Sequestration of the ionizing proton in singly charged metastable protein ions generated by MALDI

Abstract

A hypothesis is proposed for the preferential ionization/protonation of arginine residues in metastable protein ions. This phenomenon has significant consequences for detection of protein fragments resulting from polypeptide backbone cleavage. Preferential ionization at arginine (when present) is based upon MS/MS analysis of singly charged/protonated metastable protein ions: YahO, CspC, CspE, B-subunit of Shiga toxin 2 and thioredoxin, that have either zero or one arginine residue in their sequences. For protein ions having no arginine, we observe multiple b/y complementary fragment ion pairs (CFIP) by MALDI-MS/MS post-source decay demonstrating that the ionizing proton is located at multiple locations amongst the packet of protein precursor ions being analyzed. By contrast, for protein ions having one arginine, we observe little or no CFIP, and almost every fragment ion has the arginine residue in its sequence. This preferential proton sequestration is presumably due to the higher gas phase basicity of arginine compared to lysine, although lysine residues are often more numerous than arginine residues. As this phenomenon has been noted previously in the ionization of tryptic peptides by MALDI where the basic residue (K or R) is located exclusively at the C-terminus (if there are no missed proteolytic cleavages), we conclude that rapid proton transfer in the gas phase occurs via protein/matrix collisions in the MALDI plume. Upon proton transfer to the side-chain of an arginine residue, the proton is effectively immobilized by its guanidino functional group. An alternative explanation is proton exchange from the ionized side-chain of a lysine to the side-chain of the arginine in the metastable ion.