AUC cut-off values were AUC0-24: 470.8 µg/mL⋅h; AUC24-48: 473.0 µg/mL⋅h; AUC48-72: 489.7 µg/mL⋅h; AUC0-48: 910.2 µg/mL⋅h; AUC24-72: 1039.2 µg/mL⋅h; and AUC0-72: 1544.0 µg/mL⋅h. CONCLUSION: The possibility of AKI development owing to the AUC accumulation of VCM was observed (accumulation toxicity). Concentration control through early-phase blood concentration measurements and a transition to AUC0-48 <910.2 µg/mL⋅h may reduce the early-phase AKI onset.

Evaluation for Ion Heating of H2A-H2B Dimer in Ion Mobility Spectrometry-Mass Spectrometry.

Ion mobility spectrometry-mass spectrometry (IMS-MS) provides m/z values and collision cross sections (CCSs) of gas-phase ions. In our previous study, an intrinsically disordered protein, the H2A-H2B dimer, was analyzed using IMS-MS, resulting in two conformational populations of CCS. Based on experimental and theoretical approaches, this resulted from a structural diversity of intrinsically disordered regions. We predicted that this phenomenon is related to ion heating in the IMS-MS instrument. In this study, to reveal the effect of ion heating from parameters in the IMS-MS instrument on the conformational population of the H2A-H2B dimer, we investigated the arrival time distributions of the H2A-H2B dimer by changing values of three instrumental parameters, namely, cone voltage located in the first vacuum chamber, trap collision energy (trap CE) for tandem mass spectrometry, and trap bias voltage for the entrance of IMS. These results revealed that the two populations observed for the H2A-H2B dimer were due to the trap bias voltage. Furthermore, to evaluate the internal energies of the analyte ions with respect to each parameter, benzylpyridinium derivatives were used as temperature-sensitive probes. The results showed that the trap CE voltage imparts greater internal energy to the ions than the trap bias voltage. In addition, this slight change in the internal energy caused by the trap bias voltage resulted in the structural diversity of the H2A-H2B dimer. Therefore, the trap bias voltage should be set with attention to the properties of the analytes, even if the effect of the trap bias voltage on the internal energy is negligible.

Differences in the internal energies of ions in electrospray ionization mass spectrometers equipped with capillary-skimmer and capillary-RF lens interfaces.

Small metabolites are commonly analyzed using electrospray ionization mass spectrometry (ESI-MS). Although the protonated form of a compound of interest is typically the target ion in ESI-MS, the protonated forms of small metabolites occasionally undergo fragmentation during ion transmission from ambient conditions to vacuum conditions, hindering the unambiguous identification of analyte molecules. To estimate the fragmentation efficiency during ESI processes, the internal energy distribution of the ions (P(E)) must be evaluated. The common approach for the P(E) evaluation is the survival yield method, which uses thermometer ions. In this study, the P(E) of ions produced by an ESI source in a commercial triple quadrupole mass spectrometer equipped with a capillary-skimmer and capillary-RF lens interfaces was evaluated using benzyl ammonium thermometer ions. Furthermore, this study proposes the use of 3-(aminomethyl)indole and related compounds, which have the lowest Eapp values among the reported thermometer ions, to obtain P(E) values of the ions more accurately. Results showed that P(E) strongly depends on whether a capillary-skimmer interface or capillary-RF lens interface was used for ion transport to the vacuum. ESI-MS with a capillary-skimmer interface provided a considerably lower and narrower P(E) of ions than that with a capillary-RF lens interface, thereby producing intact protonated molecules without significant fragmentation of most small metabolites. However, ESI-MS equipped with capillary-RF lens interfaces provided a higher efficiency of ion transmission than ESI-MS equipped with a capillary-skimmer interface, allowing for highly sensitive analysis of metabolites.

Pentafluorobenzylpyridinium: new thermometer ion for characterizing the ions produced by collisional activation during tandem mass spectrometry.

