Improving peptide fragmentation by N‐terminal derivatization with high proton affinity

Abstract

An improved method of de novo peptide sequencing based on mass spectrometry using novel N-terminal derivatization reagents with high proton affinity has been developed. The introduction of a positively charged group into the N-terminal amino group of a peptide is known to enhance the relative intensity of b-ions in product ion spectra, allowing the easy interpretation of the spectra. However, the physicochemical properties of charge derivatization reagents required for efficient fragmentation remain unclear. In this study, we prepared several derivatization reagents with high proton affinity, which are thought to be appropriate for peptide fragmentation under low-energy collision-induced dissociation (CID) conditions, and examined their usefulness in de novo peptide sequencing. Comparison of the effects on fragmentation among three derivatization reagents having a guanidino or an amidino moiety, which differ in proton affinity, clearly indicated that there was an optimal proton affinity for efficient fragmentation of peptides. Among reagents tested in this study, derivatization with 4-amidinobenzoic acid brought about the most effective fragmentation. This derivatization approach will offer a novel de novo peptide sequencing method under low-energy CID conditions.

N-Terminal Derivatization with Structures Having High Proton Affinity for Discrimination between Leu and Ile Residues in Peptides by High-Energy Collision-Induced Dissociation

“…8) among four structures used in this study, in which d b6 -ion was also observed. is is consistent with the previous observations that an Arg residue, which has a guanidino group in its side chain, at or near the N-terminus has the most signi cant in uence on generation of d-ions compared to other basic residues such as Lys and His.…”

“…8) It is known that a guanidino group of an Arg residue has high proton a nity, which retains a proton on that residue to generate d-ions by charge-remote fragmentation under HE-CID conditions if it exists at or near the N-terminus. 9) is suggests that the N-terminal derivatization with the structures having high proton a nity may facilitate the side-chain fragmentation to discriminate between Leu and Ile under HE-CID conditions in addition to its improvement e ect on fragmentation under LE-CID conditions.…”

“…4-(Guanidinomethyl) benzoic acid, nicotinoyloxysuccinimide and 4-amidinobenzoyloxysuccinimide were synthesized as described in the literatures. 8) e peptides used in this study were synthesized in our laboratory.…”

“…is is consistent with the previous observations that an Arg residue, which has a guanidino group in its side chain, at or near the N-terminus has the most signi cant in uence on generation of d-ions compared to other basic residues such as Lys and His. 4) Since we have shown that Gmb is e ective for improving the backbone fragmentation of peptides under LE-CID conditions, 8) derivatization with Gmb was further evaluated for its usefulness in de novo sequencing in the following section.…”

Differential 14N/15N-Labeling of Peptides Using N-Terminal Charge Derivatization with a High-Proton Affinity for Straightforward de novo Peptide Sequencing

“…Of the structures tested, 4-amidinobenzoic acid (Aba) was found to have the appropriate proton affinity to serve as both a donor and an acceptor of mobile protons, which would provide "readable" fragmentation by increasing the number and intensity of b-ions. 9) However, some y-ions were still observed for cases of peptides containing highly basic residues even after derivatization, in which doubly-charged ions were selected as a precursor. This could lead to the incorrect assignment of b-ions in some cases.…”