Peptide backbone fragmentation initiated by side‐chain loss at cysteine residue in matrix‐assisted laser desorption/ionization in‐source decay mass spectrometry

Matrix‐assisted laser desorption/ionization in‐source decay (MALDI‐ISD) is initiated by hydrogen transfer from matrix molecules to the carbonyl oxygen of peptide backbone with subsequent radical‐induced cleavage leading to c′/z? fragments pair. MALDI‐ISD is a very powerful method to obtain long sequence tags from proteins or to do de novo sequencing of peptides. Besides classical fragmentation, MALDI‐ISD also shows specific fragments for which the mechanism of formation enlightened the MALDI‐ISD process. In this study, the MALDI‐ISD mechanism is reviewed, and a specific mechanism is studied in details: the N‐terminal side of Cys residue (Xxx‐Cys) is described to promote the generation of c′ and w fragments in MALDI‐ISD. Our data suggest that for sequences containing Xxx‐Cys motifs, the N–C_α bond cleavage occurs following the hydrogen attachment to the thiol group of Cys side‐chain. The c?/w fragments pair is formed by side‐chain loss of the Cys residue with subsequent radical‐induced cleavage at the N–C_α bond located at the left side (N‐terminal direction) of the Cys residue. This fragmentation pathway preferentially occurs at free Cys residue and is suppressed when the cysteines are involved in disulfide bonds. Hydrogen attachment to alkylated Cys residues using iodoacetamide gives free Cys residue by the loss of ?CH₂CONH₂ radical. The presence of alkylated Cys residue also suppress the formation of c?/w fragments pair via the (C_β)‐centered radical, whereas w fragment is still observed as intense signal. In this case, the z? fragment formed by hydrogen attachment of carbonyl oxygen followed side‐chain loss at alkylated Cys leads to a w fragment. Hydrogen attachment on peptide backbone and side‐chain of Cys residue occurs therefore competitively during MALDI‐ISD process. Copyright © 2013 John Wiley & Sons, Ltd.     

In-source decay and fragmentation characteristics of peptides using 5-aminosalicylic acid as a matrix in matrix-assisted laser desorption/ionization mass spectrometry

The use of 5-aminosalicylic acid (5-ASA) as a new matrix for in-source decay (ISD) of peptides including mono- and di-phosphorylated peptides in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is described. The use of 5-ASA in MALDI-ISD has been evaluated from several standpoints: hydrogen-donating ability, the outstanding sharpness of molecular and fragment ion peaks, and the presence of interference peaks such as metastable peaks and multiply charged ions. The hydrogen-donating ability of several matrices such as α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (2,5-DHB), 1,5-diaminonaphthalene (1,5-DAN), sinapinic acid (SA), and 5-ASA was evaluated by using the peak abundance of a reduction product [M + 2H + H]⁺ to that of non-reduced protonated molecule [M + H]⁺ of the cyclic peptide vasopressin which contains a disulfide bond (S-S). The order of hydrogendonating ability was 1,5-DAN > 5-ASA > 2,5-DHB > SA = CHCA. The chemicals 1,5-DAN and 5-ASA in particular can be classified as reductive matrices. 5-ASA gave peaks with higher sharpness for protonated molecules and fragment ions than other matrices and did not give any interference peaks such as multiply-protonated ions and metastable ions in the ISD mass spectra of the peptides used. Particularly, 1,5-DAN and 5-ASA gave very little metastable peaks. This indicates that 1,5-DAN and 5-ASA are more “cool” than other matrices. The 1,5-DAN and 5-ASA can therefore be termed “reductive cool” matrix. Further, it was confirmed that ISD phenomena such as N-Cα bond cleavage and reduction of S-S bond is a single event in the ion source. The characteristic fragmentations, which form a− and (a + 2)-series ions, [M + H − 15]⁺, [M + H − 28]⁺, and [M + H − 44]⁺ ions in the MALDI-ISD are described.     

