Transient Conformations Leading to Peptide Fragment Ion [c + 2H]+ via Intramolecular Hydrogen Bonding Using MALDI In-source Decay Mass Spectrometry of Serine-, Threonine-, and/or Cysteine-Containing Peptides.

The formation of a peptide fragment ion [c + 2H]+ was examined using ultraviolet matrix-assisted laser desorption/ionization in-source decay mass spectrometry (UV/MALDI-ISD MS). Unusually, an ISD experiment with a hydrogen-abstracting oxidative matrix 4-nitro-1-naphthol (4,1-NNL) resulted in a [c + 2H]+ ion when the analyte peptides contained serine (Ser), threonine (Thr), and/or cysteine (Cys) residues, although the ISD with 4,1-NNL merely resulted in [a]+ and [d]+ ions. The [c + 2H]+ ion observed could be rationalized through intramolecular hydrogen atom transfer (HAT), like a Type-II reaction via a seven-membered conformation involving intramolecular hydrogen bonding (HB) between the active hydrogens (-OH and -SH) of the Ser/Thr/Cys residues and the backbone carbonyl oxygen at the adjacent amino (N)-terminal side residue. The ISD of the Cys-containing peptide resulted in the [c + 2H]+ ions, which originated from cleavage at the backbone N-Cα bonds far from the Cys residue, suggesting that the peptide molecule formed 16- and 22-membered transient conformations in the gas phase. The time-dependent density functional theory (TDDFT) calculations of the model structures of the Ser and Cys residues indicated that the Cys residue did not show a constructive bond interaction between the donor thiol (-SH) and carbonyl oxygen (=CO), while the Ser residue formed a distinct intramolecular HB.

Quantum Chemical Analysis of Molecular and Fragment Ions Produced by Field Ionization of Methyl Stearate.

The formation of molecular and fragment ions observed in the field ionization mass spectrum of methyl stearate has been analyzed on the basis of quantum chemical calculations including time-dependent density functional theory (TDDFT) and natural bond orbital (NBO) analysis. The TDDFT calculations suggest that methyl stearate is ionized via two processes, namely a 7.43 eV excitation and a tunneling effect, while the high electric field of 1010 V/m enables analyte molecules to ionize at an effective 6 eV lower than the 9.26 eV ionization energy. The NBO analysis suggests that the abundances of aliphatic fragment ions [CnH2n+1]+ at m/z 29, 43, and 57 generated in the ionizing cell can be rationalized by hyperconjugation between the sigma (σ)-electrons of sp3 C-H bonds of methyl or methylene groups and the empty p-orbital of the carbocation -CH2+. The C4 periodic methyl ester fragment ions at m/z 115-269 and the complementary McLafferty rearrangement fragment ion at m/z 224 can be explained by metastable ion decay with rearrangement reactions in the ion source.

Identification of Negative Ion at m/z 20 Produced by Atmospheric Pressure Corona Discharge Ionization under Ambient Air.

The negative ion at m/z 20 observed at atmospheric pressure corona discharge ionization mass spectra has been identified by supplying the vapors of deuterium oxide (D2O) and H218O. From the mass shifts of the ion at m/z 20 observed with D2O and H218O, it was suggested that the chemical composition of the ion at m/z 20 is to be H4O. Further mass shift from m/z 20 to 22 was observed by supplying the vapor of perfluorokerocene, suggesting the chemical composition of H3F. The chemical compositions of the negative ions H4O- and H3F- were consistence with the dipole-bound complex states between hydrogen H2 and polar molecules such as H2O and hydrogen fluoride (HF) having dipole moments beyond a critical dipole moment of 1.625 D, theoretically proposed by Skurski and Simons. The ionic chemical compositions and structures of H4O- and H3F- obtained with density functional theory calculations implied that both dipole-bound complex H2O-…H2 and HF-…H2 can be formed by exothermic reactions by which H2 molecule is complexing with negative ions H2O- and HF-, respectively.

