Lau King Wai, Hart Sarah R, Lynch Jennifer A, Wong Stephen C C, Hubbard Simon J, Gaskell Simon J
Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.
Rapid Commun Mass Spectrom. 2009 May;23(10):1508-14. doi: 10.1002/rcm.4032.
Tandem mass spectrometric data from peptides are routinely used in an unsupervised manner to infer product ion sequence and hence the identity of their parent protein. However, significant variability in relative signal intensity of product ions within peptide tandem mass spectra is commonly observed. Furthermore, instrument-specific patterns of fragmentation are observed, even where a common mechanism of ion heating is responsible for generation of the product ions. This information is currently not fully exploited within database searching strategies; this motivated the present study to examine a large dataset of tandem mass spectra derived from multiple instrumental platforms. Here, we report marked global differences in the product ion spectra of protonated tryptic peptides generated from two of the most common proteomic platforms, namely tandem quadrupole-time-of-flight and quadrupole ion trap instruments. Specifically, quadrupole-time-of-flight tandem mass spectra show a significant under-representation of N-terminal b-type fragments in comparison to quadrupole ion trap product ion spectra. Energy-resolved mass spectrometry experiments conducted upon test tryptic peptides clarify this disparity; b-type ions are significantly less stable than their y-type N-terminal counterparts, which contain strongly basic residues. Secondary fragmentation processes which occur within the tandem quadrupole-time-of-flight device account for the observed differences, whereas this secondary product ion generation does not occur to a significant extent from resonant excitation performed within the quadrupole ion trap. We suggest that incorporation of this stability information in database searching strategies has the potential to significantly improve the veracity of peptide ion identifications as made by conventional database searching strategies.
来自肽段的串联质谱数据通常以无监督方式用于推断产物离子序列，进而推断其母蛋白的身份。然而，肽段串联质谱中产物离子的相对信号强度通常存在显著差异。此外，即使离子加热的共同机制导致产物离子的产生，也会观察到特定仪器的碎裂模式。目前，这些信息在数据库搜索策略中尚未得到充分利用；这促使本研究检查来自多个仪器平台的大量串联质谱数据集。在此，我们报告了由两种最常见的蛋白质组学平台（即串联四极杆-飞行时间质谱仪和四极杆离子阱仪器）产生的质子化胰蛋白酶肽段的产物离子谱存在明显的全局差异。具体而言，与四极杆离子阱产物离子谱相比，串联四极杆-飞行时间质谱显示N端b型片段的代表性明显不足。对测试胰蛋白酶肽段进行的能量分辨质谱实验澄清了这种差异；b型离子的稳定性明显低于其y型N端对应物，后者含有强碱性残基。串联四极杆-飞行时间装置内发生的二级碎裂过程解释了观察到的差异，而在四极杆离子阱内进行的共振激发在很大程度上不会产生这种二级产物离子。我们认为，将这种稳定性信息纳入数据库搜索策略有可能显著提高传统数据库搜索策略进行的肽离子鉴定的准确性。