In this study, pentafluorobenzylpyridinium (F5-BnPy+), which has the highest dissociation energy among the reported benzylpyridinium thermometer ion, is proposed to characterize the internal energy distributions of ions activated by higher energy collisional dissociation (HCD) and ion-trap collision-induced dissociation (CID) during tandem mass spectrometry. The dissociation threshold energies of F5-BnPy+ was determined using quantum chemistry calculations at the CCSD(T)/6-311++G(d,p)//M06-2X-D3/6-311++G(d,p) level of theory, and the appearance energies for ion dissociation in HCD and ion-trap CID were estimated using Rice-Ramsperger-Kassel-Marcus theory. The main differences between HCD and ion-trap CID are the collision energies used and the timescales of collisional activation. For both HCD and ion-trap CID, the average internal energy of the ions increased with increasing collision energy. In contrast, the average value for the internal energy of the ions activated by ion-trap CID was lower than that of ions activated by HCD, probably because of the smaller collisional energy and longer activation time of the ion-trap CID experiments. The reported method will aid in the determination of the optimum tandem mass spectrometry parameters for the analysis of small molecules such as metabolites.

Phenyl Sulfate Derivatives: New Thermometer Ions for Characterization of Internal Energy of Negative Ions Produced by Electrospray Ionization.

Although positive thermometer ions are widely used for evaluating the internal energy distribution of gas-phase ions, negative thermometer ions have not yet been proposed. In this study, phenyl sulfate derivatives were tested as thermometer ions to characterize the internal energy distribution of ions produced by electrospray ionization (ESI) in the negative mode because the activation of phenyl sulfate preferentially undergoes SO3 loss, providing a phenolate anion. The dissociation threshold energies for the phenyl sulfate derivatives were determined using quantum chemistry calculations at the CCSD(T)/6-311++G(2df,p)//M06-2X-D3/6-311++G(d,p) level of theory. The values for the appearance energies of the fragment ions of the phenyl sulfate derivatives depend on the dissociation time scale in the experiment; therefore, the dissociation rate constants of the corresponding ions were estimated using the Rice-Ramsperger-Kassel-Marcus theory. The phenyl sulfate derivatives were used as thermometer ions to determine the internal energy distribution of negative ions activated by the in-source collision-induced dissociation (CID) and higher-energy collisional dissociation. Both mean and full width at half-maximum values increased with increasing ion collision energy. In the in-source CID experiments, the internal energy distributions obtained by phenyl sulfate derivatives are similar to that when all voltages are mirrored, and the traditional benzylpyridinium thermometer ions are used. The reported method will aid in determining the optimum voltage for ESI mass spectrometry and the subsequent tandem mass spectrometry of acidic analyte molecules.

Fragmentation of Protonated Histamine and Histidine by Electrospray Ionization In-Source Collision-Induced Dissociation.

Electrospray ionization (ESI) generally produces intact gas-phase ions without extensive fragmentation; however, for histamine and histidine, ESI provides fragment ions through in-source collision-induced dissociation (CID). In this study, we investigated the fragmentation of these compounds both experimentally and using density functional theory calculations. We found that histamine undergoes protonation with subsequent NH3 loss by ESI in-source CID. The corresponding fragmentation mainly produces bicyclo and spiro compounds. In contrast, the ESI in-source CID of protonated histidine preferentially results in H2O loss rather than NH3 loss. However, the corresponding fragment ion is not observed in the ESI mass spectrum of histidine, because it undergoes further CO loss within 100 ps. Consequently, protonated histidine produces a fragment ion arising from a 46 Da loss, which corresponds to the masses of H2O and CO, by ESI in-source CID. The fragment ion yields of histamine and histidine produced by ESI in-source CID are then estimated from the dissociation rate constant and internal energy of the analyte ion, respectively. The dissociation rate constant and internal energy of the analyte ion were determined by double-hybrid density functional theory calculations and the survival yield method using benzylpyridinium thermometer ions, respectively. Because intense fragment ion signals are present in the ESI mass spectrum, the analysis of the fragment ions produced by ESI in-source CID facilitates the identification of metabolites originating from aromatic amino acids, such as histamine.

Characterization of the Internal Energy of Ions Produced by Electrospray Ionization Using Substituted Benzyl Ammonium Thermometer Ions.

We propose the use of substituted benzyl ammonium species as thermometer ions to characterize the internal energy distribution of the ions produced by electrospray ionization (ESI). Crucially, we found that the activation of the benzyl ammonium species preferentially provided a benzyl cation via N-Cα bond cleavage. In addition, calculations at the CCSD(T)/cc-PVTZ//M06-2X-D3/6-311++G(d,p) level of theory revealed that the threshold energies of fragmentation of the tested model ions ranged from 86 to 192 kJ mol-1, significantly lower than those of conventional 4-substituted benzylpyridinium thermometer ions. Thus, the substituted benzyl ammoniums are suitable for the characterization of the ESI process under typical experimental conditions. Further, the internal energies of the ions were found to depend on the radiofrequency voltage of the ion funnel, which is used to increase the transport efficiency of the ions from atmospheric to vacuum conditions. Our reported method will aid the determination of the optimum ion-funnel radiofrequency voltage for the analysis of small molecules such as metabolites. Furthermore, benzyl ammoniums are commercially available, which will facilitate the routine and widespread measurement of the internal energy distributions of ions.