Understanding the mechanism of CO2-Assisted electrospray ionization for parameter optimization in supercritical fluid chromatography mass spectrometry.

Supercritical fluid chromatography (SFC) is often coupled with electrospray ionization mass spectrometry (ESI-MS) for analyte detection because of its detection capability to a wide range of chemical properties. However, MS sensitivity is highly dependent on the chromatographic conditions, so that it is important to understand the ionization mechanism to determine the optimal chromatographic conditions. The ionization mechanism in SFC/ESI-MS is different to that of liquid chromatography because of the use of CO2 as a mobile phase. Some studies have suggested that alkoxycarbonic acids are formed in the mixture of CO2 and the alcohol modifier, and these species contribute to ionization in CO2-assisted SFC/ESI-MS. Therefore, in this study, we investigated CO2-assisted ESI to test this hypothesis, and we confirmed that methoxylcarbonic acid is generated in CO2/methanol mixtures and contributed to ion generation and detection because it acts as a proton donor in positive-ion mode. However, methoxylcarbonic acid interfered with ionization in negative-ion mode. Addition of ammonium acetate, which is often added to the modifier for negative ion detection in SFC/MS analysis, did not contribute to the recovery of MS sensitivity, although it tended to suppress the formation of metoxylcarbonic acid. This is likely due to ion suppression and neutralization of the negative sites of the analytes by anions or cations derived from ammonium acetate in the negative ion mode. Thus, additive-free methanol/CO2 was the most suitable mobile phase for obtaining high sensitivity in SFC/MS. To demonstrate the practicality of these findings, we tested our optimal mobile phase selection for pesticide analysis. In addition, we tested the addition of 0, 1, and 5 mM ammonium formate to the modifier and make-up solvent, and found that the addition of 1 mM ammonium formate gave the best results in pesticides analysis. In SFC/MS, salt is often added to improve separation or prevent desorption, but our findings suggest that the concentration of salt must be kept as low as possible to achieve highly sensitive MS detection. The results of this study reveal the best selection of the optimal conditions for the modifier and make-up solvent for CO2-assisted SFC/MS analysis and will be useful for the method development in SFC/MS.

Complete and selective nitration of tyrosine residue in peptides caused by ultraviolet matrix-assisted laser desorption/ionization.

Complete and highly selective nitration of tyrosine (Tyr) as a residue-specific modification in peptides was found without side reactions, using ultraviolet matrix-assisted laser desorption/ionization (UV-MALDI) with a nitroaromatic reagent 3, 5-dinitrosalicylic acid (3,5-DNSA). The tyrosine nitration supported two propositions, namely, the UV-induced. NO2 attack reaction mechanism by Long et al. and the C-NO2 homolysis as a thermal process by Wiik et al. and Furman et al. With the UV-MALDI of peptides, a residue-specific reaction was observed in glycine (Gly) residue, i.e., an oxidation of the alpha-carbon of Gly due to attack of hydroxyl radical (.OH).

Sensitive and resistant of the homologous disulfide-bridged proteins α-lactalbumin and lysozyme to attack of hydrogen-atoms, dithiothreitol and trifluoroacetic acid, examined by matrix-assisted laser desorption/ionization mass spectrometry.

