Protein cross-links: universal isolation and characterization by isotopic derivatization and electrospray ionization mass spectrometry.
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 alpha-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 alpha-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 (VTCG)(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.
Chen
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School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106-4935, USA.
Chen
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Anderson
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D000595:Amino Acid Sequence; D001704:Biopolymers / Q000737:chemistry / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D003903:Deuterium; D004139:Dinitrofluorobenzene; D013058:Mass Spectrometry / Q000379:methods*; D008745:Methylation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D025821:Polyubiquitin; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D014452:Ubiquitins / Q000737:chemistry / Q000302:isolation & purification
D001704:Biopolymers; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D011506:Proteins; D014452:Ubiquitins; D025821:Polyubiquitin; D003903:Deuterium; D004139:Dinitrofluorobenzene