Solid-phase N-terminal peptide enrichment study by optimizing trypsin proteolysis on homoarginine-modified proteins by mass spectrometry.
Solid-phase N-terminal peptide enrichment study by optimizing trypsin proteolysis on homoarginine-modified proteins by mass spectrometry.
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DOI：
10.1002/rcm.6820
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发表时间：
2014-03-30
影响因子：
2
通讯作者：
Bruce, James E.
中科院分区：
文献类型：
--
作者：
Chowdhury, Saiful M.;Munske, Gerhard R.;Yang, Jonathon;Zhukova, Daria;Hamilton Nguyen;Bruce, James E.
蛋白水解裂解通过在蛋白中产生新的n -末端产生活性前体蛋白。最近发表了一些关于利用赖氨酸残基和胺反应试剂的胍基化来富集原始或新形成的n端肽的策略。为了有效富集n端肽，必须了解胰蛋白酶对同源精氨酸修饰蛋白的水解效率，并开发简单通用的固相n端捕获策略。我们在此提出了一项基于质谱的研究，以评估和优化胰蛋白酶对胍修饰蛋白的蛋白水解。用不同的胰蛋白酶与修饰蛋白的比例研究了胰蛋白酶的蛋白水解。为了捕获原始的n -末端，在蛋白质的胍基化后，将原始的n -末端乙酰化，并用胰蛋白酶消化蛋白质。新形成的n端色氨酸肽用一种新的胺反应性酸可切割固相试剂捕获。然后从溶液的上清液中收集原始的n端肽。我们展示了胰蛋白酶对精氨酸修饰蛋白的效率的详细研究。我们观察到，同型精氨酸残基的水解速率比赖氨酸/精氨酸对应部分要慢，但根据蛋白质与蛋白酶的浓度比，在隔夜消化期后通常会发生裂解。通过从两种标准蛋白泛素和rna酶中富集原始n端肽，研究了固相n端试剂的选择性。我们发现胰蛋白酶在胍基化形式的蛋白质中是有活性的，这取决于酶对蛋白质的浓度、时间和精氨酸残基在序列中的接近程度。新型固相捕获试剂也成功富集了标准蛋白混合物中的n端肽。我们相信胰蛋白酶对同精氨酸修饰蛋白的蛋白水解研究和我们简单通用的固相捕获策略将对质谱富集和蛋白n端序列测定非常有用。
Proteolytic cleavages generate active precursor proteins by creating new N-termini in the proteins. A number of strategies recently published regarding the enrichment of original or newly formed N-terminal peptides using guanidination of lysine residues and amine reactive reagents. For effective enrichment of N-terminal peptides, the efficiency of trypsin proteolysis on homoarginine (guanidinated) modified proteins must be understood and simple and versatile solid-phase N-terminal capture strategies should be developed. We present here a mass spectrometry-based study to evaluate and optimize the trypsin proteolysis on a guanidinated modified protein. Trypsin proteolysis was studied using different amount of trypsin to modified protein ratios. To capture the original N-termini, after guanidination of proteins, original N-termini were acetylated and the proteins were digested with trypsin. The newly formed N-terminal tryptic peptides were captured with a new amine reactive acid-cleavable solid-phase reagent. The original N-terminal peptides were then collected from the supernatant of the solution. We demonstrated a detailed study of the efficiency of enzyme trypsin on homoarginine modified proteins. We observed that the rate of hydrolysis of homoarginine residues compared to their lysine/arginine counter parts were slower but generally cleaved after an overnight digestion period depending on the protein to protease concentration ratios. Selectivity of the solid-phase N-terminal reagent was studied by enrichment of original N-terminal peptides from two standard proteins, ubiquitin and RNaseS. We found enzyme trypsin is active in guanidinated form of protein depending on enzyme to protein concentrations, time and the proximity of arginine residues in the sequence. The novel solid-phase capture reagent also successfully enriched N-terminal peptides from the standard protein mixtures. We believe this trypsin proteolysis study on homoarginine modified proteins and our simple and versatile solid- phase capture strategy could be very useful for enrichment and sequence determination of proteins N-termini by mass spectrometry.
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影响因子：
7.4
作者：
Kim, Jong-Seo;Dai, Ziyu;Aryal, Uma K.;Moore, Ronald J.;Camp, David G., II;Baker, Scott E.;Smith, Richard D.;Qian, Wei-Jun
通讯作者：
Qian, Wei-Jun
影响因子：
3.5
作者：
CHAUVET, J;ACHER, R
通讯作者：
ACHER, R
影响因子：
7.4
作者：
Beardsley, RL;Reilly, JP
通讯作者：
Reilly, JP
影响因子：
48
作者：
McDonald, L;Robertson, DHL;Beynon, RJ
通讯作者：
Beynon, RJ
影响因子：
2
作者：
Chowdhury, SM;Munske, GR;Bruce, JE
通讯作者：
Bruce, JE