Kapp Eugene A, Schütz Frédéric, Reid Gavin E, Eddes James S, Moritz Robert L, O'Hair Richard A J, Speed Terence P, Simpson Richard J
Joint ProteomicS Laboratory, Ludwig Institute for Cancer Research and The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia.
Anal Chem. 2003 Nov 15;75(22):6251-64. doi: 10.1021/ac034616t.
A database of 5500 unique peptide tandem mass spectra acquired in an ion trap mass spectrometer was assembled for peptides derived from proteins digested with trypsin. Peptides were identified initially from their tandem mass spectra by the SEQUEST algorithm and subsequently validated manually. Two different statistical methods were used to identify sequence-dependent fragmentation patterns that could be used to improve fragmentation models incorporated into current peptide sequencing and database search algorithms. The currently accepted "mobile proton" model was expanded to derive a new classification scheme for peptide mass spectra, the "relative proton mobility" scale, which considers peptide ion charge state and amino acid composition to categorize peptide mass spectra into peptide ions containing "nonmobile", "partially mobile", or "mobile" protons. Quantitation of amide bond fragmentation, both N- and C-terminal to any given amino acid, as well as the positional effect of an amino acid in a peptide and peptide length on such fragmentation, has been determined. Peptide bond cleavage propensities, both positive (i.e., enhanced) and negative (i.e., suppressed), were determined and ranked in order of their cleavage preferences as primary, secondary, or tertiary cleavage effects. For example, primary positive cleavage effects were observed for Xaa-Pro and Asp-Xaa bond cleavage for mobile and nonmobile peptide ion categories, respectively. We also report specific pairwise interactions (e.g., Asn-Gly) that result in enhanced amide bond cleavages analogous to those observed in solution-phase chemistry. Peptides classified as nonmobile gave low or insignificant scores, below reported MS/MS score thresholds (cutoff filters), indicating that incorporation of the relative proton mobility scale classification would lead to improvements in current MS/MS scoring functions.
构建了一个包含5500个独特肽段串联质谱的数据库，这些质谱是在离子阱质谱仪中获取的，肽段来源于用胰蛋白酶消化的蛋白质。肽段最初通过SEQUEST算法从其串联质谱中鉴定出来，随后进行人工验证。使用了两种不同的统计方法来识别序列依赖性断裂模式，这些模式可用于改进当前肽段测序和数据库搜索算法中纳入的断裂模型。目前被接受的“移动质子”模型得到扩展，以推导一种新的肽质谱分类方案，即“相对质子迁移率”尺度，该尺度考虑肽离子电荷状态和氨基酸组成，将肽质谱分类为包含“非移动”、“部分移动”或“移动”质子的肽离子。已确定了任何给定氨基酸N端和C端酰胺键断裂的定量，以及肽段中氨基酸的位置效应和肽段长度对这种断裂的影响。确定了肽键断裂倾向，包括正向（即增强）和负向（即抑制），并按其作为一级、二级或三级断裂效应的断裂偏好顺序进行排序。例如，对于移动和非移动肽离子类别，分别观察到Xaa-Pro和Asp-Xaa键断裂的一级正向断裂效应。我们还报告了导致酰胺键断裂增强的特定成对相互作用（例如Asn-Gly），类似于在溶液相化学中观察到的情况。分类为非移动的肽段得分较低或不显著，低于报告的MS/MS得分阈值（截止过滤器），这表明纳入相对质子迁移率尺度分类将导致当前MS/MS评分函数的改进。