$$ {{\text{C}}_{\alpha }} - {\text{C}} $$ Bond Cleavage of the Peptide Backbone in MALDI In-Source Decay Using Salicylic Acid Derivative Matrices

The use of 5-formylsalicylic acid (5-FSA) and 5-nitrosalicylic acid (5-NSA) as novel matrices for in-source decay (ISD) of peptides in matrix-assisted laser desorption/ionization (MALDI) is described. The use of 5-FSA and 5-NSA generated a- and x-series ions accompanied by oxidized peptides [M – 2 H + H]⁺. The preferential formation of a- and x-series ions was found to be dependent on the hydrogen-accepting ability of matrix. The hydrogen-accepting ability estimated from the ratio of signal intensity of oxidized product [M – 2 H + H]⁺ to that of non-oxidized protonated molecule [M + H]⁺ of peptide was of the order 5-NSA > 5-FSA > 5-aminosalicylic acid (5-ASA) ≒ 2,5-dihydroxyl benzoic acid (2,5-DHB) ≒ 0. The results suggest that the hydrogen transfer reaction from peptide to 5-FSA and 5-NSA occurs during the MALDI-ISD processes. The hydrogen abstraction from peptides results in the formation of oxidized peptides containing a radical site on the amide nitrogen with subsequent radical-induced cleavage at the \textC_a - \textC {{\text{C}}_{\alpha }} - {\text{C}} bond, leading to the formation of a- and x-series ions. The most significant feature of MALDI-ISD with 5-FSA and 5-NSA is the specific cleavage of the \textC_a - \textC {{\text{C}}_{\alpha }} - {\text{C}} bond of the peptide backbone without degradation of side-chain and post-translational modifications (PTM). The matrix provides a useful complementary method to conventional MALDI-ISD for amino acid sequencing and site localization of PTMs in peptides.     

Study on the redox reactions for organic dyes and S‐nitrosylated peptide in electrospray droplet impact

Reduction of analytes in ionization processes often obscures the determination of molecular structure. The reduction of analytes is found to take place in various desorption/ionization methods such as fast atom bombardment (FAB), secondary ion mass spectrometry (SIMS), matrix‐assisted laser desorption/ionization (MALDI) and desorption ionization on porous silicon (DIOS). To examine the extent of the reduction reactions taking place in electrospray droplet impact (EDI) processes, reduction‐sensitive dyes and S‐nitrosylated peptide were analyzed by EDI. No reduction was observed for methylene blue. While methyl red has a lower reduction potential than methylene blue, the reduction product ions were detected. For S‐nitrosylated peptide, protonated molecule ion [M + H]⁺ and NO‐eliminated molecular ion [M − NO + H]^+• were observed but reduction reactions are largely suppressed in EDI compared with that in MALDI. As such, the analytes examined suffer from little reduction reactions in EDI. Copyright © 2008 John Wiley & Sons, Ltd.     

Sodiation as a tool for enhancing the diagnostic value of MALDI‐TOF/TOF‐MS spectra of complex astaxanthin ester mixtures from Haematococcus pluvialis

The microalga Haematococcus pluvialis produces the pigment astaxanthin mainly in esterified form with a multitude of fatty acids, which results in a complex mixture of carotenol mono‐ and diesters. For rapid fingerprinting of these esters, matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF/TOF‐MS) might be an alternative to traditional chromatographic separation combined with MS. Investigation of ionization and fragmentation of astaxanthin mono‐ and diester palmitate standards in MALDI‐TOF/TOF‐MS showed that sodium adduct parent masses [M + Na]⁺ gave much simpler MS² spectra than radical / protonated [M]^+● / [M + H]⁺ parents. [M + Na]⁺ fragments yielded diagnostic polyene‐specific eliminations and fatty acid neutral losses, whereas [M]^+● / [M + H]⁺ fragmentation resulted in a multitude of non‐diagnostic daughters. For diesters, a benzonium fragment, formed by polyene elimination, was required for identification of the second fatty acid attached to the astaxanthin backbone. Parents were forced into [M + Na]⁺ ionization by addition of sodium acetate, and best signal‐to‐noise ratios were obtained in the 0.1 to 1.0 mM range. This method was applied to fingerprinting astaxanthin esters in a crude H. pluvialis extract. Prior to MALDI‐TOF/TOF‐MS, the extract was fractionated by normal phase Flash chromatography to obtain fractions enriched in mono‐ and diesters and to remove pheophytin a, which compromised monoester signals. All 12 types of all‐trans esterified esters found in LC were identified with MALDI‐TOF/TOF‐MS, with the exception of two minor monoesters. Copyright © 2013 John Wiley & Sons, Ltd.     