Experimental and Theoretical Investigation of MALDI In-Source Decay of Peptides with a Reducing Matrix: What Is the Initial Fragmentation Step?

Matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) with a reducing matrix is believed to be initiated by hydrogen transfer from the matrix to the peptide. Several new matrices have recently been developed to achieve more efficient MALDI-ISD. In particular, the use of matrices containing aniline groups facilitates MALDI-ISD to a greater extent than that of matrices containing phenol groups, although the N-H bond in aniline is stronger than the O-H bond in phenol. In this study, photoelectron yield spectroscopy of matrix solids revealed that conversion of the phenol group to the aniline group decreased the ionization energy of the matrix solids. Crucially, the use of a matrix with lower ionization energy has been found to result in efficient cleavage at N-Cα and disulfide bonds by MALDI-ISD. Therefore, electron association with the peptide rather than the fragmentation mechanism involving hydrogen atom attachment is proposed as the initial step of the MALDI-ISD process. In this mechanism, electron transfer from the reducing matrix to the peptide produces a peptide anion radical, which provides either a [cn + H]/[zm]• or [an]•/[ym + H] fragment pair. Fragmentation of the peptide anion radical strongly depends on the gas-phase acidity of the matrix used. Subsequently, the resultant fragments/radicals underwent a reaction in the MALDI plume, producing observable even-electron ions. Consequently, MALDI-ISD fragments are observed as both positive and negative ions, even though MALDI-ISD with a reducing matrix involves fragmentation of peptide anion radicals. The proposed mechanism is suitable for obtaining a better understanding of the MALDI-ISD process.

Hot Hydrogen Atom Irradiation of Protonated/Deprotonated Peptide in an Ion Trap Facilitates Fragmentation through Heated Radical Formation.

Tandem mass spectrometry with fragmentation involving the reaction with hydrogen atoms is expected to be useful for the analysis of peptides and proteins. In general, hydrogen atoms preferentially react with odd-electron radicals. The attachment of hydrogen atoms to even-electron peptide ions is barely observed because of their low reaction rate. To date, only the methodology developed by our group has successfully induced the fragmentation of even-electron peptide ions by reacting with hydrogen atoms. In the present study, we focused on the temperature of the peptide ions and hydrogen atoms in an ion trap mass spectrometer to understand the mechanism of the corresponding reaction. Because the reaction between even-electron peptide ions and hydrogen atoms has a significant transition state barrier, the use of hot hydrogen atoms is required to initiate the reaction. The reaction contributes to increase the internal energy of the resultant peptide radicals because the heat of reaction and kinetic energy of the hydrogen atom are converted to the internal energy of the product. The resultant oxygen- and carbon-centered peptide radicals undergo radical-induced fragmentation with sub-picosecond and sub-millisecond time scales, respectively.

Fragmentation efficiency of phenethylamines in electrospray ionization source estimated by theoretical chemistry calculation.

Small molecules with polar functional groups, including substituted phenethylamines, are commonly analyzed by liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization (ESI). Analyte molecules are mostly detected in protonated and cation-adducted forms through positive-ion electrospray ionization-mass spectrometry (ESI-MS). However, the ESI of substituted phenethylamines commonly provides an intense signal of fragment ions by ESI in-source collision-induced dissociation (IS-CID), which hinders the unambiguous identification of phenethylamines. This phenomenon was approximated as a unimolecular dissociation model, and the dissociation efficiency was evaluated by various quantum chemistry calculations to determine the ESI IS-CID efficiency. The calculated results were consistent with the experimental data, when the dissociation threshold energy of phenethylamines was calculated using the post-Hartree-Fock (post-HF) method, CCSD(t)/cc-pVTZ//MP2(full)/6-311++G(d,p). In contrast to post-HF methods, the utilization of density functional theory calculations with a suitable functional is recognized as an accurate and competitive low-cost approach. In particular, ωB97-XD, M06-2X-D3, and recently developed Minnesota functionals, such as M11, MN12-SX, and MN15, provided reliable results, as in the case of the post-HF method. The results obtained by the recently developed double hybrid functionals, DSD-PEBP86-D3(BJ), PBE0-DH, and PBE-QIDH, were also reliable. The consideration of ESI IS-CID can facilitate the identification of analyte molecules because most phenethylamines, except for N-methylated analogs, provide an intense signal in the ESI mass spectrum.