BACKGROUND: Evolutionarily homologous proteins bovine α-lactoalbumin (αLA) and hen egg-white lysozyme (HEL) are very similar in primary, secondary and tertiary structures involving the location of disulfide-bridges (S-S), and are resistant to the action of hydrolytic enzymes and reagents. It is of interest to examine and compare the difference in backbone cleavage characteristics, by using reductive and hydrolytic reagents. METHODS: In-source decay (ISD) combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), reductive treatment of αLA and HEL with dithiothreitol (DTT) and acid hydrolysis with trifluoroacetic acid (TFA) were employed to examine the difference in the backbone cleavage characteristics of αLA and HEL. RESULTS: The treatment of αLA and HEL with DTT/AcOHNH3 resulted in similar cleavage behaviors of the backbone N-Cα and S-S bonds, i.e., the enhancements of the intensity and m/z range of sequence-reflected fragment ions were very similar. However, the treatment of αLA with DTT/TFA resulted in unexpected residue-specific degradation at the peptide bond of the Asp-Xxx, Xxx-Ser/Thr, Gln-Xxx, Xxx-Gly and Gly-Xxx residues, while HEL did not occur such degradation. CONCLUSIONS: The results obtained above indicate that acidic αLA is very sensitive to acidic additive such as TFA, while basic HEL is resistance to acidic additives. GENERAL SIGNIFICANCE: The study demonstrates the sensitive and resistant of evolutionary homologous proteins αLA and HEL to the acid hydrolysis and these characters come from acidic and basic nature of the proteins.

Matrix-dependent a/x pair and overdegraded w/y/z ions generated by radical-directed dissociation of peptide radical cations [M]+ in matrix-assisted laser desorption/ionization in-source decay.

Fragmentation of peptide radical cations [M]. + has been examined using matrix-assisted laser desorption/ionization (MALDI) in-source decay (ISD) with hydrogen-abstracting nitro-substituted matrices. The ISD spectra of peptides containing an arginine (Arg) residue at carboxyl (C)-termini showed preferential [w]+ ions when 4-nitro-1-naphthol (4,1-NNL) matrix was used, whereas the use of 3,5-dinitrosalicylic acid (3,5-DNSA) resulted in preferential [x]+ ions. Minor or some [d]+ , [x]+ , [y]+ , and [z]+ ions were also observed. For peptides containing Arg residue at amino (N)-termini, the ISD spectra showed preferential [a]+ ions independent of matrix used. The observed [a]+ , [w]+ , [x]+ , [y]+ , and [z]+ ions can be rationally explained by radical-directed dissociation (RDD) of the peptide radical cations [M]. + , although [d]+ ions may be formed via Norrish Type I cleavage and/or by RDD of [M]. + ions. The formation of overdegraded [d]+ , [w]+ , [y]+ , and [z]+ ions is discussed from the standpoint of the internal energy of radical cations [M]. + and radical fragment ions [a + H]. + and [x + H]. + deposited via collisional interactions with excited matrix molecules in the MALDI plume. The radical site of the peptide cations [M]. + was presumed to be backbone amide nitrogen, from MALDI-ISD data with three different deuterated amino acids.

Influence of hydrophilic additives on the signal intensity in electrospray ionization of flavonoid glycosides.

RATIONALE: The influence of hydrophilic additives glycine, glucose, and glycerol on electrospray ionization (ESI) signal intensity of flavonoid glycosides and a nonreducing disaccharide is examined. The addition of excess glycine to the ESI solution would affect signal intensity more than glucose and glycerol due to its strong hydration capability. METHODS: The ESI signal response upon the addition of excess additives prepared was estimated in both selected ion monitoring and scan mode. All the mass spectrometry data were acquired in negative ion mode, because negative ion mode is recommended for saccharide compounds. RESULTS: The addition of glycine to the ESI solution of flavonoid glycosides and trehalose enhanced signal intensity, whereas the addition of glucose and glycerol had little effect. The signal intensity of rutin was higher than that of naringin and hesperidin, in accordance with their solubility in ESI solution. Trehalose molecules specifically interacted with glycine molecules to form a 1:1 trehalose-glycine complex, whereas the flavonoid glycosides did not produce such complex ions. CONCLUSIONS: The ESI signal enhancement of the saccharides with the additive glycine can be explained by its strong hydration capability, with the deprotonated carboxylic oxygens of zwitterionic glycine molecules strongly interacting with water hydrogen atoms resulting in strong hydration enthalpy. Consequently, glycine molecules set the analytes free from solvation with water molecules in the ESI droplets.

Multiple Hydrogen Loss from [M + H]+ and [a]+ ions of Peptides in MALDI In-Source Decay Using a Dinitro-Substituted Matrix.

