VIB-UGent Center for Medical Biotechnology, Ghent, Belgium.
Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.
Methods Mol Biol. 2023;2718:1-10. doi: 10.1007/978-1-0716-3457-8_1.
Mass spectrometry-based proteomics combining more than one protease in parallel facilitates the identification of more peptides and proteins than when a single protease is used. Trypsin cleaves proteins C-terminally to arginine and lysine, while its mirroring protease Tryp-N cleaves N-terminally to the same amino acids. Here, we combine trypsin and Tryp-N with the commercially available S-Trap columns, which purify protein samples and catalyze digestion. Comparison of trypsin or Tryp-N coupled with S-Trap columns demonstrates plasma and cell lysate proteins unique to one protease. We thus suggest the use of both proteases in a complementary manner to obtain deeper proteome coverage.
基于质谱的蛋白质组学结合了多种蛋白酶平行处理，可以比使用单一蛋白酶识别更多的肽和蛋白质。胰蛋白酶从 C 端切割蛋白质到精氨酸和赖氨酸，而其镜像蛋白酶 Tryp-N 从 N 端切割到相同的氨基酸。在这里，我们将胰蛋白酶和 Tryp-N 与市售的 S-Trap 柱结合使用，S-Trap 柱可以纯化蛋白质样品并催化消化。胰蛋白酶或 Tryp-N 与 S-Trap 柱偶联的比较表明，一种蛋白酶具有独特的血浆和细胞裂解物蛋白质。因此，我们建议使用两种蛋白酶以互补的方式获得更深入的蛋白质组覆盖。
