Protein Cross-Links: Universal Isolation and Characterization by Isotopic Derivatization and Electrospray Ionization Mass Spectrometry

✍ Scribed by Xiaohui Chen; Yong Hong Chen; Vernon E. Anderson

• Book ID: 102559898
• Publisher: Elsevier Science
• Year: 1999
• Tongue: English
• Weight: 156 KB
• Volume: 273
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1999.4243

✦ Synopsis

A general method of unequivocally identifying and obtaining sequence information on cross-linked peptides derived by proteolytic digestion of cross-linked proteins has been developed. The method is based on isotopic labeling of ␣-amino groups with 2,4-dinitrofluorobenzene (DNFB) coupled with electrospray ionization mass spectrometry. Proteins containing covalent cross-link(s) are reductively methylated to convert lysine residues to dimethyl lysine. The methylated protein is partially hydrolyzed and the liberated ␣-amino termini are derivatized with an equimolar mixture of DNFB and [ 2 H 3 ]DNFB. Dinitrophenyl (DNP)-labeled peptides may be fractionated into mono-and bis-DNP pools by chromatography on phenyl media. The bis-DNP peptides are further separated by reverse-phase HPLC and analyzed by electrospray ionization mass spectrometry. The molecular ions of cross-linked peptides are unambiguously identified as 1:2:1 triplets in the mass spectrum resulting from the binomial distribution of isotopic label in the bis-DNP derivative. Sequence information can be elucidated from the unique product ion patterns which are generated from in-source fragmentation at an elevated cone voltage. Analysis of the disulfide cross-linked peptide (VTC ¸G) 2 was undertaken as a proof of concept and the generality of the method was demonstrated by isolating and sequencing the isopeptide bond of polyubiquitin.