Large-Scale Quantitative Assessment of Different In-Solution Protein Digestion Protocols Reveals Superior Cleavage Efficiency of Tandem Lys-C/Trypsin Proteolysis over Trypsin Digestion
Large-Scale Quantitative Assessment of Different In-Solution Protein Digestion Protocols Reveals Superior Cleavage Efficiency of Tandem Lys-C/Trypsin Proteolysis over Trypsin Digestion
复制标题
DOI：
10.1021/pr300273g
复制
发表时间：
2012-11-01
影响因子：
4.4
通讯作者：
Schmidt, Alexander
中科院分区：
文献类型：
--
作者：
Glatter, Timo;Ludwig, Christina;Schmidt, Alexander
将蛋白质样品完全和特异性地蛋白水解切割成肽对于每个鸟枪式LC-MS/MS实验的成功至关重要。特别是，流行的基于肽的无标记和靶向质谱方法依赖于完全切割肽的有效生成，以确保准确和灵敏的蛋白质定量。与以前的研究相比，我们使用酵母细胞裂解物、无标记定量和统计分析，全面和定量地评估了不同消化策略的效率。消化条件包括双重胰蛋白酶消化、表面活性剂辅助消化和串联组合Lys-C/胰蛋白酶消化。与胰蛋白酶解酶相比，发现Lys-C/胰蛋白酶解酶最有效地产生完全切割的肽，同时减少错误切割的肽的丰度。随后的序列背景分析显示，Lys-C/胰蛋白酶对两侧为带电碱性和特定酸性残基的错切序列段的消化性能得到改善。此外，有针对性的MS分析表明，只有在Lys-C/胰蛋白酶消化后，才能进行更全面的蛋白质切割，从而实现更准确的绝对蛋白质定量，并扩大了适用于SRM检测开发的肽的数量。因此，我们得出结论，连续Lys-C/胰蛋白酶消化对于基于溶液中消化方案的定量MS蛋白质组学中的大多数应用都非常有吸引力。
The complete and specific proteolytic cleavage of protein samples into peptides is crucial for the success of every shotgun LC-MS/MS experiment. In particular, popular peptide-based label-free and targeted mass spectrometry approaches rely on efficient generation of fully cleaved peptides to ensure accurate and sensitive protein quantification. In contrast to previous studies, we globally and quantitatively assessed the efficiency of different digestion strategies using a yeast cell lysate, label-free quantification, and statistical analysis. Digestion conditions include double tryptic, surfactant-assisted, and tandem-combinatorial Lys-C/trypsin digestion. In comparison to tryptic digests, Lys-C/trypsin digests were found most efficient to yield fully cleaved peptides while reducing the abundance of miscleaved peptides. Subsequent sequence context analysis revealed improved digestion performances of Lys-C/trypsin for miscleaved sequence stretches flanked by charged basic and particulary acidic residues. Furthermore, targeted MS analysis demonstrated a more comprehensive protein cleavage only after Lys-C/trypsin digestion, resulting in a more accurrate absolute protein quantification and extending the number of peptides suitable for SRM assay development Therefore, we conclude that a serial Lys-C/trypsin digestion is highly attractive for most applications in quantitative MS-based proteomics building on in-solution digestion schemes.