The formation and radical-directed dissociation of multiple hydrogen-abstracted peptide cations [M + H - mH]·+ has been reported using MALDI-ISD with dinitro-substituted matrices. The MALDI-ISD of synthetic peptides using 3,5-dinitrosalicylic acid (3,5-DNSA) and 3,4-dinitrobenzoic acid (3,4-DNBA) as matrices resulted in multiple hydrogen abstraction from the analyte [M + H]+ and fragment [a]+ ions, i.e., [M + H - mH]+ and [a - mH]+ (m = 1-8). All of the ISD spectra showed unusually intense [a]+ ions originating from cleavage at the Cα-C bond of the Leu-Xxx residues when peptides without Phe/Tyr/His/Cys residues were used. The intensity of the [an]+ series ions generated using 3,5-DNSA and 3,4-DNBA rapidly decreased with increasing residue number n, suggesting cleavage at multiradical sites of [M + H - mH]•+. It was suggested that multiple hydrogen abstraction from protonated peptides [M + H]+ mainly takes place from the backbone amide nitrogen.

Estimation of Flexible and Rigid Residues of Disulfide-Bridged and Phosphorylated Proteins Using Matrix-Assisted Laser Desorption/Ionization in-Source Decay Mass Spectrometry.

Flexible and rigid residues in disulfide-bridged and phosphorylated proteins have been estimated by using MALDI in-source decay mass spectrometry (ISD MS). The MALDI-ISD spectra of bovine α-lactoalbumin, ß-lactoglobulin A, and ß-casein predict that the backbone amide of Xxx-Asp/Asn/Cys/Ser/pSer and Gly-Xxx residues has higher hydrogen accessibility than other residues, while Xxx-Ile/Val residues have less accessibility. The higher hydrogen-accessible and lower accessible residues as measured by MALDI-ISD are consistent with the flexible and rigid residues determined by X-ray, nuclear magnetic resonance, and fluorescence decay methods. The disulfide bridges and phosphate groups do not prevent the estimation of flexible or rigid residues, whereas some other disulfide bridges inhibit the identification because of decreased sensitivity of ISD fragment ions. The estimation of flexible and rigid residues by means of the matrix-hydrogen accessibility can be explained by exposure or lack thereof to the hydrogen-accessible sites of intact proteins. It is proposed that MALDI-ISD is a powerful tool for identifying flexible and rigid residues of posttranslational modified proteins without the conformation information of the protein data bank.

Atmospheric Pressure Dark-Current Argon Discharge Ionization with Comparable Performance to Direct Analysis in Real Time Mass Spectrometry.

Herein, a dark-current discharge state created by combining argon flow with a needle electrode in ambient air is described that has an ionization efficiency and mechanism comparable to those of conventional helium direct analysis in real time (DART), without requiring dopants and DART glow discharge. Using this method, polar compounds such as α-amino acids were ionized in the dark-current argon discharge via (de)protonation, molecular anion formation, fragmentation, (de)protonation with the attachment of oxygen, deprotonation with hydrogen loss and negative ion attachment. In contrast, nonpolar compounds (e.g., n-alkanes) were detected as positive ions via hydride abstraction and oxidation. Major background ions observed were H3O+(H2O) n , O2 ·+, O2 ·-(H2O) n and CO3 ·-. These results indicate that the present dark-current discharge efficiently generates resonance-state argon with an internal energy of ∼14.2 eV, higher than that of the well-known metastable state (∼11.6 eV). It is therefore suggested that ionization reactions occurring there can be attributed to the Penning ionization of atmospheric H2O and O2 by resonance-state argon, analogous to helium DART.

Intramolecular Hydrogen Transfer from the Alpha-Carbon (Cα) and Backbone Amide Nitrogen (Nb) to Form c- and y-Ions in Negative-Ion CID of Peptides.