Rapid chiral discrimination of oncometabolite dl-2-hydroxyglutaric acid using derivatization and field asymmetric waveform ion mobility spectrometry/mass spectrometry.

2-Hydroxyglutaric acid is a chiral metabolite whose enantiomers specifically accumulate in different diseases. An enantiomeric excess of the d-form in biological specimens reflects the existence of various pathogenic mutations in cancer patients, however, conventional methods using gas or liquid chromatography and capillary electrophoresis had not been used for large clinical studies because they require multiple analytical instruments and a long run time to separate the enantiomers. Here, we present a rapid separation method for dl-2-hydroxyglutaric acid using a chiral derivatizing reagent and field asymmetric waveform ion mobility spectrometry/mass spectrometry, which requires a single analytical instrument and <1 s for the separation. We compared three derivatization methods and found that a method using (S)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine enables the separation. In addition, we were able to detect dl-2-hydroxyglutaric acid in standard solution at lower concentrations than that previously reported for the serum. These results show the potential of the method to be used in clinical analysis.

Study of Substituted Phenethylamine Fragmentation Induced by Electrospray Ionization Mass Spectrometry and Its Application for Highly Sensitive Analysis of Neurotransmitters in Biological Samples.

Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) equipped with electrospray ionization (ESI) is widely employed for metabolite analysis, substituted phenethylamines commonly undergo fragmentation during ESI in-source collision-induced dissociation (CID). Unexpected fragmentation hampers not only unambiguous identification but also accurate metabolite quantification. ESI in-source CID induces N-Cα bond dissociation in substituted phenethylamines lacking a ß-hydroxy group to produce fragment ions with a spiro[2.5]octadienylium motif. In contrast, phenethylamines with a ß-hydroxy group generate substituted 2-phenylaziridium through ESI in-source CID-induced H2O loss. The fragment ion yield produced by ESI in-source CID can be estimated by the dissociation rate constant and internal energy of the analyte ion, determined by employing density functional theory calculations and the survival yield method using a thermometer ion, respectively. Fragmentation is strongly enhanced by the presence of an ß-hydroxy group, whereas N-methylation suppresses fragmentation. In particular, octopamine and noradrenaline, which contain an ß-hydroxy and primary amine groups, produce more intense fragment ion signals than protonated molecules. Regarding the quantitative analysis of phenethylamines present in the mouse brain, the noradrenaline fragment ion used as the precursor in multiple reaction monitoring (MRM) provided a higher signal-to-noise ratio in the resulting spectra than protonated noradrenaline. The present method allows for the quantitative analysis of substituted phenethylamines with high sensitivity.

Fragmentation study of tryptophan-derived metabolites induced by electrospray ionization mass spectrometry for highly sensitive analysis.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is interfaced with electrospray ionization (ESI), which generally produces intact gas-phase ions of biomolecules. However, ESI induces the fragmentation of tryptophan-derived metabolites, which are known to act as neurotransmitters and psychoactive drugs. Tryptophan-derived metabolites undergo N-Cα bond dissociation during ESI, producing a fragment ion with a spiro[cyclopropane-indolium] backbone. Fragmentation is suppressed by the presence of an α-carboxyl group and the modification of amino groups. In particular, tryptamine and serotonin, which lack such functional groups, produce more intense fragment-ion signals than protonated molecules. The multiple reaction monitoring (MRM)-based quantitative analysis of tryptamine and serotonin used the fragment ions produced from in-source collision-induced dissociation as the precursor ions, which improved the signal-to-noise ratio of the resulting spectra. The present method allows for the quantitative analysis of tryptamine and serotonin with high sensitivity.

Cooperative dissociation of peptide backbones and side-chains during matrix-assisted laser desorption/ionization in-source decay mediated by hydrogen abstraction.

Matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) causes the selective cleavage of Cα -C peptide bonds when an oxidizing matrix is used, and the fragmentation involves the hydrogen abstraction from a peptide by a matrix. The hydrogen abstraction from either an amide nitrogen or ß-carbon atom has been proposed to be the initial step leading to the Cα -C bond cleavage. In this regard, the production of [a]+ fragments originated upon bond cleavage at the C-terminal side of phenylglycine residues strongly suggested that that the Cα -C bond cleavage occurred through a nitrogen-centered radical intermediate and that the fragmentation through a ß-carbon-centered radical intermediate can be ruled out from the MALDI-ISD process, because phenylglycine residues do not contain ß-carbon atoms. The Cα -C bond cleavage of such nitrogen-centered radical initially produced an [a]•/[x - H] fragment pair, and then the [a]• radical either reacted with the matrix or underwent loss of the side-chain, leading to [a - H] or [d - H] fragment. The Cα -C bond cleavage at the C-terminal side of phenylglycine and phenylalanine residues only generated [a]+ fragments, whereas that of homophenylalanine and S-methylated cysteine residues provided both [a]+ and [d]+ fragments. The yield of [d]+ fragments was dependent on the chemical stability of the resultant radicals formed upon side-chain loss. MALDI-ISD produced [M - H + matrix]+ , [M - 16 + H]+ , [M - 32 + H]+ , and [d]+ fragments, when the analyte peptide contained a methionine residue. These fragments were formed upon abstraction of a hydrogen atom from the side-chain of a methionine residue and its subsequent reaction with the matrix. The oxidation of methionine residues suppressed the hydrogen abstraction from their side-chain.

Gas-Phase Peptide Fragmentation Induced by Hydrogen Attachment, from Principle to Sequencing of Amide Nitrogen-Methylated Peptides.

Tandem mass spectrometry (MS/MS) with radical-based fragmentation was developed recently, which involves the reaction of hydrogen atoms and peptides in a process called hydrogen attachment/abstraction dissociation (HAD). HAD mainly produces [cn + 2H]+ and [zm + 2H]+ via hydrogen attachment to the carbonyl oxygen on the peptide backbone. In addition, HAD often generates [an + 2H]+ and [xm + 2H]+. To explain the formation of [an + 2H]+ and [xm + 2H]+, hydrogen attachment to the carbonyl carbon atom on the peptide backbone is proposed to initiate Cα-C bond cleavage. The resultant hydrogen-abundant oxygen-centered radical intermediate undergoes radical-induced dissociation to give [an + H]+• and [xm + 2H]+. Subsequently, [an + 2H]+ was produced by the reaction of [an + H]+• and a hydrogen atom. The fragment ions formed by the cleavage of N-Cα and Cα-C bonds are observed in the HAD-MS/MS spectra, and the mass differences of these fragment ions correspond to the mass of peptide bonds. Consequently, HAD-MS/MS allows the identification of post-translational modifications on the peptide backbone. In addition, HAD-MS/MS provides a consecutive series of [cn + 2H]+ and [an + 2H]+ as the N-terminal fragments, as well as [zm + 2H]+ and [xm + 2H]+, which enables the sequencing of peptides with post-translational modification, including the discrimination of modifications on the side chain and backbone.

In-Source Fragmentation of Phenethylamines by Electrospray Ionization Mass Spectrometry: Toward Highly Sensitive Quantitative Analysis of Monoamine Neurotransmitters.

Electrospray ionization mass spectrometry (ESI-MS) is widely used to analyze biomolecules, which are usually detected as protonated and cation-adducted molecules in the positive-ion mode. However, phenethylamine derivatives, which are known as neurotransmitters and psychoactive drugs, undergo the protonation and subsequently lose NH3 during ESI. As a result, intense fragment-ion signals are observed in their ESI-MS spectra, which hamper the unambiguous identification of phenethylamine derivatives. To understand the mechanism of the loss of NH3 from these phenethylammoniums, the fragmentations of model 4-substituted phenethylamines were investigated and the fragment ions were identified as spiro[2.5]octadienyliums. Fragmentation was enhanced by the presence of electron-donating groups, and most substituted phenethylamines generated spiro[2.5]octadienyliums as fragment ions during ESI-MS, except those with strong electron-withdrawing groups. The quantitative analysis of phenethylamines by liquid chromatography tandem mass spectrometry is typically performed by multiple reaction monitoring using protonated molecules as the precursor. In contrast, the conversion of precursor ions from the protonated molecules into the spiro[2.5]octadienylium fragment improved the signal-to-noise ratio, allowing the quantitative analysis of phenethylamines with high sensitivity and accuracy.