Chen X, Chen Y H, Anderson V E
School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106-4935, USA.
Anal Biochem. 1999 Sep 10;273(2):192-203. doi: 10.1006/abio.1999.4243.
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.
已经开发出一种明确鉴定交联蛋白经蛋白水解消化得到的交联肽并获取其序列信息的通用方法。该方法基于用2,4 -二硝基氟苯（DNFB）对α-氨基进行同位素标记并结合电喷雾电离质谱法。含有共价交联的蛋白质进行还原甲基化，将赖氨酸残基转化为二甲基赖氨酸。甲基化的蛋白质进行部分水解，释放出的α-氨基末端用DNFB和[(2)H(3)]DNFB的等摩尔混合物进行衍生化。二硝基苯基（DNP）标记的肽可以通过在苯基介质上的色谱法分离成单-DNP和双-DNP组分。双-DNP肽通过反相高效液相色谱进一步分离，并用电喷雾电离质谱分析。在双-DNP衍生物中，由于同位素标记的二项式分布，交联肽的分子离子在质谱中明确鉴定为1:2:1三联体。序列信息可以从在升高的锥电压下源内裂解产生的独特产物离子模式中阐明。对二硫键交联肽（VTCG）(2)进行了分析作为概念验证，并且通过分离和测序多聚泛素的异肽键证明了该方法的通用性。
