The Effect Of Basic Amino Acid On The Protonated Peptide Fragmentation Mechanism

Proteomics is the frontier area in modern life science research field, its core analytical technique and common used approach for protein identification—mass spectrometry has gained great improvement in throughput, sensitivity, resoltuion and automation, so in return it promote a rapid development in peptide sequencing, protein identification and proteomics research. However the lack knowledge of peptide fragmentation mechanism and some selective cleavage in gas phase prevent the mass spectrometry application in sequencing of peptides or identification of proteins even in proteomics study. So clarify and elucidate the peptide fragmentation mechanism in mass spectrometry condition (collsion-induced dissociation condition) is very necessary for both mass spectrometry itself and proteomics research.Peptides with basic residue are widely used in life science researches and can always provide a strong signal in positive mass spectrometry. The investigation on the effect of basic amino acid on the protonated peptide fragmentation mechanism can contribute to a increase accuracy in peptide sequencing, a new design in mass spectrometry database searching algorithm and so on.Mobile proton model is a major peptide fragmentation theory that shaped by previous studies, it claims that the processes of protonated peptide fragmentation are charge-directed. Based on this model, we explore the effect of basic amino acid on the protonated peptide fragmentation mechanism, especially on charge-directed mechanism, from three aspects:different gas phase basicity, different arginine position and different chemical modification using electrospray ionization ion trap mass spectrometry combined with quantum chemical calculations. The main content of this work can be summarized as follows:1、There are three amino acids with basic residue in natural amino acids-arginine, lysine and histidine, and their gas phase basicities are different. In chapter2, we investigate the effect of basic amino acid with varying basicity on the protonated peptide VARML、VAKML and VAHML fragmentation mechanism. The experiment results showed the lower basicity was, the more loosely proton was sequestered by basic amino acid side chain, the easier the peptide fragmentation became. When the arginine turned into lysine in the peptide, the cleavage pattern of amino bond Met-Leu in VAKML was changed and became easily, compared to that of Met-Leu in VARML. When the lysine turned into histidine, the fragmentation of amino bond Ala-His in VAHML became easily compared to Ala-His in VAKML. The quantum chemical calculation results revealed that the relative charge ratios Qc/Qh in amino bonds of peptide VARML、VAKML and VAHML could provide a charge-directed mechanism. An increase of the Qc/Qh value in amino bonds and a decrease of amino acid basicity both mean that a peptide fragment easily.2、Arginine has a most basic side-chain in natural amino acids. In chapter3, we investigate the effect of basic amino acid-arginine with varying position on the protonated peptide RVAML、VRAML、VARML、VAMRL and VAMLR fragmentation mechanism. The experiment results showed that the peptides fragmentation became difficult if the position of arginine moved from terminal to the middle of peptides. When the arginine was localized at N-terminal, the N-terminal ions (an. bn ions) frequently formed from amino bond cleavage. When the arginine was localized at C-terminal, the C-terminal ions-yn ions often formed in peptide fragmentation. When the arginine was localized at the middle of peptide sequence, both N-terminal and C-terminal ions were observed in peptide fragmentation. The quantum chemical calculation results revealed that the relative charge QC+O+N-H in amino bonds of peptide RVAML、VRAML、VARML、VAMRL and VAMLR provide a charge-directed mechanism. An increase of the QC+O+N+H value in amino bonds means a peptide fragment easily. Compared to other peptides, the charge QC+O+N+H of peptide VARML was the highest value, so amino bond cleavage of VARML was most difficult.3、The acetylation and amidation are common post-translational modifications. In chapter4, we investigate the effect of acetylation and amidation on the protonated peptide Ac-GRMG、GRMG-NH2and GRMG-OH fragmentation mechanism. The experiment results showed the amino bonds of acetylated peptide Ac-GRMG were difficult to cleave than that of original peptide GRMG-OH, and the fragmentation extent of amidated peptide GRMG-NH2was similar with that of original peptide GRMG-OH. The fragment ions b2*and MH*0formation from Ac-GRMG were suppressed by acetylation modification, and the fragment ions formed from GRMG-NH2were not impacted by amidation modification. The quantum chemical calculation results revealed that the relative charge Qc+o in amino bonds of peptide Ac-GRMG、GRMG-NH2and GRMG-OH provide a charge-directed mechanism. When the Qc+o value in amino bonds increase, the peptides become difficult to cleave.This paper explores the effect of basic amino acid on the protonated peptide fragmentation mechanism from three aspects:different basicity, different arginine position and different chemical modification, substantiating and refining the charge-directed mechanism of mobile proton model for peptide fragmentation. The experimental and computational strategies consisting of electrospray ionization ion trap tandem mass spectrometry combining with quantum chemical calculations for investigation on charge-directed mechanism were also established in this paper. Though the conclusions about charge-directed mechanism in amino bonds were different, the results described here can enrich the peptide fragmentation database and may provide some reference gists for peptide/protein identification algorithms based on charge-directed mechanism.