Specific Cα-C Bond Cleavage of β-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 β-carbon (Cβ)-centered radical peptide ions [M???Hβ?+?H]^·+ are predominantly produced in MALDI conditions. RDD reactions from the peptide ions [M???Hβ?+?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???Hβ?+?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???Hβ?+?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???H_N?+?H]^·+, while ab initio calculations indicate that the formation of [M???Hα?+?H]^·+ is energetically most favorable.

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Graphical Abstract ?

     

The unexpected formation of [M − H]+ species during MALDI and dopant‐free APPI MS analysis of novel antineoplastic curcumin analogues

Unusual ionization behavior was observed with novel antineoplastic curcumin analogues during the positive ion mode of matrix‐assisted laser desorption ionization (MALDI) and dopant‐free atmospheric pressure photoionization (APPI). The tested compounds produced an unusual significant peak designated as [M ? H]⁺ ion along with the expected [M + H]⁺ species. In contrast, electrospray ionization, atmospheric pressure chemical ionization and the dopant‐mediated APPI (dopant‐APPI) showed only the expected [M + H]⁺ peak. The [M ? H]⁺ ion was detected with all evaluated curcumin analogues including phosphoramidates, secondary amines, amides and mixed amines/amides. Our experiments revealed that photon energy triggers the ionization of the curcumin analogues even in the absence of any ionization enhancer such as matrix, solvent or dopant. The possible mechanisms for the formation of both [M ? H]⁺ and [M + H]⁺ ions are discussed in this paper. In particular, three proposed mechanisms for the formation of [M ? H]⁺ were evaluated. The first mechanism involves the loss of H₂ from the protonated [M + H]⁺ species. The other two mechanisms include hydrogen transfer from the analyte radical cation or hydride abstraction from the neutral analyte molecule. Copyright © 2014 John Wiley & Sons, Ltd.     

Atmospheric pressure laser desorption/ionization using a 6–7 µm‐band mid‐infrared tunable laser and liquid water matrix

Due to the characteristic absorption peaks in the IR region, various molecules can be used as a matrix for infrared matrix‐assisted laser desorption/ionization (IR‐MALDI). Especially in the 6–7 µm‐band IR region, solvents used as the mobile phase for liquid chromatography have absorption peaks that correspond to their functional groups, such as O–H, CO, and CH₃. Additionally, atmospheric pressure (AP) IR‐MALDI, which is applicable to liquid‐state samples, is a promising technique to directly analyze untreated samples. Herein we perform AP‐IR‐MALDI mass spectrometry of a peptide, angiotensin II, using a mid‐IR tunable laser with a tunable wavelength range of 5.50–10.00 µm and several different matrices. The wavelength dependences of the ion signal intensity of [M + H]⁺ of the peptide are measured using a conventional solid matrix, α‐cyano‐4‐hydroxycinnamic acid (CHCA) and a liquid matrix composed of CHCA and 3‐aminoquinoline. Other than the O–H stretching and bending vibration modes, the characteristic absorption peaks are useful for AP‐IR‐MALDI. Peptide ions are also observed from an aqueous solution of the peptide without an additional matrix, and the highest peak intensity of [M + H]⁺ is at 6.00 µm, which is somewhat shorter than the absorption peak wavelength of liquid water corresponding to the O–H bending vibration mode. Moreover, long‐lasting and stable ion signals are obtained from the aqueous solution. AP‐IR‐MALDI using a 6–7 µm‐band IR tunable laser and solvents as the matrix may provide a novel on‐line interface between liquid chromatography and mass spectrometry. Copyright © 2015 John Wiley & Sons, Ltd.     

