High-Efficiency Microflow and Nanoflow Negative Electrospray Ionization of Peptides Induced by Gas-Phase Proton Transfer Reactions

Marija Nišavić, Amela Hozić, Zdenko Hameršak, Martina Radić, Ana Butorac, Marija Duvnjak, Mario Cindrić

Liquid chromatography coupled to electrospray ionization mass spectrometry is routinely used in proteomics research. Mass spectrometry-based peptide analysis is de facto performed in positive ion mode, except for the analysis of some post-translationally modified peptides (e.g. phosphorylation and glycosylation). Collected mass spectrometry data after peptide negative ionization analysis is scarce because of a lack of negatively charged amino acid side chain residues that would enable efficient ionization (i.e. in average, every tenth amino acid residue is negatively charged). Also, several phenomena linked to negative ionization such as corona discharge, arcing and electrospray destabilization due to the presence of polar mobile-phase solutions or acidic mobile phase additives (e.g. formic or trifluoroacetic acid) reduce its use. Named phenomena influence micro- and nano-flow electrospray ionization of peptides in a way that prevents formation of negatively charged peptide ions. In this work we have investigated the effects of post-column addition of isopropanol solutions of formaldehyde, 2,2-dimethylpropanal, ethyl methanoate and 2-phenyl-2-oxoethanal as the negative ion mode mobile phase modifiers for the analysis of peptides. According to the obtained data, all four modifiers exhibited significant enhancement of peptide negative ionization, while ethyl methanoate showed the best results. The proposed mechanism of action of the modifiers includes proton transfer reactions trough oxonium ion formation. In this way, mobile phase protons are prevented from interfering with the process of negative ionization. To the best of our knowledge, this is the first study that describes the use and reaction mechanism of aforementioned modifiers for enhancement of peptide negative ionization.