Iglesias Amadeu H, Santos Luiz F A, Gozzo Fabio C
Center for Structural and Molecular Biology, Brazilian Synchrotron Light Source, Campinas, Brazil.
J Am Soc Mass Spectrom. 2009 Apr;20(4):557-66. doi: 10.1016/j.jasms.2008.11.009. Epub 2008 Nov 21.
The use of chemical crosslinking is an attractive tool that presents many advantages in the application of mass spectrometry to structural biology. The correct assignment of crosslinked peptides, however, is still a challenge because of the lack of detailed fragmentation studies on resultant species. In this work, the fragmentation patterns of intramolecular crosslinked peptides with disuccinimidyl suberate (DSS) has been devised by using a set of versatile, model peptides that resemble species found in crosslinking experiments with proteins. These peptides contain an acetylated N-terminus followed by a random sequence of residues containing two lysine residues separated by an arginine. After the crosslinking reaction, controlled trypsin digestion yields both intra- and intermolecular crosslinked peptides. In the present study we analyzed the fragmentation of matrix-assisted laser desorption/ionization-generated peptides crosslinked with DSS in which both lysines are found in the same peptide. Fragmentation starts in the linear moiety of the peptide, yielding regular b and y ions. Once it reaches the cyclic portion of the molecule, fragmentation was observed to occur either at the following peptide bond or at the peptide crosslinker amide bond. If the peptide crosslinker bond is cleaved, it fragments as a regular modified peptide, in which the DSS backbone remains attached to the first lysine. This fragmentation pattern resembles the fragmentation of modified peptides and may be identified by common automated search engines using DSS as a modification. If, on the other hand, fragmentation happens at the peptide bond itself, rearrangement of the last crosslinked lysine is observed and a product ion containing the crosslinker backbone and lysine (m/z 222) is formed. The detailed identification of fragment ions can help the development of softwares devoted to the MS/MS data analysis of crosslinked peptides.
化学交联的应用是一种颇具吸引力的工具，在质谱技术应用于结构生物学领域具有诸多优势。然而，由于对交联肽段的详细裂解研究匮乏，交联肽段的正确归属仍是一项挑战。在本研究中，通过使用一组通用的模型肽设计出了用辛二酸二琥珀酰亚胺酯（DSS）交联的分子内交联肽段的裂解模式，这些模型肽类似于在蛋白质交联实验中发现的肽段。这些肽段含有一个乙酰化的N端，随后是一个随机的残基序列，其中包含两个被一个精氨酸隔开的赖氨酸残基。交联反应后，通过可控的胰蛋白酶消化可产生分子内和分子间交联的肽段。在本研究中，我们分析了用DSS交联的基质辅助激光解吸/电离产生的肽段的裂解情况，其中两个赖氨酸位于同一肽段中。裂解始于肽段的线性部分，产生常规的b离子和y离子。一旦到达分子的环状部分，观察到裂解要么发生在接下来的肽键处，要么发生在肽交联剂酰胺键处。如果肽交联剂键被裂解，它会像一个常规的修饰肽段一样裂解，其中DSS主链仍与第一个赖氨酸相连。这种裂解模式类似于修饰肽段的裂解，并且可以通过使用DSS作为修饰的常见自动搜索引擎来识别。另一方面，如果裂解发生在肽键本身，则会观察到最后一个交联赖氨酸的重排，并形成一个包含交联剂主链和赖氨酸（m/z 222）的产物离子。碎片离子的详细鉴定有助于开发专门用于交联肽段的MS/MS数据分析的软件。
