使用注射器进行加压酶解。

Pressure-assisted tryptic digestion using a syringe.

机构信息

Department of Chemistry, Chungnam National University, Daejeon, South Korea.

出版信息

Rapid Commun Mass Spectrom. 2010 Apr 15;24(7):901-8. doi: 10.1002/rcm.4467.

PMID:20196188

Abstract

A simple and effective digestion method was developed using a syringe. A 3 mL syringe was used to apply a pressure of 6 atm to expedite tryptic digestion. Application of a pressure of 6 atm during digestion resulted in better digestion efficiency than digestion under atmospheric pressure. The protein peaks in the matrix-assisted laser desorption/ionization mass spectra of three model proteins (cytochrome c, horse heart myoglobin, and bovine serum albumin (BSA)) completely disappeared within 30 min at 37 degrees C under a pressure of 6 atm, with greater numbers of peptides observed in 30 min pressure-assisted digestion than in overnight atmospheric pressure digestion. This is mostly due to the miscleaved peptides. Similar sequence coverages were obtained for 30 min pressure-assisted digestion and overnight atmospheric pressure digestion of the three model proteins (92% vs. 88% for cytochrome c, 100% vs. 97% for horse heart myoglobin, and 53% vs. 53% for BSA).

摘要

开发了一种简单有效的消化方法，使用注射器进行。使用 3 毫升注射器施加 6 个大气压的压力，以加快胰蛋白酶消化。与在大气压下消化相比，在消化过程中施加 6 个大气压的压力可提高消化效率。在 37°C 下施加 6 个大气压的压力，3 种模型蛋白（细胞色素 c、马心血红蛋白和牛血清白蛋白（BSA））的基质辅助激光解吸/电离质谱中的蛋白峰在 30 分钟内完全消失，在 30 分钟加压消化中观察到的肽数量多于过夜大气压消化。这主要是由于肽的错误切割。对 3 种模型蛋白（细胞色素 c 为 30 分钟加压消化和过夜大气压消化分别为 92%和 88%，马心血红蛋白为 100%和 97%，BSA 为 53%和 53%），获得了相似的序列覆盖率。