New ionization tags based on the structure of the 5-azoniaspiro[4.4]nonyl tag for a sensitive peptide sequencing by mass spectrometry

Abstract

Quaternary ammonium salts (QAS), both linear and bicyclic, are often utilized to improve the mass spectrometry (MS) analysis of peptides by fixing a permanent positive charge on the analyzed molecule. However, during collision-induced dissociation (CID) experiments, QAS undergo unwanted side reactions-Hofmann elimination as well as a tertiary amine loss- rendering the data interpretation complicated. In this work, we present 2-thia- and 2-oxa-5-azoniaspiro[4.4]nonyl groups as heterocyclic derivatives of the highly stable ionization group, 5-azoniaspiro[4.4]nonyl, for a sensitive peptide analysis by MS. Due to the permanent positive charge, labeled peptides are characterized by enhanced ionization efficiency during electrospray mass spectrometry (ESI-MS) conditions. Moreover, interpretation of the CID fragmentation of labeled peptides is facilitated since a series of generated fragmentation ions enable a complete sequence coverage. Introduction of a heteroatom into the 5-azoniaspiro[4.4]nonyl scaffold allows for liberation of a stable reporter ion which could be used in selected reaction monitoring (SRM)-targeted quantification experiments. Additionally, we synthesized a deuterated analog of the tag for LC-SRM-targeted quantitative analysis. The obtained results indicate the general usefulness of the proposed heterocyclic quaternary ammonium ionization tag for sequencing and quantification of peptides. Graphical abstract New reagents based on the structure of the 5-azoniaspiro[4.4]nonyl tag for peptide analysis by tandem mass spectrometry.

Keywords: Derivatization of peptides; Ionization tag; Peptide sequencing; Tandem mass spectrometry.

Graphical abstract

New reagents based on the structure of the 5-azoniaspiro[4.4]nonyl tag for peptide analysis by tandem mass spectrometry

Fig. 1

Synthesis of the model QAS-peptide derivatives on the solid support. DIPEA, N,N-diisopropylethylamine, DMF, N,N-dimethylformamide, TFA, trifluoroacetic acid, TIS, triisopropylsilane, X-sulfur or oxygen atom. Yellow ball represents polystyrene solid support

Fig. 2

a ESI-MS/MS spectrum of the ASN^Thz+-CO-DVYT-NH₂, m/z 665.270 (collision energy, 25 V); b ESI-MS/MS spectrum of the BASN^Thz+-CO-DVYT-NH₂, m/z 713.292 (collision energy, 25 V); c ASN^Oxa+-CO-DVYT-NH₂, m/z 649.316 (collision energy, 25 V); d BASN^Oxa+-CO-DVYT-NH₂, m/z 697.316 (collision energy, 25 V). Asterisk is added to denote the presence of the derivative moiety that represents a charged (non-protonated) fragment. Diamond symbol denotes the parent peaks

Fig. 3

Derivatization reaction with ionization tag based on the heterocyclic analogue of the benzo-5-azoniaspiro[4.4]nonyl scaffold

Fig. 4

a ESI-MS/MS spectrum of the LVTDLTK[BASN^Oxa+-4-Abz], m/z 555.299 (collision energy, 20 V); b ESI-MS/MS spectrum of the LVNELTEFAK[BASN^Oxa+-4-Abz], m/z 742.382 (collision energy, 20 V). Asterisk is added to denote the presence of the derivative moiety that represents a charged (non-protonated) fragment. Diamond symbol denotes the parent peaks

Fig. 5

Pair of isotopologues of ionization tag based on the 2-oxa-benzo-5-azoniaspiro[4.4]nonyl scaffold. Light tag (left) and heavy tag (right)

Fig. 6

Schematic diagram of the protein quantification by a newly developed pair of light and heavy heterocyclic ionization tags. Based on the peptide sequence: LFTGHPETLEK