超声辅助酶切法用于溶液内蛋白质样品酶切特点分析

目的分析超声辅助酶切法对溶液内蛋白质样品酶切的特点。方法分别应用超声辅助酶切法与传统酶切法对牛血清白蛋白进行溶液内酶切,通过飞行时间质谱进行分析,对数据库检索后得到的多肽进行比较分析。结果超声辅助酶切法的酶切时间可缩短到10 min,酶切后多肽的回收率比传统酶切法低8%,但飞行时间质谱鉴定评分(MASCOT评分)比传统方法高约1倍,肽段的覆盖率高10%,匹配的多肽数多40%以上。对超声辅助酶切法鉴定出的多肽进一步分析表明,这些多肽的误切率较传统方法高22%,比传统方法多鉴定出的多肽分子质量主要在2～3 ku之间,且大部分为含有误切位点的多肽。结论超声辅助酶切可明显缩短蛋白质酶切时间,得到更好的多肽鉴定,但部分多肽酶切不完全,样品损失会更多。

Analysis of the characteristics of ultrasound-assisted digestion method for in-solution protein sample

Objective Analyze the characteristics of ultrasound-assisted digestion for in-solution protein sample. Methods Bovine serum albumin was in-solution digested by ultrasound-assisted digestion and overnight digestion methods, and the digested peptides were analyzed by MALDI-TOF mass spectrometry. After database searching the identified peptides from two methods were analyzed. Results The digestion time of ultrasound-assisted method can be shortened to 10 minutes, but the peptide recovery was 8% lower than overnight method. For peptide identifications the MASCOT score, protein coverage and peptide number of ultrasound-assisted method was about 1 fold, 10% and 40% higher than that of overnight method, but the miscleavage peptide rate was 22% higher. Analysis of the peptides only identified from ultrasound-assisted method showed that molecular weight distribution was 2~3ku and most of them was miscleavage peptides. Conclusions Ultrasound-assisted digestion method can significantly shorten protein digestion time and obtain better peptide identifications, but the sample loss will be increased and part of the peptides were not completely digested.