Department of Chemistry, University of Kansas, Lawrence, KS 66045, USA.
J Am Soc Mass Spectrom. 2011 Mar;22(3):492-8. doi: 10.1007/s13361-010-0064-x. Epub 2011 Feb 5.
Disulfide bonds are a post-translational modification (PTM) that can be scrambled or shuffled to non-native bonds during recombinant expression, sample handling, or sample purification. Currently, mapping of disulfide bonds is not easy because of various sample requirements and data analysis difficulties. One step towards facilitating this difficult work is developing a better understanding of how disulfide-bonded peptides fragment during collision induced dissociation (CID). Most automated analysis algorithms function based on the assumption that the preponderance of product ions observed during the dissociation of disulfide-bonded peptides result from the cleavage of just one peptide bond, and in this report we tested that assumption by extensively analyzing the product ions generated when several disulfide-bonded peptides are subjected to CID on a quadrupole time of flight (QTOF) instrument. We found that one of the most common types of product ions generated resulted from two peptide bond cleavages, or a double cleavage. We found that for several of the disulfide-bonded peptides analyzed, the number of double cleavage product ions outnumbered those of single cleavages. The influence of charge state and precursor ion size was investigated, to determine if those parameters dictated the amount of double cleavage product ions formed. It was found in this sample set that no strong correlation existed between the charge state or peptide size and the portion of product ions assigned as double cleavages. These data show that these ions could account for many of the product ions detected in CID data of disulfide bonded peptides. We also showed the utility of double cleavage product ions on a peptide with multiple cysteines present. Double cleavage products were able to fully characterize the bonding pattern of each cysteine where typical single b/y cleavage products could not.
二硫键是一种翻译后修饰（PTM），在重组表达、样品处理或样品纯化过程中，可能会发生非天然键的混乱或错配。目前，由于各种样品要求和数据分析困难，二硫键的定位并不容易。朝着这个方向迈进的一步是更好地了解二硫键结合的肽在碰撞诱导解离（CID）过程中如何发生片段化。大多数自动分析算法的功能基于这样的假设，即在二硫键结合肽的解离过程中观察到的大多数产物离子是由于仅仅一个肽键的裂解产生的，在本报告中，我们通过广泛分析几个二硫键结合肽在四极杆飞行时间（QTOF）仪器上进行 CID 时产生的产物离子来检验这一假设。我们发现，产生的最常见类型的产物离子之一来自两个肽键的裂解，或双裂解。我们发现，对于分析的几个二硫键结合肽，双裂解产物离子的数量超过了单裂解产物离子的数量。我们研究了电荷状态和前体离子大小的影响，以确定这些参数是否决定了双裂解产物离子的形成量。在这个样本集中发现，在电荷状态或肽大小与被指定为双裂解的产物离子的比例之间没有很强的相关性。这些数据表明，这些离子可能解释了在 CID 数据中二硫键结合肽中检测到的许多产物离子。我们还展示了在存在多个半胱氨酸的肽上使用双裂解产物离子的实用性。双裂解产物能够充分表征每个半胱氨酸的键合模式，而典型的单 b/y 裂解产物则不能。