Positive chemical ionization triple‐quadrupole mass spectrometry and ab initio computational studies of the multi‐pathway fragmentation of phthalates

We report the first positive chemical ionization (PCI) fragmentation mechanisms of phthalates using triple‐quadrupole mass spectrometry and ab initio computational studies using density functional theories (DFT). Methane PCI spectra showed abundant [M + H]⁺, together with [M + C₂H₅]⁺ and [M + C₃H₅]⁺. Fragmentation of [M + H]⁺, [M + C₂H₅]⁺ and [M + C₃H₅]⁺ involved characteristic ions at m/z 149, 177 and 189, assigned as protonated phthalic anhydride and an adduct of phthalic anhydride with C₂H₅⁺ and C₃H₅⁺, respectively. Fragmentation of these ions provided more structural information from the PCI spectra. A multi‐pathway fragmentation was proposed for these ions leading to the protonated phthalic anhydride. DFT methods were used to calculate relative free energies and to determine structures of intermediate ions for these pathways. The first step of the fragmentation of [M + C₂H₅]⁺ and [M + C₃H₅]⁺ is the elimination of [R? H] from an ester group. The second ester group undergoes either a McLafferty rearrangement route or a neutral loss elimination of ROH. DFT calculations (B3LYP, B3PW91 and BPW91) using 6‐311G(d,p) basis sets showed that McLafferty rearrangement of dibutyl, di(‐n‐octyl) and di(2‐ethyl‐n‐hexyl) phthalates is an energetically more favorable pathway than loss of an alcohol moiety. Prominent ions in these pathways were confirmed with deuterium labeled phthalates. Copyright © 2010 John Wiley & Sons, Ltd.     

Mass spectrometric characterization of phosphorylated peptides using MALDI in‐source decay via redox reactions

Matrix‐assisted laser desorption/ionization in‐source decay (MALDI‐ISD) has been used for characterization of a phosphorylated peptides and proteins because labile phosphate group is not lost during the MALDI‐ISD process. The conventional MALDI‐ISD is initiated by the hydrogen transfer from reducing matrix molecules to peptide backbone, leading to c′‐ and z′‐series ions. In contrast, when an oxidizing chemical 5‐nitrosalicylic acid (5‐NSA) is served as the MALDI‐ISD matrix, a‐ and x‐series ions are specifically generated by hydrogen abstraction from peptide backbone to matrix molecule. The 5‐NSA provides useful complementary information to the conventional MALDI‐ISD for the analysis of amino acid sequencing and site localization of phosphorylation in peptides. The MALDI‐ISD with reducing and oxidizing matrix could be a useful method for the de novo peptide sequencing. Copyright © 2012 John Wiley & Sons, Ltd.     

A comparative study of in- and post-source decays of peptide and preformed ions in matrix-assisted laser desorption ionization time-of-flight mass spectrometry: Effective temperature and matrix effect

In-source decay (ISD) and post-source decay (PSD) of a peptide ion ([Y₆ + H]⁺) and a preformed ion (benzyltriphenylphosphonium, BTPP) generated by matrix-assisted laser desorption ionization (MALDI) were investigated with time-of-flight mass spectrometry. α-Cyano-4-hydroxycinammic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) were used as matrices. For both ions, ISD yield was unaffected by delay time, indicating rapid termination of ISD. This was taken as evidence for rapid expansion cooling of hot “early” plume formed in MALDI. CHCA was hotter than DHB for [Y₆ + H]⁺ while the matrix effect was insignificant for BTPP. The “early” plume temperature estimated utilizing previous kinetic results was 800–900 K, versus 400–500 K for “late” plume. The results support our previous finding that the temperature of peptide ions interrogated by tandem mass spectrometry was lower than most rough estimates of MALDI temperature.     