The source of hydrogen in the formation of c- and y-ions produced by intramolecular hydrogen transfer in negative-ion CID experiments with peptides has been examined using Cα-, Cß-, and backbone amide (Nb)-deuterated peptides AAA(d3)AA, AAG(d2)AA, AAAG(d2)A, and AAAAA-d7, as well as five other peptides. The c- and y-ions produced by deuterium transfer from the deuterated residues were detected and identified by the exact m/z values obtained with a high-resolution orbitrap mass spectrometer. The rate of deuterium transfer obtained indicates that over 50% of the hydrogen was originated from the backbone amide nitrogen, with the residual hydrogen coming from the backbone Cα. It is clear that the hydrogen does not originate from the side chain Cß. It is hypothesized that the intramolecular hydrogen transfer to form negative c- and y-ions takes place via 3-, 4-, 6-, 7-, 8-, and 9-membered ring transition states.

Influence of Solvent Composition and Surface Tension on the Signal Intensity of Amino Acids in Electrospray Ionization Mass Spectrometry.

The influence of solvent composition and surface tension on the signal intensity of deprotonated molecules [M-H]- in electrospray ionization mass spectrometry (ESI MS) was evaluated using alanine (Ala), threonine (Thr) and phenylalanine (Phe), which have differing levels of hydrophobicity. The surface tension of the ESI solution was varied by changing the ratio of the organic solvents methanol (MeOH) and acetonitrile (MeCN) in water (H2O). In ESI MS, the signal intensity of all the amino acids was increased with decreasing surface tension for the two solutions, H2O/MeOH and H2O/MeCN. The use of H2O/MeCN was more favorable for achieving a strong signal for the analytes compared to H2O/MeOH. The smaller vaporization enthalpy of MeCN compared to MeOH was proposed as one of the most plausible explanation for this. The order of the signal intensity of amino acids was Phe>Thr>Ala, the same order as their hydrophobicity. It can be practically concluded that the use of solutions with lower surface tensions and lower vaporization enthalpies would result in higher signal intensities in ESI MS.

Specific Cα-C Bond Cleavage of ß-Carbon-Centered Radical Peptides Produced by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

Radical-driven dissociation (RDD) of hydrogen-deficient peptide ions [M - H + H]·+ has been examined using matrix-assisted laser dissociation/ionization in-source decay mass spectrometry (MALDI-ISD MS) with the hydrogen-abstracting matrices 4-nitro-1-naphthol (4,1-NNL) and 5-nitrosalicylic acid (5-NSA). The preferential fragment ions observed in the ISD spectra include N-terminal [a] + ions and C-terminal [x]+, [y + 2]+, and [w]+ ions which imply that ß-carbon (Cß)-centered radical peptide ions [M - Hß + H]·+ are predominantly produced in MALDI conditions. RDD reactions from the peptide ions [M - Hß + H]·+ successfully explains the fact that both [a]+ and [x]+ ions arising from cleavage at the Cα-C bond of the backbone of Gly-Xxx residues are missing from the ISD spectra. Furthermore, the formation of [a]+ ions originating from the cleavage of Cα-C bond of deuterated Ala(d3)-Xxx residues indicates that the [a]+ ions are produced from the peptide ions [M - Hß + H]·+ generated by deuteron-abstraction from Ala(d3) residues. It is suggested that from the standpoint of hydrogen abstraction via direct interactions between the nitro group of matrix and hydrogen of peptides, the generation of the peptide radical ions [M - Hß + H]·+ is more favorable than that of the α-carbon (Cα)-centered radical ions [M - Hα + H]·+ and the amide nitrogen-centered radical ions [M - HN + H]·+, while ab initio calculations indicate that the formation of [M - Hα + H]·+ is energetically most favorable. Graphical Abstract ᅟ.

pH Dependence of the Number of Discrete Conformers of Carbonic Anhydrase 2, as Evaluated from Collision Cross-Section Using Ion Mobility Coupled with Electrospray Ionization.

