Development of methods for the analysis of protein post-translational modifications: isoaspartic acid and protein crosslinking.

Permanent URL: http://hdl.handle.net/2047/d20128343
Title:
Development of methods for the analysis of protein post-translational modifications : isoaspartic acid and protein crosslinking
Creator:
Liu, Min (Author)
Contributor:
Zhou, Zhaohui Sunny (Advisor)
Ren, Da (Advisor)
Vouros, Paul (Committee member)
Hancock, William (Committee member)
Cheetham, Janet (Committee member)
Publisher:
Boston, Massachusetts : Northeastern University, 2014
Date Accepted:
September 2014
Date Awarded:
January 2015
Type of resource:
Text
Genre:
Dissertations
Format:
electronic
Digital origin:
born digital
Abstract/Description:
Analysis of protein posttranslational modifications (PTMs) plays pivotal roles for the understanding of their biological importance. Isoaspartic acid (isoAsp) as the smallest PTM is observed in vivo and in vitro. No mass difference and subtle difference in physiochemical property between isoAsp and Asp pose a great challenging for sensitive detection and ambiguous location of isoAsp site in complex samples. A novel assay of isoAsp by exploiting methylation specificity of protein isoaspartate methyltransferase (PIMT) at isoAsp and subsequent 18O-incorporation during methyl ester hydrolysis is presented for sensitive detection and unambiguous site location of several isoAsp residues in IgG1 (Anal Chem 2012, 84, 1056-1062). The method can be applied to biological samples to understand the isoAsp process and identify biomarkers.

Ubiquitous protein crosslinks in biological systems and biopharmaceuticals are reported to result in loss of bioactivity and immunogenicity, but their characterization is poor, especially when the crosslink chemistry is undefined, due to their intrinsic structural complexity and a lack of a systematic analytical approach. A comprehensive methodology, XChem-Finder, has been developed to break down the analytical challenge via 18O labeling and mass spectrometry, leading to the discovery of a total of 14 cross-linked thioether peptides in IgG2, including those that have not been previously reported (Anal Chem 2013, 85, 5900-5908). Furthermore, a novel Histidine-Histidine (His-His) crosslink in IgG1 was successfully discovered and characterized via our XChem-Finder (Anal Chem 2014, 86, 4940-4948). This again demonstrates the broad applicability and utility of our XChem-Finder. The further improvement of XChem-Finder is discussed. The discovery of more novel crosslinks in protein by XChem-Finder will be successful without any doubt.
Subjects and keywords:
18O-labeling
IgG
degradants
characterization
Isoaspartic acid (isoAsp)
protein crosslinks
mass spectrometry
MS
method development for analysis and structure elucidation
protein posttranslational modifications
PTMs
Biochemistry
Biology
Chemistry
Post-translational modification
Isoaspartic acid -- Analysis
Proteins -- Crosslinking -- Analysis
Methylation
Peptides
DOI:
https://doi.org/10.17760/d20128343
Permanent URL:
http://hdl.handle.net/2047/d20128343
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