HPLC‐DAD and HPLC‐ESI‐MS separation,determination and identification of the spin‐labeled diastereoisomers of podophyllotoxin

Spin‐labeled nitroxide derivatives of podophyllotoxin had better antitumor activity and less toxicity than that of the parent compounds. However, the 2‐H configurations of these spin‐labeled derivatives cannot be determined by nuclear magnetic resonance (NMR) methods. In the present paper, a high‐performance liquid chromatography‐diode array detection (HPLC‐DAD) and a high‐performance liquid chromatography‐electrospray ionization tandem mass spectrometry (HPLC‐ESI/MS/MS) method were developed and validated for the separation, identification of four pairs of diastereoisomers of spin‐labeled derivatives of podophyllotoxin at C‐2 position. In the HPLC‐ESI/MS spectra, each pair of diastereoisomers of the spin‐labeled derivatives in the mixture was directly confirmed and identified by [M+H]⁺ ions and ion ratios of relative abundance of [M‐ROH+H]⁺ (ion 397) to [M+H]⁺. When the [M‐ROH+H]⁺ ions (at m/z 397) were selected as the precursor ions to perform the MS/MS product ion scan. The product ions at m/z 313, 282, and 229 were the common diagnostic ions. The ion ratios of relative abundance of the [M‐ROH+H]⁺ (ion 397) to [M+H]⁺, [A+H]⁺ (ion 313) to [M‐ROH+H]⁺, [A+H‐OCH₃]⁺ (ion 282) to [M‐ROH+H]⁺ and [M‐ROH‐ArH+H]⁺ (ion 229) to [M‐ROH+H]⁺ of each pair of diastereoisomers of the derivatives specifically exhibited a stereochemical effect. Thus, by using identical chromatographic conditions, the combination of DAD and MS/MS data permitted the separation and identification of the four pairs of diastereoisomers of spin‐labeled derivatives of podophyllotoxin at C‐2 in the mixture.     

Mass spectrometric monitoring of oxidation of aliphatic C6–C8 hydrocarbons and ethanol in low pressure oxygen and air plasmas

Experimental and theoretical studies on the oxidation of saturated hydrocarbons (n‐hexane, cyclohexane, n‐heptane, n‐octane and isooctane) and ethanol in 28 Torr O₂ or air plasma generated by a hollow cathode discharge ion source were made. Ions corresponding to [M + 15]⁺ and [M + 13]⁺ in addition to [M ? H]⁺ and [M ? 3H]⁺ were detected as major ions where M is the sample molecule. The ions [M + 15]⁺ and [M + 13]⁺ were assigned as oxidation products, [M ? H + O]⁺ and [M ? 3H + O]⁺, respectively. By the tandem mass spectrometry analysis of [M ? H + O]⁺ and [M ? 3H + O]⁺, H₂O, olefins (and/or cycloalkanes) and oxygen‐containing compounds were eliminated from these ions. Ozone as one of the terminal products in the O₂ plasma was postulated as the oxidizing reagent. As an example, the reactions of C₆H₁₄^+? with O₂ and of C₆H₁₃⁺ (CH₃CH₂CH⁺CH₂CH₂CH₃) with ozone were examined by density functional theory calculations. Nucleophilic interaction of ozone with C₆H₁₃⁺ leads to the formation of protonated ketone, CH₃CH₂C(=OH⁺)CH₂CH₂CH₃. In air plasma, [M ? H + O]⁺ became predominant over carbocations, [M ? H]⁺ and [M ? 3H]⁺. For ethanol, the protonated acetic acid CH₃C(OH)₂⁺ (m/z 61.03) was formed as the oxidation product. The peaks at m/z 75.04 and 75.08 are assigned as protonated ethyl formate and protonated diethyl ether, respectively, and that at m/z 89.06 as protonated ethyl acetate. Copyright © 2016 John Wiley & Sons, Ltd.     