Ion mobility experiments coupled with electrospray ionization (ESI) were conducted to evaluate the folding states of bovine carbonic anhydrase 2 (CA2) under three different pH conditions. Collision cross-section (CCS) of the CA2 ions generated by ESI revealed the presence of six discrete conformers in the gas phase under the conditions employed in this study. The CCS of the most extended conformer was three times larger than that of the most compact one. The charge state distribution of the CA2 ions was indicative of three conformers being present. Although there was consistency in conformer assignment conducted by CCS and charge state distribution, the CCS measurement was shown to be more effective because the information obtained provided more detailed knowledge of the conformation of the protein.

Collision-Induced Dissociation Study of Strong Hydrogen-Bonded Cluster Ions Y-(HF) n (Y=F, O2) Using Atmospheric Pressure Corona Discharge Ionization Mass Spectrometry Combined with a HF Generator.

Hydrogen fluoride (HF) was produced by a homemade HF generator in order to investigate the properties of strong hydrogen-bonded clusters such as (HF) n . The HF molecules were ionized in the form of complex ions associated with the negative core ions Y- produced by atmospheric pressure corona discharge ionization (APCDI). The use of APCDI in combination with the homemade HF generator led to the formation of negative-ion HF clusters Y-(HF) n (Y=F, O2), where larger clusters with n≥4 were not detected. The mechanisms for the formation of the HF, F-(HF) n , and O2-(HF) n species were discussed from the standpoints of the HF generator and APCDI MS. By performing energy-resolved collision-induced dissociation (CID) experiments on the cluster ions F-(HF) n (n=1-3), the energies for the loss of HF from F-(HF)3, F-(HF)2, and F-(HF) were evaluated to be 1 eV or lower, 1 eV or higher, and 2 eV, respectively, on the basis of their center-of-mass energy (ECM). These ECM values were consistent with the values of 0.995, 1.308, and 2.048 eV, respectively, obtained by ab initio calculations. The stability of [O2(HF) n ]- (n=1-4) was discussed on the basis of the bond lengths of O2H-F-(HF) n and O2-H-F(HF) n obtained by ab initio calculations. The calculations indicated that [O2(HF)4]- separated into O2H and F-(HF)3.

Photochemical Reactions of Aminonaphthols Caused by Laser Desorption/Ionization.

The formation of monomeric and dimeric ions of seven different aminonaphthols (ANLs) has been studied by using laser desorption/ionization (LDI) with a nitrogen laser. The positive-ion data of all the ANLs merely showed molecular ion M(·+) without protonated molecule [M+H](+), while 1-amino-2-naphthol (1,2-ANL) and 2-amino-1-naphthol (2,1-ANL) showed an intense dimeric ion [2 M-2H2O+H](+). The negative-ion data showed deprotonated molecule [M-H](-) in common, while the spectra of 1,2-ANL, 2,1-ANL and 8-amino-2-naphthol (8,2-ANL) accompanied an intense peak corresponding to negative molecular ion M(·-) and the 8,2-ANL and 4-amino-1-naphthol (4,1-ANL) accompanied dehydrogenated anion [M-2H](·-). The formation of monomeric ions was discussed from the standpoints of thermochemical properties such as ionization energy, gas-phase acidity, electron affinity, and bond dissociation energy. The formation of dimeric ions [2 M-2H2O+H](+) observed in the 1,2-ANL and 2,1-ANL could be explained by the radical combination in the amino groups. An isomer 5-amino-1-naphthol (1-ANL) did not give any dimeric ions in the both positive- and negative-ion spectra. The influence of laser fluence upon the appearance of the monomeric ions such as M(·+), [M+H](+), [M-H](-) and [M-2H](·-) of the 5,1-ANL has been examined.

Intracellular Metabolism of α,ß-Unsaturated Carbonyl Compounds, Acrolein, Crotonaldehyde and Methyl Vinyl Ketone, Active Toxicants in Cigarette Smoke: Participation of Glutathione Conjugation Ability and Aldehyde-Ketone Sensitive Reductase Activity.

