Protein sequencing by matrix-assisted laser desorption ionization-postsource decay mass spectrometry analysis of the N-tris(2,4,6-trimethoxyphenyl)phosphine-acetylated tryptic digests
Protein sequencing by matrix-assisted laser desorption ionization-postsource decay mass spectrometry analysis of the N-tris(2,4,6-trimethoxyphenyl)phosphine-acetylated tryptic digests
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DOI：
10.1006/abio.1998.3085
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发表时间：
1999-03-15
影响因子：
2.9
通讯作者：
Watson, JT
中科院分区：
文献类型：
--
作者：
Huang, ZH;Shen, TL;Watson, JT
我们最近报道了一种简单的方法，通过该方法可以将低皮摩尔量的肽修饰成相应的N-三（2，4，6-三甲氧基苯基）膦-乙酰基（TMPP-Ac）衍生物（Z. H Huang，J. Wu，D. A.盖奇和J. T.沃森，阿纳尔。69，137-144，1997）。这种修改显着促进序列的解释，通过提供专门的N-末端产物离子（主要是a-型离子）的快原子轰击-MS/MS和基质辅助激光解吸电离源后衰变（MALDI-PSD）-MS光谱。TMPP-Ac衍生化方法现在已经扩展到直接衍生化胰蛋白酶，其源自1-5 μ g分子量为10-120 kDa的蛋白质。我们的新方法包括在用磷酸盐或Tris-HCl缓冲至pH 8-8.2的水溶液中进行胰蛋白酶消化，然后与TMPP-乙酸N-羟基琥珀酰亚胺酯反应（TMPP-AcOSu溴化物，2-4 nmol试剂/μ g蛋白，室温，20分钟）以提供N-末端衍生化产物，而赖氨酸中的ε-NH 2基团保持不变。随后通过MALDI-PSD-MS使用0.5-至1-pmol等分试样分析所得衍生化肽混合物或其部分分离的HPLC级分，产生易于解释的产物离子光谱。由于不需要材料转移和更换缓冲介质，串联酶-化学反应/MS分析过程通常以非常高的通量进行（消化，1 h;反应，1/3 h; HPLC，1 h; MALDI-PSD，3-4个片段/h）。该程序将有可能用于直接从混合物中获得序列信息或作为肽质量图谱的辅助手段，以提供高置信度的蛋白质鉴定。(C)北京：科学出版社.
We have recently reported a simple procedure by which low picomole quantities of peptides can be modified to the corresponding N-Tris(2,4,6-trimethoxyphenyl)phosphonium-acetyl (TMPP-Ac) derivatives (Z. H Huang, J. Wu, D. A. Gage, and J. T. Watson, Anal. Chem. 69, 137-144, 1997). This modification significantly facilitates sequence interpretation by providing exclusively N-terminal product ions (mainly a-type ions) in the fast-atom bombardment-MS/MS and matrix-assisted laser desorption ionization-postsource decay(MALDI-PSD)-MS spectra. The TMPP- Ac derivatization approach has been extended now for the direct derivatization of tryptic digests originating from 1-5 mu g of proteins with molecular weights from 10-120 kDa, Our new procedure involves tryptic digestion in aqueous solution buffered to pH 8-8.2 with phosphate or Tris-HCl, followed by reaction with TMPP-acetic acid N-hydroxysuccinimide ester (TMPP-AcOSu bromide, 2-4 nmol reagent/mu g protein, rt, 20 min) to provide N-terminally derivatized products, while the epsilon-NH2 groups in lysine remain unchanged. The resultant derivatized peptide mixture or its partially separated HPLC fractions are subsequently analyzed by MALDI-PSD-MS using 0.5- to 1-pmol aliquots, giving rise to product ion spectra that are easily interpretable. As there is no need for material transfer and change of buffer media, the tandem enzymatic-chemical reaction/MS analysis process is usually carried out with very high throughput (digestion, 1 h; reaction, 1/3 h; HPLC, 1 h; MALDI-PSD, 3-4 fragments/h). This procedure will be of potential use for obtaining sequence information directly from mixtures or as an adjunct of peptide mass mapping to provide protein identification with high confidence. (C) 1999 Academic Press.