Mitsuo Takayama

N-Cα bond cleavage of the peptide backbone via hydrogen abstraction

The specific cleavage of N-Cα bonds on the peptide backbone to form the so-called ‘c’ and ‘z + 2’ products, which can be used for the rapid determination of protein amino-acid sequences, has been examined to clarify the mechanism(s) that occur during hydrogen abstraction induced by bombardment with 337-nm laser photons in matrix-assisted laser desorption/ionization (MALDI) method. Intramolecular hydrogen abstraction, which results from the hydrogen(s) on the Cα or Cβ carbon, did not occur with a deuterium-labeled dodecapeptide. To confirm a proposition that intermolecular hydrogen abstraction occurs between the peptide and the MALDI matrix, a deuterium dodecapeptide embedded in a deuterium 2,5-dihydroxybenzoic acid matrix at a molar ratio of 1:7000 was analyzed. The resulting deuterium c product ions suggested that c ions form via intermolecular hydrogen abstraction, although the results obtained did not deny any other possibilities such as intramolecular transfer of labile hydrogen. A mechanism for the N-Cα bond cleavage has been proposed that the formation of hypervalent radical species and subsequent prompt bond cleavages occur. The proposed mechanism successfully rationalizes the formation of both the z + 2 and the c product ions.

In-source decay characteristics of peptides in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

In-source decay (ISD) of peptides, coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, has been examined to determine the influence of the matrix, the susceptibility of amino-acid residues to ISD, and the effect of extraction delay times. Out of nine di- and tri-hydroxybenzoic acids and three cinnamic derivatives tested, the most suitable matrix for ISD was 2,5-dihydroxybenzoic acid. The amine bond at Xxx-Gly and Xxx-Val residues was less susceptible than other amino-acid residues to ISD; however, the more sensitive residue(s) were not as clear. Using a peptide that gave the yn- and (zn + 2)-series product ions, it was confirmed that amide-bond cleavage (formation of the yn-series ions) accompanied metastable peaks, whereas metastable peaks were never observed with amine-bond cleavage [formation of the (zn + 2)-series ions]. Furthermore, abundant cn-series ions, which originate from amine-bond cleavage on the peptide backbone, were observed whenever a minimum delay time of 38 ns or continuous extraction was used to obtain spectra. These data indicate that amine-bond cleavage in ISD takes place on the ionization time scale before the energy randomization is completed.

Does in-source decay occur independent of the ionization process in matrix-assisted laser desorption?

Abstract The influence of the acidic and basic characters of constituent amino acid residues on the peptide fragment ions produced by in-source decay under matrix assisted laser desorption/ionization (MALDI) conditions has been studied using positive- and negative-ion experiments. Whereas the in-source decay spectra of peptides containing basic Arg and/or Lys residues near the N-terminus showed so-called c n - and a n -series ions in positive-ion mode, a peptide that has an acidic amino acid cluster near the N-terminus and a basic residue near the C-terminus characteristically formed y n - and z n -series ions in the positive-ion in-source decay spectrum. These results indicated that fragment ion series produced by in-source decay depend strongly upon the acidic and basic characters of the constituent amino acid residues and the near N- and C-termini. It was suggested that in-source decay processes occur intrinsically at NH–C α and CO–NH bonds independent of the formation of molecular-related ions, and that the cleavages at the NH–C α and CO–NH bonds occurred independently and were dependent on the matrix used.

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.

Mass Spectrometry of Prenylated Flavonoids

The fragmentation patterns originating from the degradation of prenyl group(s) in positive ion electron ionization (EI), fast-atom bombardment (FAB) and chemical ionization (CI) mass spectrometry (MS) of prenylated flavonoids were reviewed. The EI spectra showed the characteristic fragmentation patterns reflecting the location of prenyl group in the flavonoid compounds, whereas the FAB and CI spectra showed relatively monotonous patterns. It was described how the EI and FAB fragmentation patterns are useful for the identification of prenylated flavonoids

Sequence information of peptides and proteins with in‐source decay in matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry

In‐source decay coupled with matrix assisted laser desorption/ionization‐mass spectrometry, which is a mass spectrometric degradation method for the sequencing of peptides and proteins, has been applied to several different polypeptides and proteins. The influence of the nature of the constituent amino acids on positively charged product ions is described. Relatively small molecular mass peptides produced c‐, b‐, and/or a‐series ions usable for C‐terminal sequencing as well as y‐ and/or z‐series ions usable for N‐terminal sequencing. The formation of the C‐terminal sequencing ions (c, b and a) and the N‐terminal sequencing ions (y and z) was strongly dependent on the location(s) of basic arginine and lysine residues. The presence of the arginine and/or lysine residues at the N‐terminal region was one‐sided in the formation of c‐, b‐, and/or a‐series ions, while the presence of those at the C‐terminal region was favorable for the formation of y‐ and z‐series ions. In‐source decay experiments of intact proteins, apomyoglobin and two viral coat proteins, led to large amounts of c‐series ions and small amounts of y‐series ions, which reflected internal sequences.