The major toxicants in cigarette smoke, α,ß-unsaturated aldehydes, such as acrolein (ACR) and crotonaldehyde (CA), and α,ß-unsaturated ketone, methyl vinyl ketone (MVK), are known to form Michael-type adducts with glutathione (GSH) and consequently cause intracellular GSH depletion, which is involved in cigarette smoke-induced cytotoxicity. We have previously clarified that exposure to cigarette smoke extract (CSE) of a mouse melanoma cell culture medium causes rapid reduction of intracellular GSH levels, and that the GSH-MVK adduct can be detected by LC/MS analysis while the GSH-CA adduct is hardly detected. In the present study, to clarify why the GSH-CA adduct is difficult to detect in the cell medium, we conducted detailed investigation of the structures of the reaction products of ACR, CA, MVK and CSE in the GSH solution or the cell culture medium. The mass spectra indicated that in the presence of the cells, the GSH-CA and GSH-ACR adducts were almost not detected while their corresponding alcohols were detected. On the other hand, both the GSH-MVK adducts and their reduced products were detected. In the absence of the cells, the reaction of GSH with all α,ß-unsaturated carbonyls produced only their corresponding adducts. These results show that the GSH adducts of α,ß-unsaturated aldehydes, CA and ACR, are quickly reduced by certain intracellular carbonyl reductase(s) and excreted from the cells, unlike the GSH adduct of α,ß-unsaturated ketone, MVK. Such a difference in reactivity to the carbonyl reductase might be related to differences in the cytotoxicity of α,ß-unsaturated aldehydes and ketones.

Improvement in ionization efficiency of direct analysis in real time-mass spectrometry (DART-MS) by corona discharge.

Herein it is shown that a combination of direct analysis in real time (DART) with a corona discharge system consisting of only a needle electrode easily improves DART ionization efficiency. Positive and negative DC corona discharges led to a formation of abundant excited helium atoms as well as the reactant ions H3O(+)(H2O)n and O2˙(-) in the DART analyte ionization area. These phenomena resulted in an increase in the absolute intensities of (de)protonated analytes by a factor of 2-20 over conventional DART. The other analyte ions detected in this corona-DART system (i.e., molecular ions, fragment ions, oxygenated (de)protonated analytes, dehydrogenated deprotonated analytes, and negative ion adducts) were quite similar to those obtained from DART alone. This indicates a lack of side reactions due to the corona discharge. The change in the relative intensities of individual analyte-related ions due to the combination of a corona discharge system with DART suggests that there is no effect of the abundant excited helium in the analyte ionization area on the fragmentation processes or enhancement of oxidation due to hydroxyl radicals HO˙. Furthermore, it was found that the corona-DART combination can be applied to the highly sensitive analysis of n-alkanes, in which the alkanes are ionized as positive ions via hydride abstraction and oxidation, independent of the type of alkane or the mass spectrometer used.

MALDI In-Source Decay of Protein: The Mechanism of c-Ion Formation.

The in-source decay (ISD) phenomenon, the fragmentation at an N-Cα bond of a peptide backbone that occurs within several tens of nanoseconds in the ion-source in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), is discussed from the standpoints of the discovery and early publications dealing with MALDI-ISD, the formation of c-ions in energy-sudden desorption/ionization methods, the formation of radical species in a MALDI, model construction for ISD, and matrix materials that are suitable for use in MALDI-ISD. The formation of c-ions derived from peptides and proteins in MALDI-ISD can be rationalized by a mechanism involving intermolecular hydrogen transfer, denoted as the "Takayama's model" by De Pauw's group (Anal. Chem. 79: 8678-8685, 2007). It should be emphasized that the model for MALDI-ISD was constructed on the basis of X-ray crystallography and scanning probe microscopy (SPM) analyses of matrix crystals, as well as the use of isotopically-labelled peptides.