Characteristic neutral loss of CH3CHO from Thr‐containing sodium‐associated peptides

A characteristic neutral loss of 44 Da is observed in the MS/MS spectra of Thr‐containing sodiated peptides. A combination of tandem mass spectrometry and quantum chemical calculations calculated at the B3LYP/6‐311G (d, p) level of ab initio theory is used to elucidate this fragmentation pathway. The high resolution mass spectrometry data indicate this neutral loss is acetaldehyde lost from the side chain of Thr rather than CO₂. The intensity of this neutral loss can be enhanced when Thr residue is far from the C‐terminus and when the C‐terminus is esterified as well. The mechanism of the acetaldehyde loss is proposed to adopt a McLafferty‐type rearrangement reaction, which involves a proton transfer from the hydroxyl of Thr side chain to its C‐terminal neighboring carbonyl oxygen inducing the cleavage of the C_a–C_β bond. This mechanism is further supported by examining the fragmentation of a [GT(tBu)G + Na]⁺ peptide derivative and by comparing the product ion spectra of [M + Na‐44]⁺ of [GTGA + Na]⁺ with [M + Na]⁺ of [GGGA + Na]⁺. A similar neutral loss of HCHO can also be detected in Ser‐containing peptides. Our computational results reveal that the most stable [GTG + Na]⁺ ion is present as a tridentate charge‐solvated structure and the dissociation leading to the 44 loss is dynamically and energetically favorable. Copyright © 2015 John Wiley & Sons, Ltd.     

Disulfide bond cleavage in TEMPO-free radical initiated peptide sequencing mass spectrometry

The gas‐phase free radical initiated peptide sequencing (FRIPS) fragmentation behavior of o‐TEMPO‐Bz‐conjugated peptides with an intra‐ and intermolecular disulfide bond was investigated using MSⁿ tandem mass spectrometry experiments. Investigated peptides included four peptides with an intramolecular cyclic disulfide bond, Bactenecin (RLC RIVVIRVC R), TGF‐α (C HSGYVGVRC ), MCH (DFDMLRC MLGRVFRPC WQY) and Adrenomedullin (16–31) (C RFGTC TVQKLAHQIY), and two peptides with an intermolecular disulfide bond. Collisional activation of the benzyl radical conjugated peptide cation, which was generated through the release of a TEMPO radical from o‐TEMPO‐Bz‐conjugated peptides upon initial collisional activation, produced a large number of peptide backbone fragments in which the S? S or C? S bond was readily cleaved. The observed peptide backbone fragments included a‐, c‐, x‐ or z‐types, which indicates that the radical‐driven peptide fragmentation mechanism plays an important role in TEMPO‐FRIPS mass spectrometry. FRIPS application of the linearly linked disulfide peptides further showed that the S? S or C? S bond was selectively and preferentially cleaved, followed by peptide backbone dissociations. In the FRIPS mass spectra, the loss of ?SH or ?SSH was also abundantly found. On the basis of these findings, FRIPS fragmentation pathways for peptides with a disulfide bond are proposed. For the cleavage of the S? S bond, the abstraction of a hydrogen atom at C_β by the benzyl radical is proposed to be the initial radical abstraction/transfer reaction. On the other hand, H‐abstraction at C_α is suggested to lead to C? S bond cleavage, which yields [ion ± S] fragments or the loss of ?SH or ?SSH. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous Determination Baicalin and Forsythin in Shuanghuanglian Oral Liquid by HPLC/DAD and LC‐ESI‐MS