Further study of the matrix effect on the extent of fragmentation in molecular ions M+ produced under fast atom bombardment (FAB) conditions

Abstract The extents of fragmentation in the molecular ions M + of prenylated flavonoids produced under FAB conditions using various liquid matrices are evaluated by comparison with the corresponding electron ionization (EI) mass spectral patterns at electron impact energies of 15–70 eV. The extents of fragmentation in the M + ions produced with m -nitrobenzyl alcohol as a FAB matrix are always lower than those produced with glycerol and thioglycerol as the matrices. A few FAB spectral data obtained here indicate that the extends of FAB fragmentation in the M + ions correspond to those of the EI fragmentation in the M + ions produced at the electron impact energy of more than 70 eV. Consequently, it is concluded that FAB is an EI-like hard ionization process even when a liquid matrix is used.

Fragmentation processes of hydrogen-deficient peptide radicals in matrix-assisted laser desorption/ionization in-source decay mass spectrometry.

The mechanism of in-source decay (ISD) in matrix-assisted laser desorption/ionization (MALDI) has been described. The MALDI-ISD with an oxidizing matrix is initiated by hydrogen abstraction from peptides to matrix molecules, leading to hydrogen-deficient peptide radicals. Subsequently, the C(α)-C and C(α)-H bonds are cleaved, forming the a•/x fragment pair and [M-2H], respectively. Those reactions competitively occur during MALDI-ISD processes. Our results suggest that the C(α)-H bond cleavage to form [M-2H] was induced by collisions between hydrogen-deficient peptide radicals and matrix molecules in the MALDI plume. In contrast, the C(α)-C bond cleavages occur via a unimolecular dissociation process and independently of the collision rate in the MALDI plume. The formation mechanism of the a-, b-, and d-series fragments are also described. We report 2,5-bis(2-hydroxyethoxy)-7,7,8,8-tetracyanoquinodimethane (bisHE-TCNQ), being known as an organic semiconductor and an electron acceptor, as a novel suitable matrix for the MALDI-ISD of peptides via hydrogen abstraction.

Specific cleavage at peptide backbone Cα–C and CO–N bonds during matrix‐assisted laser desorption/ionization in‐source decay mass spectrometry with 5‐nitrosalicylic acid as the matrix

The use of 5-nitrosalicylic acid (5-NSA) as a matrix for in-source decay (ISD) of peptides during matrix-assisted laser desorption/ionization (MALDI) is described herein. Mechanistically, the decay process is initiated by a hydrogen abstraction from a peptide backbone amide nitrogen by 5-NSA. Hydrogen abstraction results in formation of an oxidized peptide containing a radical amide nitrogen. Subsequently, the C(α)-C bond N-terminal to the peptide bond is cleaved to form an a·/x fragment pair. The C(α)-C bonds C-terminal to Gly residues were less susceptible to cleavage than were those of other residues. C(α)-C bonds N-terminal to Pro and Sar residues were not cleaved by the aforementioned mechanism; instead, after hydrogen abstraction from a Pro or Sar C(α)-H bond, the peptide bond N-terminal to the Pro was cleaved yielding b- and y-series ions. We also show that fragments produced by MALDI 5-NSA-induced ISD were formed independently of the ionization process.

Influence of Charge State and Amino Acid Composition on Hydrogen Transfer in Electron Capture Dissociation of Peptides

Although conventional N-Cα bond cleavage in electron capture dissociation (ECD) of multiply-charged peptides generates a complementary c′ and z′ fragment pair, the N-Cα cleavage followed by hydrogen transfer from c′ to z′ fragments produces other fragments, namely c′ and z′. In this study, the influence of charge state and amino acid composition on hydrogen transfer in ECD is described using sets of peptides. Hydrogen transferred ionic species such as c′ and z′ were observed in ECD spectra of doubly-protonated peptides, while the triply-protonated form did not demonstrate hydrogen transfer. The extent of hydrogen transfer in ECD of doubly-protonated peptides was dependent on constituent amino acids. The ECD of doubly-protonated peptides possessing numerous basic sites showed extensive hydrogen transfer compared with ECD of less basic peptides. The extent of hydrogen transfer is discussed from the viewpoints of the structure of peptide ions, the possibility of internal hydrogen bonding and intermediate lifetime of complex [c′+z′].