Article

Enhanced responses in matrix-assisted laser desorption/ionization mass spectrometry of peptides derivatized with arginine via a C-terminal oxazolone

Department of Chemistry, Nara Women's University, Nara, Nara, Japan
Rapid Communications in Mass Spectrometry (Impact Factor: 2.64). 04/2004; 18(7):799-807. DOI: 10.1002/rcm.1409
Source: PubMed

ABSTRACT We have developed a novel method for enhancing the response of a peptide in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) by activating the C-terminal carboxyl group through an oxazolone with which is coupled an amine containing a functional group to help ionize the peptide. The reactions consist of dehydration with acetic anhydride to give an oxazolone, followed by aminolysis with an appropriate amino acid derivative such as arginine methyl ester. The MALDI signal of Ac-Tyr-Gly-Gly-Phe-Leu-Arg-OMe, thus converted from leucine-enkephalin, was detected while completely excluding the responses of arginine-deficient peptides coexisting in the reaction mixture. Some less intense peaks corresponding to a few sequential degradation products, also terminated with the arginine derivative, were also observed. The side-chain groups potentially that are reactive were conveniently protected by acetylation simultaneous with the C-terminal activation, and those that remained unprotected were reduced to virtually negligible proportions when the reaction was conducted in a peptide solution of concentration less than 1 mM. The greatly increased responses of such arginine-terminated peptides could possibly be exploited to discern the C-terminal tryptic peptide of a protein that is otherwise almost insensitive to MALDI-MS in general. The simplicity of the post-source decay spectrum of enkephalin derivatized by arginine methyl ester characteristically accentuated z- and b-type ions, and this should facilitate sequencing of such derivatized peptides. Remaining problems with practical applications of this approach are discussed.

0 Bookmarks
 · 
94 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: C-termini of proteins often play an important role in various biological processes. The determination of protein C-terminus is crucial since it provides not only distinct functional annotation, but also a way to monitor the proteolysis-modified proteins. In this study, an isotopic labeling approach based on oxazolone chemistry was developed to achieve the identification and quantification of C-termini. Aminolysis reagent such as arginine selectively reacts with the α-carboxyl group at peptide C-terminus via oxazolone-like intermediate. Side chain carboxyl groups does not participate this reaction. When an isotopic mixture consisting of 50% arginine (0Arg) and 50% C6-arginine (6Arg) was introduced to react with C-terminus of protein and followed by proteolysis, C-terminal peptide could be directly recognized in the mass spectrum due to its unique isotopic paired peaks, and the sequence could be interpreted in MS2. Besides, the incorporation of an additional basic amino acid in C-terminal peptide greatly enhanced the signal intensity for C-termini detection. Moreover, the isotopic arginine labeling strategy could be applied for relative C-termini quantitation. Our method showed an excellent correlation of the measured ratios to theoretical ratios and high reproducibility within 2 order of magnitude of dynamic range. The correlation coefficients (R2) were higher than 0.99, with the coefficients of variation (CVs) ranging from 1.16% to 10.91%. Finally, the approach was used to analyze the C-termini from Thermoanaerobacter tengcongensis which was cultured under different temperature. As a result, 68 C-termini have been identified and 53 of them were quantified in total using our strategy, and 24 neo-C-terminal peptides have also been discovered.
    Analytical Chemistry 10/2013; 85(22). DOI:10.1021/ac401647m · 5.83 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Due to almost identical chemical properties of C-terminal and side-chain carboxylic groups, selective C-terminal derivatization has been difficult. Although oxazolone-based C-terminal derivatization is the only selective C-terminal modification available, it has not been used widely because of its low derivatization efficiency. In this paper, an improved oxazolone chemistry for incorporation of Br signature to C-terminus is reported. MS/MS analysis of the brominated peptides led to a series of y ions with Br signature, facilitating de novo C-terminal sequencing.
    Analytical Biochemistry 08/2011; 419(2):211-6. DOI:10.1016/j.ab.2011.08.011 · 2.31 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We present here an approach to C-terminal sequencing of proteins by the procedure consisting of the following: (1) derivatization of the C-terminal α-carboxyl group with 3-aminopropyltris(2,4,6-trimethoxyphenyl)-phosphonium bromide (TMPP-propylamine) through oxazolone chemistry, (2) enzymatic proteolysis of the TMPP-derivatized protein, and (3) MALDI-MS/MS analysis of the peptide mixture, in which the C-terminal peptide incorporating the TMPP group is preferably detected. In this protocol, it is possible to choose any endoproteinase such as trypsin, GluC, and AspN for digestion so that a C-terminal peptide with length appropriate for mass spectrometric sequencing could be generated. The peptide labeled with TMPP-propylamine at the C terminus tends to exhibit y-type ions in MS/MS spectra, allowing manual sequence interpretation with the simplified fragmentation pattern. The efficacy of the method was verified with five proteins, which demonstrated that the C-terminal peptides were readily distinguishable by their peak intensity and characteristic mass signature peak in MALDI-PSD analysis.
    Analytical and Bioanalytical Chemistry 06/2012; 404(1):125-32. DOI:10.1007/s00216-012-6093-5 · 3.66 Impact Factor