Development of peptide de novo sequencing methods using bromine isotope pattern
thesis · 2012
| Date | 2012-01-01 |
| Type | thesis |
| Publisher | PhD thesis |
| Contribution | algorithm |
| Link | http://dcollection.snu.ac.kr:80/jsp/common/DcLoOrgPer.jsp?sItemId=000000002031 |
| Citations (OpenAlex) | 0 |
Abstract
De novo sequencing of peptides by tandem mass spectrometry is subject to complications arising from various fragmentations of peptide ions. Thus, it is necessary to simplify tandem mass spectra by selectively tagging or removing b- or y-ions. In this thesis, new de novo methods using selective derivatization of the N-terminus with bromine-containing reagent are presented. \nIn Chapter 1, it is demonstrated that bromopicolinamidination of the N-terminus leads to MS/MS spectrum showing consecutive b-ions with bromine signature. The b-ions could be used for de novo N-terminal sequencing, which provides key information about N-terminal proteolytic processing of proteins involved in many cellar processes. We synthesized ethyl bromopicolinimidate and used it for bromopicolinamidination of the peptide or protein N-terminal amine. The dual advantage of MALDI signal enhancement by the basic picolinamidine and b-ion selection aided by Br signature is demonstrated using a variety of peptides. Identification of phosphorylation site as well as N-terminal sequencing of a phosphopeptide was straightforward. The b-ions with Br signature in the MS/MS spectra were used for N-terminal sequencing of myoglobin and hemoglobin. The N-terminal peptide was selected for MS/MS analysis from the MS spectrum, obtained from chymotryptic digest of the derivatized proteins, based on the Br signature introduced to the N-terminal amine.\nIn Chapter 2, it is shown that the b-series ions can be distinguished from y-series ions in the MALDI TOF-TOF spectra by incorporating bromine-tag to the N-terminal amino group through rapid and selective acetylation using bromoacetic anhydride without blocking the lysine and tyrosine residues. The 51:49 ratio of Br-79 and Br-81 isotopes facilitated identification of ions carrying the tag. With the Br-tag in the b-series ions, N-terminal sequencing of tryptic peptides from hemoglobin as well as model peptides was straightforward. When the b-ions were low in intensity, ions without the Br-tag were identified as y-ions and used for sequencing.
Methods and tools
- Oxazolone C-terminal derivatization: Selective C-terminal derivatization, which is hard because C-terminal and side-chain carboxyls behave alike, improved in efficiency and given a bromine signature so the C-terminal fragment can be picked out and sequenced.