Shao Chen, Zhang Yang, Sun Wei
National Key Laboratory of Medical Molecular Biology, Department of Physiology and Pathophysiology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, China.
Department of Neurosurgery/China National Clinical Research Center for Neurological Diseases, Beijing Tiantan Hospital, Capital Medical University, 6 Tian Tan Xi Li, Beijing 100050, China.
J Proteomics. 2014 Sep 23;109:26-37. doi: 10.1016/j.jprot.2014.06.012. Epub 2014 Jun 27.
High-energy collisional dissociation (HCD) is an efficient peptide fragmentation method that is widely used in Orbitrap mass spectrometers. A greater understanding of HCD fragmentation patterns would benefit the development of proteomic data analysis algorithms. In this study, b and y ion fragmentation patterns and residue-specific cleavage effects in HCD mode were statistically characterized on a LTQ Orbitrap Velos mass spectrometer. We compared HCD and CID spectra collected in an Orbitrap for the same doubly and triply charged tryptic peptides. Our analytical results revealed novel statistical features of HCD spectra. The intensity of y ions reached a maximum in the 60-70% and 40-50% relative mass bins of HCD spectra from doubly and triply charged peptides, respectively. The HCD mode showed a slight preference for generating y ions with lower charges than did CID mode. Singly charged fragment ions dominated the five fragment ions with the highest intensity in HCD spectra. Hydrophobic residues for b ions were the primary differences in cleavage selectivity between the two modes, while residues for y ions showed a similar cleavage preference. These results will assist with the development of database search engines and the design of proper transitions for targeted proteomic analysis.
Orbitrap mass spectrometry is becoming the popular instrument for proteomic analysis, and HCD mode is becoming the main analysis mode for its high resolution and high sensitivity. This study characterizes the features of HCD spectrum by comparing with CID mode on LTQ Velos Orbitrap. The patterns of b and y ions in HCD and CID modes were systematically compared for the first time, including the charge state, ion frequency and intensity and cleavage selectivity. These results will help develop database search engines and design proper transitions for targeted proteomic analysis in the future.
高能碰撞解离（HCD）是一种高效的肽段碎裂方法，广泛应用于轨道阱质谱仪中。更深入地了解HCD碎裂模式将有助于蛋白质组数据分析算法的开发。在本研究中，在LTQ Orbitrap Velos质谱仪上对HCD模式下的b离子和y离子碎裂模式以及残基特异性裂解效应进行了统计表征。我们比较了在轨道阱中针对相同的双电荷和三电荷胰蛋白酶肽段收集的HCD和CID谱图。我们的分析结果揭示了HCD谱图的新统计特征。y离子的强度分别在双电荷和三电荷肽段的HCD谱图的相对质量区间60 - 70%和40 - 50%处达到最大值。与CID模式相比，HCD模式在生成较低电荷的y离子方面表现出轻微偏好。单电荷碎片离子在HCD谱图中强度最高的五个碎片离子中占主导。两种模式之间裂解选择性的主要差异在于b离子的疏水残基，而y离子的残基表现出相似的裂解偏好。这些结果将有助于数据库搜索引擎的开发以及靶向蛋白质组分析中合适跃迁的设计。
轨道阱质谱正成为蛋白质组分析的常用仪器，而HCD模式因其高分辨率和高灵敏度正成为主要的分析模式。本研究通过在LTQ Velos轨道阱上与CID模式比较来表征HCD谱图的特征。首次系统比较了HCD和CID模式下b离子和y离子的模式，包括电荷状态、离子频率和强度以及裂解选择性。这些结果将有助于未来数据库搜索引擎的开发以及靶向蛋白质组分析中合适跃迁的设计。
