Fragmentation Pathways Of Sodium-associated Peptides Under Low-energy Collision Induced Dissociation In Electrospray Ionization Mass Spectrometry

Na+plays many critical roles in the biological processes such as enzymesactivation, cellular metabolism, stabilization of protein structure, etc. Additionalproduct ions obtained by sodiated peptides can provide useful sequence informationsupplementary to protonated peptides and improve protein identification. Therefore,the interaction between Na+and amino acids and their structures in gas-phase haveattracted increased interest. In this work, we investigated influence of residuecomposition, conformation of a peptide on the fragmentation pathways of sodium-associated peptides upon CID with support of tandem mass spectrometry experimentsand quantum chemical calculations. We also compared different dissociationpathways of sodiated and protonated peptides and found characteristic dissociationpathways of sodiated a3ions as well as peptides containing some special amino acidresidues. This thesis contains the following three parts:(1) Formation and dissociation pathways of sodiated a3ions. A3ions are reportedto be seldom observed relative to other anions such as a2and a4due to their reducedkinetic stability. However, the sodium-ion-associated analogues of the a3ions aresignificant for most peptides we tested. By synthesizing reaction intermediates,tandem mass spectrometry experiment and theoretical calculations, we demonstratethat the most stable [a3+Na-H]+ion is present as a bidentate linear structure. Furtherfragmentation of the [a3+Na-H]+ion needs to overcome several relatively highenergetic barriers to form [b2+Na-H]+ion with a diketopiperazine structure. Presentwork provides a mechanistic insight into how a sodium ion affects the fragmentationbehaviors of peptides.(2) Characteristic neutral loss of CH3CHO from Thr-containing sodium-associated peptides. By far, the dissociation pathways of sodiated Thr-containing peptides have received little focus although it has been reported to exhibit acharacteristic neutral loss of44Da. However, how the ion is formed is currentlyunknown. There are few reports giving structural information of a sodiated Thr-containing peptide so far. In this work, we investigate the characteristic neutral loss of44Da. The results reveal that this neutral loss is acetaldehyde from the side chain ofThr through a Mclafferty type rearrangement. This phenomenon can also be observedin sodiated Ser-containing peptides.(3) Characteristic neutral loss of NH3+CO from Gln-containing sodium-associated peptides. Loss of NH3upon CID is common for Gln containing peptides. Itis reported that when Gln residue is located at the N-terminus, the loss of of NH3canbe induced by the nucleophilic attack of N-terminal amino group; When Gln residueis located at other sites along the peptide, it can be attacked by amide carbonyl oxygenor nitrogen atom. In our work, we found different fragmentation behaviors of sodiatedGln containing peptides. The loss of NH3is followed by the loss of CO. We comparedthe influence of the two different charge carriers on the neutral loss and proposed amechanism of the loss of NH3and CO involving a6-membered anhydrideintermediate.Considering such specific fragment ions as additional markers after regulardatabase searches may eliminate false positives and boost the confidence of proteinidentification. We hope these characteristic sodium-ion-associated product ions can beimplemented into bioinformatics algorithms to improve the accuracy of proteinidentification.