Protein cross-links: Universal isolation and characterization by isotopic derivatization and electrospray ionization mass spectrometry

A general method of unequivocally identifying and obtaining sequence inform ation on cross-linked peptides derived by proteolytic digestion of cross-li nked proteins has been developed. The method is based on isotopic labeling of alpha-amino groups with 2,4-dinitrofluorobenzene (DNFB) coupled with ele ctrospray ionization mass spectrometry. Proteins containing covalent cross- link(s) are reductively methylated to convert lysine residues to dimethyl l ysine. The methylated protein is partially hydrolyzed and the liberated alp ha-amino termini are derivatized with an equimolar mixture of DNFB and [H-2 (3)]DNFB. Dinitrophenyl (DNP)-labeled peptides may be fractionated into mon o- and bis-DNP pools by chromatography on phenyl media, The bis-DNP peptide s are further separated by reverse-phase RPLC and analyzed by electrospray ionization mass spectrometry, The molecular ions of cross-linked peptides a re unambiguously identified as 1:2:1 triplets in the mass spectrum resultin g from the binomial distribution of isotopic label in the bis-DNP derivativ e. Sequence information can be elucidated from the unique product ion patte rns which are generated from in-source fragmentation at an elevated cone vo ltage, Analysis of the disulfide cross-linked peptide (VTCG)(2) was underta ken as a proof of concept and the generality of the method was demonstrated by isolating and sequencing the isopeptide bond of polyubiquitin. (C) 1999 Academic Press.