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

Bartosz Setner,Zbigniew Szewczuk

doi:10.1007/s00216-017-0771-2

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

Highlights

Made progress in both instrumentation as well as methodology of mass spectrometry (MS) makes it a method of choice for analysis of complex peptide mixtures [2]
Bąchor et al observed that polyethylene glycol-modified Quaternary ammonium salts (QAS) peptide conjugates undergo internal fragmentation within the bicyclic moiety during collision-induced dissociation (CID) experiment [22]
The QAS groups were introduced to the side chain of the Cterminal lysine residue

Summary

Introduction

Made progress in both instrumentation as well as methodology of mass spectrometry (MS) makes it a method of choice for analysis of complex peptide mixtures [2]. A commonly utilized strategy is a shotgun approach. In this technique, protein samples are enzymatically digested into peptides which are analyzed by MS [3]. Only qualitative information about the peptides present in the prepared samples could be obtained by this approach. The simplest method for MS-based peptides quantification strategy is based on eXtracted Ion Chromatograms (XIC) derived from LC-MS measurements. In this approach a peak height or a peak area are used to determine the analyte abundance. Due to a limited sensitivity as well as a poor reproducibility of LC-MS measurements, a new strategy—selected reaction monitoring (SRM) was introduced [4]

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