Charging and dissociation of peptides and intact proteins formed by electrospray ionisation

Abstract

Electrospray ionisation (ESI) mass spectrometry (MS) has emerged as a central technique for protein sequence analysis. Protein analysis by mass spectrometry typically proceeds by either the (i) top-down method, where an intact protein is ionised and fragmented inside the mass spectrometer; or (ii) the bottom-up method, where a protein is chemically digested to form peptides, which are subsequently measured by liquid chromatography (LC)-MS. However, protein and peptide ions are usually formed in low charge states and are often not ionized efficiently, which reduces the performance of MS based techniques for protein identification. Using small chemical additives (e.g. 1,2-butylene carbonate), proteins and peptides can be ‘supercharged’ to significantly improve the performance of ESI mass spectrometry for protein analysis.For top-down protein analysis, the use of 1,2-butylene carbonate and ESI can result in the formation of protein ions in sufficiently high charge states that they can protonate atmospheric gases (e.g., N2, O2, and Ar) in room-temperature ion-molecule reactions. These results suggest that protein ion charging is limited by proton transfer reactions between multiply charged protein ions and atmospheric gases. By performing ESI at reduced pressure, protein ion charge states can be increased by over 40% compared to the use of conventional chemical supercharging. The most highly charged protein ions can transfer a proton to helium, which indicates that such ions are the most acidic entities isolated to date. For bottom-up protein analysis, a T-junction was used to introduced 1,2-hexylene carbonate into the LC eluent in LC-MS measurements to significantly increase peptide ion abundances. For a whole protein HeLa digest, the average ion abundances improved by ~5.5 times from 2.2 x106 (no additive) to 1.2 x 107 (1,2-hexylene carbonate). This results in a 100% and 50% increase in the respective number of peptide and protein identifications. Using 1,2-hexylene carbonate, peptides can be identified in sub-nanogram protein loadings. These data suggesting that 1,2-hexylene carbonate will be beneficial for improving bottom-up protein analysis by LC-MS