Hennrich Marco L, Boersema Paul J, van den Toorn Henk, Mischerikow Nikolai, Heck Albert J R, Mohammed Shabaz
Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Anal Chem. 2009 Sep 15;81(18):7814-22. doi: 10.1021/ac901108g.
In proteomics, proteolytic peptides are often chemically modified to improve MS analysis, peptide identification, and/or to enable protein/peptide quantification. It is known that such chemical modifications can alter peptide fragmentation in collision induced dissociation MS/MS. Here, we investigated the fragmentation behavior of such chemically modified peptides in MS/MS using the relatively new activation method electron transfer dissociation (ETD). We generated proteolytic peptides using the proteases Lys-N and trypsin and compared the fragmentation behavior of the unlabeled peptides with that of their chemically modified cognates. We investigated the effect of several commonly used modification reactions, namely, guanidination, dimethylation, imidazolinylation, and nicotinylation (ICPL). Of these guanidination and imidazolinylation specifically target the epsilon-amino groups of lysine residues in the peptides, whereas dimethylation and nicotinylation modify both N-termini and epsilon-amino groups of lysine residues. Dimethylation, guanidination, and particularly imidazolinylation of doubly charged Lys-N peptides resulted in a significant increase in peptide sequence coverage, resulting in more reliable peptide identification using ETD. This may be rationalized by the increased basicity and resulting positive charge at the N-termini of these peptides. Nicotinylation of the peptides, on the other hand, severely suppressed backbone fragmentation, hampering the use of this label in ETD based analysis. Doubly charged C-terminal lysine containing tryptic peptides also resulted in an enhanced observation of a single type of fragment ion series when guanidinated or imidazolinylated. These labels would thus facilitate the use of de novo sequencing strategies based on ETD for both arginine and lysine containing tryptic peptides. Since isotopic analogues of the labeling reagents applied in this work are commercially available, one can combine quantitation with improved ETD based peptide sequencing for both Lys-N and trypsin digested samples.
在蛋白质组学中，蛋白水解肽段常经过化学修饰以改善质谱分析、肽段鉴定和/或实现蛋白质/肽段定量。已知此类化学修饰会改变碰撞诱导解离串联质谱（MS/MS）中的肽段碎裂情况。在此，我们使用相对较新的激活方法——电子转移解离（ETD），研究了此类化学修饰肽段在MS/MS中的碎裂行为。我们使用Lys-N蛋白酶和胰蛋白酶生成蛋白水解肽段，并将未标记肽段与其化学修饰对应物的碎裂行为进行了比较。我们研究了几种常用修饰反应的效果，即胍基化、二甲基化、咪唑啉化和烟酰胺化（ICPL）。其中，胍基化和咪唑啉化专门针对肽段中赖氨酸残基的ε-氨基，而二甲基化和烟酰胺化则修饰赖氨酸残基的N端和ε-氨基。双电荷Lys-N肽段的二甲基化、胍基化，尤其是咪唑啉化导致肽段序列覆盖率显著增加，从而使用ETD能更可靠地鉴定肽段。这可以通过这些肽段N端碱性增加以及由此产生的正电荷来解释。另一方面，肽段的烟酰胺化严重抑制了主链碎裂，妨碍了该标记物在基于ETD的分析中的使用。当胍基化或咪唑啉化时，含双电荷C端赖氨酸的胰蛋白酶肽段也导致对单一类型碎片离子系列的观察增强。因此，这些标记物将有助于基于ETD的从头测序策略用于含精氨酸和赖氨酸的胰蛋白酶肽段。由于本工作中使用的标记试剂的同位素类似物有商业供应，对于Lys-N和胰蛋白酶消化的样品，人们可以将定量与基于ETD的改进肽段测序相结合。