Rapid, simple and reliable HPLC/DAD and LC‐ESI‐MS methods for the simultaneous determination of baicalin and forsythin in the traditional Chinese medicinal preparation Shuanghuanglian oral liquid were described and validated. The separation condition for HPLC/DAD was optimized using a BDS hypersil C18 column (Thermo, 2.1 × 150 mm, particle size 5 μm) by gradient elution using methanol‐0.2 % ammonium acetate as the mobile phase. The suitable detection wavelength was set at 277 nm for the quantitative analysis of baicalin and forsythin in this method. Some operational parameters of the ESI interface were optimized, negative m/z 445[M?H]^? for baicalin and negative m/z 593[M+CH₃COO]^? for forsythin, positive m/z 447[M+H]⁺ for baicalin and positive m/z 552[M+NH 4]⁺ for forsythin, respectively. These HPLC/DAD and LC‐ESI‐MS methods were validated in terms of recovery, linearity, accuracy and precision (intra‐ and inter‐day validation). These methods can be used as a complementary method for the commercial quality control of Shuanghuanglian oral liquid and its pharmaceutical preparations.     

Characterization of N‐Boc/Fmoc/Z‐N′‐formyl‐gem‐diaminoalkyl derivatives using electrospray ionization multi‐stage mass spectrometry

N‐Boc/Fmoc/Z‐N′‐formyl‐gem‐diaminoalkyl derivatives, intermediates particularly useful in the synthesis of partially modified retro‐inverso peptides, have been characterized by both positive and negative ion electrospray ionization (ESI) ion‐trap multi‐stage mass spectrometry (MSⁿ). The MS² collision induced dissociation (CID) spectra of the sodium adduct of the formamides derived from the corresponding N‐Fmoc/Z‐amino acids, dipeptide and tripeptide acids show the [M + Na‐NH₂CHO]⁺ ion, arising from the loss of formamide, as the base peak. Differently, the MS² CID spectra of [M + Na]⁺ ion of all the N‐Boc derivatives yield the abundant [M + Na‐C₄H₈]⁺ and [M + Na‐Boc + H]⁺ ions because of the loss of isobutylene and CO₂ from the Boc protecting function. Useful information on the type of amino acids and their sequence in the N‐protected dipeptidyl and tripeptidyl‐N′‐formamides is provided by MS² and subsequent MSⁿ experiments on the respective precursor ions. The negative ion ESI mass spectra of these oligomers show, in addition to [M‐H]^?, [M + HCOO]^? and [M + Cl]^? ions, the presence of in‐source CID fragment ions deriving from the involvement of the N‐protecting group. Furthermore, MSⁿ spectra of [M + Cl]^? ion of N‐protected dipeptide and tripeptide derivatives show characteristic fragmentations that are useful for determining the nature of the C‐terminal gem‐diamino residue. The present paper represents an initial attempt to study the ESI‐MS behavior of these important intermediates and lays the groundwork for structural‐based studies on more complex partially modified retro‐inverso peptides. Copyright © 2013 John Wiley & Sons, Ltd.     

Dissociation of disulfide‐intact somatostatin ions: the roles of ion type and dissociation method

The dissociation chemistry of somatostatin‐14 was examined using various tandem mass spectrometry techniques including low‐energy beam‐type and ion trap collision‐induced dissociation (CID) of protonated and deprotonated forms of the peptide, CID of peptide‐gold complexes, and electron transfer dissociation (ETD) of cations. Most of the sequence of somatostatin‐14 is present within a loop defined by the disulfide linkage between Cys‐3 and Cys‐14. The generation of readily interpretable sequence‐related ions from within the loop requires the cleavage of at least one of the bonds of the disulfide linkage and the cleavage of one polypeptide backbone bond. CID of the protonated forms of somatostatin did not appear to give rise to an appreciable degree of dissociation of the disulfide linkage. Sequential fragmentation via multiple alternative pathways tended to generate very complex spectra. CID of the anions proceeded through CH₂? S cleavages extensively but relatively few structurally diagnostic ions were generated. The incorporation of Au(I) into the molecule via ion/ion reactions followed by CID gave rise to many structurally relevant dissociation products, particularly for the [M+Au+H]²⁺ species. The products were generated by a combination of S? S bond cleavage and amide bond cleavage. ETD of the [M+3H]³⁺ ion generated rich sequence information, as did CID of the electron transfer products that did not fragment directly upon electron transfer. The electron transfer results suggest that both the S? S bond and an N? C_α bond can be cleaved following a single electron transfer reaction. Copyright © 2009 John Wiley & Sons, Ltd.     

