Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia.
Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark.
Rapid Commun Mass Spectrom. 2023 Jul 30;37(14):e9534. doi: 10.1002/rcm.9534.
Selective derivatization of peptide N-terminus with 4-formyl-benzenesulfonic acid (FBSA) enables chemically activated fragmentation in positive and negative ion modes (ESI+/-) under charge reduction conditions. Overlapped positive and negative tandem mass spectra show b-ions making the assignment of b-ion series fragments easy and accurate.
We developed an FBSA-peptide microwave-assisted derivatization procedure. Derivatized and nonderivatized bovine serum albumin tryptic peptides and insulin non-tryptic peptide were compared after tandem mass spectrometry (MS/MS) analysis in positive and negative ion modes. A high-quality data set of sulfonated b-ions obtained in negative tandem mass spectra of singly charged FBSA-peptides were matched to detected b-ions in positive MS/MS spectra. Moreover, negative spectra signals were converted and matched against y-ions in positive tandem mass spectra to identify complete peptide sequences.
The FBSA derivatization procedure produced a significantly improved MS/MS data set (populated by high-intensity signals of b- and y-ions) compared to commonly used N-terminal sulfonation reagents. Undesired side reactions almost do not occur, and the procedure reduces the derivatization time. It was found that b-ion intensities comprise 15% and 13% compared to combined ion intensities generated in positive- and negative ion modes, respectively. High visibility of b-ion series in negative ion mode can be attributed to N-terminal sulfonation that had no negative effect on the production of b- and y-ion series in positive ion mode.
The FBSA derivatization and de novo sequencing approach outlined here is a reliable method for accurate peptide sequence assignment. Increased production of b-ions in positive- and negative ion modes greatly improves peak assignment and thus enables accurate sequence reconstruction. Implementation of the named methodology would improve the quality of de novo sequencing data and reduce the number of misinterpreted spectra.
通过 4-甲酰基苯磺酸（FBSA）选择性衍生化肽的 N 端，在电荷还原条件下，可以在正离子和负离子模式（ESI+/-）下进行化学活化碎裂。重叠的正离子和负离子串联质谱显示 b-离子，使 b-离子系列碎片的分配变得容易和准确。
我们开发了一种 FBSA-肽微波辅助衍生化程序。在正离子和负离子模式下进行串联质谱（MS/MS）分析后，比较了衍生化和未衍生化牛血清白蛋白胰蛋白酶肽以及胰岛素非胰蛋白酶肽。从单电荷 FBSA-肽的负离子串联质谱中获得的高质量磺化 b-离子数据集与正 MS/MS 谱中检测到的 b-离子相匹配。此外，将负离子谱信号转换并与正串联质谱中的 y-离子匹配，以鉴定完整的肽序列。
与常用的 N 端磺化试剂相比，FBSA 衍生化程序产生了显著改善的 MS/MS 数据集（由 b-和 y-离子的高强度信号组成）。几乎不会发生不需要的副反应，并且该程序缩短了衍生化时间。发现 b-离子强度分别占正离子和负离子模式下产生的总离子强度的 15%和 13%。负离子模式下 b-离子系列的高可见度归因于 N 端磺化，它对正离子模式下 b-和 y-离子系列的产生没有负面影响。
本文概述的 FBSA 衍生化和从头测序方法是一种可靠的方法，可用于准确的肽序列分配。正离子和负离子模式中 b-离子的产量增加大大改善了峰分配，从而能够进行准确的序列重建。实施该命名方法将提高从头测序数据的质量，并减少错误解释的谱图数量。
