CaltechTHESIS
  A Caltech Library Service

New reagents and methods for mass spectrometry-based proteomics investigations

Citation

Sohn, Chang Ho (2011) New reagents and methods for mass spectrometry-based proteomics investigations. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:02162011-180032183

Abstract

New chemical reagents and methods have been employed for mass spectrometry (MS)-based proteomics investigations. Many chemical reagents are synthesized to be covalently attached to biomolecules, especially peptides and proteins. The properties of the resulting peptide conjugates are characterized by various tandem mass spectrometric techniques (e.g., collision-induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), infrared multiphoton dissociation (IRMPD), and free radical initiated peptide sequencing (FRIPS)). In Chapter 2, the effect of high electron affinity tags in ECD and ETD is investigated using their peptide conjugates. The initial intramolecular electron transfer from the high-lying Rydberg states to the covalently attached high electron affinity tag occurs in competition with the Coulomb stabilized π* orbitals of the amide bonds in the model peptides. This leads to the inhibition of the normal sequence of ECD and ETD processes, yielding no backbone fragmentations. In Chapter 3, selective disulfide bond cleavages are observed by the FRIPS method. A newly prepared TEMPO-based FRIPS reagent is labeled to model peptides containing disulfide bonds and subject to CID to monitor free radical induced cleavages. Highly selective C–S and S–S bond cleavages are observed and their reaction mechanisms are proposed. In Chapter 4, novel Caltech isobaric tags (CITs) for protein quantification are developed and validated using various model samples. A newly discovered low-energy gas-phase fragmentation pathway, a nucleophilic substitution of the N3 in the 1,2,3-triazole ring generated by copper-catalyzed azide-alkyne cycloaddition (CuAAC) inspired us to create CITs. This selective cleavage is applied to the formation of the reporter ions to quantify protein expression level in cells. Chapter 5 describes clickable cross-linkers (CXLs) developed for elucidation of three-dimensional protein structures and protein-protein interactions (PPIs). In CXLs, cross-linking reactions are separated from the conjugation of affinity tags, avoiding steric hindrance. Cross-linked peptides are enriched from the complex mixture of yeast lysate and cross-linked ubiquitin digests using avidin affinity chromatography, showing high sensitivity of the CXL-based analysis. The low-energy pathway used for CIT reagents is also adopted to produce the reporter ion, filtering MS/MS scans of cross-linked peptides from those of unmodified peptides.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Ion Chemistry, Mass Spectrometry, Electron Capture Dissociation, Electron Transfer Dissociation, Disulfide Bond, Free Radical Initiated Peptide Sequencing, Isobaric Tag, Cross-Linker
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Beauchamp, Jesse L.
Thesis Committee:
  • Blake, Geoffrey A. (chair)
  • Heath, James R.
  • Rees, Douglas C.
  • Beauchamp, Jesse L.
Defense Date:9 May 2011
Author Email:changho.sohn (AT) gmail.com
Funders:
Funding AgencyGrant Number
Kwanjeong Educational FoundationUNSPECIFIED
Record Number:CaltechTHESIS:02162011-180032183
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:02162011-180032183
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:6247
Collection:CaltechTHESIS
Deposited By: Chang Ho Sohn
Deposited On:27 May 2011 22:14
Last Modified:16 Apr 2013 23:27

Thesis Files

[img]
Preview
PDF (Full thesis) - Final Version
See Usage Policy.

7Mb
[img]
Preview
PDF (Front Matter and Ch. 1) - Final Version
See Usage Policy.

470Kb
[img]
Preview
PDF (Ch. 2) - Final Version
See Usage Policy.

1081Kb
[img]
Preview
PDF (Ch. 3) - Final Version
See Usage Policy.

2893Kb
[img]
Preview
PDF (Ch. 4) - Final Version
See Usage Policy.

1101Kb
[img]
Preview
PDF (Ch. 5) - Final Version
See Usage Policy.

2902Kb
[img]
Preview
PDF (Bibliography) - Final Version
See Usage Policy.

194Kb

Repository Staff Only: item control page