Zhang Mingxuan, Kaltashov Igor A
Department of Chemistry, University of Massachusetts at Amherst, Amherst, Massachusetts 01003, USA.
Anal Chem. 2006 Jul 15;78(14):4820-9. doi: 10.1021/ac060132w.
Fast mapping of disulfide bonds in proteins containing multiple cysteine residues is often required in order to assess the integrity of the tertiary structure of proteins prone to degradation and misfolding or to detect distinct intermediate states generated in the course of oxidative folding. A new method of rapid detection and identification of disulfide-linked peptides in complex proteolytic mixtures utilizes the tendency of collision-activated peptide ions to lose preferentially side chains of select amino acids in the negative ion mode. Cleavages of cysteine side chains result in efficient dissociation of disulfide bonds and produce characteristic signatures in the fragment ion mass spectra. While cleavages of other side chains result in insignificant loss of mass and full retention of the peptide ion charge, dissociation of external disulfide bonds results in physical separation of two peptides and, therefore, significant changes of both mass and charge of fragment ions relative to the precursor ion. This feature allows the fragment ions generated by dissociation of external disulfide bonds to be easily detected and identified even if multiple precursor ions are activated simultaneously. Such broadband selection of precursor ions for consecutive activation is achieved by lowering the dc/rf amplitude ratio in the first quadrupole filter of a hybrid quadrupole time-of-flight mass spectrometer. The feasibility of the new method is demonstrated by partial mapping of disulfide bridges within a 37-kDa protein containing 16 cysteine residues and complete disulfide mapping within a lysozyme (14.5 kDa) containing 8 cysteine residues. In addition to detecting peptide pairs connected by a single external disulfide, the new method is also shown to be capable of identifying peptides containing both external and internal disulfide bonds. The two major factors determining the efficiency of disulfide mapping using the new methodology are the effectiveness of proteolysis and the ability of the resulting proteolytic fragments to form multiply charged negative ions.
为了评估易于降解和错误折叠的蛋白质三级结构的完整性，或检测氧化折叠过程中产生的不同中间状态，通常需要对含有多个半胱氨酸残基的蛋白质中的二硫键进行快速定位。一种在复杂蛋白水解混合物中快速检测和鉴定二硫键连接肽段的新方法，利用了碰撞激活肽离子在负离子模式下优先丢失特定氨基酸侧链的趋势。半胱氨酸侧链的裂解导致二硫键有效解离，并在碎片离子质谱中产生特征信号。虽然其他侧链的裂解导致质量损失不显著且肽离子电荷完全保留，但外部二硫键的解离导致两个肽段物理分离，因此，相对于前体离子，碎片离子的质量和电荷都发生了显著变化。这一特性使得即使同时激活多个前体离子，由外部二硫键解离产生的碎片离子也能很容易地被检测和鉴定。通过降低混合四极杆飞行时间质谱仪第一四极杆过滤器中的直流/射频幅度比，可以实现对前体离子的这种宽带选择以进行连续激活。通过对含有16个半胱氨酸残基的37 kDa蛋白质中二硫键桥的部分定位以及对含有8个半胱氨酸残基的溶菌酶（14.5 kDa）中完整二硫键的定位，证明了该新方法的可行性。除了检测由单个外部二硫键连接的肽段对之外，该新方法还能够鉴定同时含有外部和内部二硫键的肽段。使用新方法确定二硫键定位效率的两个主要因素是蛋白水解的有效性以及所得蛋白水解片段形成多电荷负离子的能力。
