Han Xuemei, Jin Mi, Breuker Kathrin, McLafferty Fred W
Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, NY 14853, USA.
Science. 2006 Oct 6;314(5796):109-12. doi: 10.1126/science.1128868.
For characterization of sequence and posttranslational modifications, molecular and fragment ion mass data from ionizing and dissociating a protein in the mass spectrometer are far more specific than are masses of peptides from the protein's digestion. We extend the approximately 500-residue, approximately 50-kilodalton (kD) dissociation limitation of this top-down methodology by using electrospray additives, heated vaporization, and separate noncovalent and covalent bond dissociation. This process can cleave 287 interresidue bonds in the termini of a 1314-residue (144-kD) protein, specify previously unidentified disulfide bonds between 8 of 27 cysteines in a 1714-residue (200-kD) protein, and correct sequence predictions in two proteins, one with 2153 residues (229 kD).
为了表征序列和翻译后修饰，在质谱仪中使蛋白质电离和解离所获得的分子和碎片离子质量数据，比蛋白质消化产生的肽段质量更具特异性。我们通过使用电喷雾添加剂、加热汽化以及分别进行非共价键和共价键解离，扩展了这种自上而下方法约500个残基、约50千道尔顿（kD）的解离限制。该过程能够切割1314个残基（144 kD）蛋白质末端的287个残基间键，确定1714个残基（200 kD）蛋白质中27个半胱氨酸里8个之间先前未鉴定的二硫键，并校正两种蛋白质（其中一种有2153个残基（229 kD））的序列预测。
