Robotham Scott A, Kluwe Christien, Cannon Joe R, Ellington Andrew, Brodbelt Jennifer S
Department of Chemistry, University of Texas , Austin, Texas 78712, United States.
Anal Chem. 2013 Oct 15;85(20):9832-8. doi: 10.1021/ac402309h. Epub 2013 Oct 3.
Although in silico database search methods remain more popular for shotgun proteomics methods, de novo sequencing offers the ability to identify peptides derived from proteins lacking sequenced genomes and ones with subtle splice variants or truncations. Ultraviolet photodissociation (UVPD) of peptides derivatized by selective attachment of a chromophore at the N-terminus generates a characteristic series of y ions. The UVPD spectra of the chromophore-labeled peptides are simplified and thus amenable to de novo sequencing. This method resulted in an observed sequence coverage of 79% for cytochrome C (eight peptides), 47% for β-lactoglobulin (five peptides), 25% for carbonic anhydrase (six peptides), and 51% for bovine serum albumin (33 peptides). This strategy also allowed differentiation of proteins with high sequence homology as evidenced by de novo sequencing of two variants of green fluorescent protein.
尽管在鸟枪法蛋白质组学方法中，基于计算机的数据库搜索方法仍然更受欢迎，但从头测序能够识别来自缺乏测序基因组的蛋白质以及具有细微剪接变体或截短的蛋白质的肽段。通过在N端选择性连接发色团衍生化的肽段进行紫外光解离（UVPD）会产生一系列特征性的y离子。发色团标记肽段的UVPD光谱得以简化，因此适合从头测序。该方法在细胞色素C（8个肽段）上观察到的序列覆盖率为79%，在β-乳球蛋白（5个肽段）上为47%，在碳酸酐酶（6个肽段）上为25%，在牛血清白蛋白（33个肽段）上为51%。这种策略还能够区分具有高度序列同源性的蛋白质，绿色荧光蛋白的两种变体的从头测序证明了这一点。