The effect of temperature on the stability of compounds used as UV‐MALDI‐MS matrix: 2,5‐dihydroxybenzoic acid, 2,4,6‐trihydroxyacetophenone, α‐cyano‐4‐hydroxycinnamic acid, 3,5‐dimethoxy‐4‐hydroxycinnamic acid,nor‐harmane and harmane

The thermal stability of several commonly used crystalline matrix‐assisted ultraviolet laser desorption/ionization mass spectrometry (UV‐MALDI‐MS) matrices, 2,5‐dihydroxybenzoic acid (gentisic acid; GA), 2,4,6‐trihydroxyacetophenone (THA), α‐cyano‐4‐hydroxycinnamic acid (CHC), 3,5‐dimethoxy‐4‐hydroxycinnamic acid (sinapinic acid; SA), 9H‐pirido[3,4‐b]indole (nor‐harmane; nor‐Ho), 1‐methyl‐9H‐pirido[3,4‐b]indole (harmane; Ho), perchlorate of nor‐harmanonium ([nor‐Ho + H]⁺) and perchlorate of harmanonium ([Ho + H]⁺) was studied by heating them at their melting point and characterizing the remaining material by using different MS techniques [electron ionization mass spectrometry (EI‐MS), ultraviolet laserdesorption/ionization‐time‐of‐flight‐mass spectrometry (UV‐LDI‐TOF‐MS) and electrospray ionization‐time‐of‐flight‐mass spectrometry (ESI‐TOF‐MS)] as well as by thin layer chromatography analysis (TLC), electronic spectroscopy (UV‐absorption, fluorescence emission and excitation spectroscopy) and ¹H nuclear magnetic resonance spectroscopy (¹H‐NMR). In general, all compounds, except for CHC and SA, remained unchanged after fusion. CHC showed loss of CO₂, yielding the trans‐/cis‐4‐hydroxyphenylacrilonitrile mixture. This mixture was unambiguously characterized by MS and ¹H‐NMR spectroscopy, and its sublimation capability was demonstrated. These results explain the well‐known cluster formation, fading (vanishing) and further recovering of CHC when used as a matrix in UV‐MALDI‐MS. Commercial SA (SA 98%; trans‐SA/cis‐SA 5 : 1) showed mainly cis‐ to‐trans thermal isomerization and, with very poor yield, loss of CO₂, yielding (3′,5′‐dimethoxy‐4′‐hydroxyphenyl)‐1‐ethene as the decarboxilated product. These thermal conversions would not drastically affect its behavior as a UV‐MALDI matrix as happens in the case of CHC. Complementary studies of the photochemical stability of these matrices in solid state were also conducted. Copyright © 2008 John Wiley & Sons, Ltd.     

Evaluation of the α‐phenylvinyl cation as a chemical ionization reagent for the differentiation of isomeric substituted phenols in an ITMS

Ion–molecule reactions between the α‐phenylvinyl cation and isomeric naturally occurring phenols were investigated using a quadruple ion trap mass spectrometer. The α‐phenylvinyl cation m/z 103, generated by chemical ionization from phenylacetylene, reacts with neutral aromatic compounds to form the characteristic species: [M + 103]⁺ adduct ions and the trans‐vinylating product ions [M + 25]⁺, which correspond to [M + 103]⁺ adduct after the loss of benzene. Isomeric differentiation of several ring‐substituted phenols was achieved by using collision‐induced dissociation of the [M + 103]⁺ adduct ions. This method also showed to be effective in the differentiation of 4‐ethylguaiacol from one of its structural isomers that displays identical EI and EI/MS/MS spectra. The effects of gas‐phase alkylation with phenylvinyl cation on the dissociation behavior were examined using mass spectrometryⁿ and labeled derivatives. Copyright © 2015 John Wiley & Sons, Ltd.