Identification and Sequencing of N-Terminal Peptides in Proteins by LC-Fluorescence-MS/MS: An Approach to Replacement of the Edman Degradation,Analytical Chemistry

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Identification and Sequencing of N-Terminal Peptides in Proteins by LC-Fluorescence-MS/MS: An Approach to Replacement of the Edman Degradation
Analytical Chemistry ( IF 6.7 ) Pub Date : 2019-10-15 , DOI: 10.1021/acs.analchem.9b02754
Malgorzata Monika Vecchi ₁ , Yongsheng Xiao ₁ , Dingyi Wen ₁

Affiliation

Automated Edman degradation for sequencing is the classic tool for determining the N-terminal sequences of proteins. Although it has been complemented or largely replaced by a variety of mass spectrometric methods in recent years, there is no simple mass spectrometric method for identification and de novo determination of the N-terminal sequence of a protein. Here, we present a simple and reliable approach which identifies N-terminal peptides of proteins by selectively labeling the N-terminal α-amino groups with a fluorescent reagent followed by enzyme digestion and tandem mass spectrometric analysis using an LC-MS/MS system coupled with a fluorescence detector. This approach also includes a differential peptide mapping step for identification of pyroglutamate-blocked N-terminal peptides. In addition, the strategy can be applied to proteins in gel. With this approach, complete N-terminal sequences of proteins can be obtained precisely and efficiently.

中文翻译：

LC-荧光-MS / MS对蛋白质中N末端肽的鉴定和测序：一种替代Edman降解的方法

用于测序的自动Edman降解是确定蛋白质N端序列的经典工具。尽管近年来它已被各种质谱方法补充或很大程度上替代了，但没有简单的质谱方法可用于识别和重新鉴定确定蛋白质的N端序列。在这里，我们提出了一种简单可靠的方法，该方法通过使用荧光试剂选择性标记N端α-氨基基团，然后使用LC-MS / MS系统进行酶消化和串联质谱分析来鉴定蛋白质的N端肽段带有荧光检测器。该方法还包括用于鉴定焦谷氨酸封闭的N-末端肽的差异肽作图步骤。另外，该策略可以应用于凝胶中的蛋白质。通过这种方法，可以精确而有效地获得蛋白质的完整N末端序列。

更新日期：2019-10-16

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