Polyphenol Identification Based on Systematic and Robust High-Resolution Accurate Mass Spectrometry Fragmentation

High-mass resolution multi-stage mass spectrometry (MSn) fragmentation was tested for differentiation and identification of metabolites, using a series of 121 polyphenolic molecules. The MSn fragmentation approach is based on the systematic breakdown of compounds, forming a so-called spectral tree. A chip-based nanoelectrospray ionization source was used combined with an ion-trap, providing reproducible fragmentation, and accurate mass read-out in an Orbitrap Fourier transform (FT) MS enabling rapid assignment of elemental formulas to the molecular ions and all fragment ions derived thereof. The used protocol resulted in reproducible MSn fragmentation trees up to MS5. Obtained results were stable over a 5 month time period, a concentration change of 100-fold, and small changes in normalized collision energy, which is key to metabolite annotation and helpful in structure and substructure elucidation. Differences in the hydroxylation and methoxylation patterns of polyphenolic core structures were found to be reflected by the differential fragmentation of the entire molecule, while variation in a glycosylation site displayed reproducible differences in the relative intensities of fragments originating from the same aglycone fragment ion. Accurate MSn-based spectral tree data are therefore a powerful tool to distinguish metabolites with similar elemental formula, thereby assisting compound identification in complex biological samples such as crude plant extracts.

本研究选取121种多酚类分子作为研究对象，对高分辨多级质谱（MSⁿ）碎裂技术用于代谢物的区分与鉴定进行了验证。该多级质谱碎裂方法基于化合物的系统性裂解过程，可生成所谓的谱图树。本研究采用基于芯片的纳米电喷雾电离源与离子阱联用，实现了可重复的碎裂过程；同时搭配Orbitrap傅里叶变换（FT）质谱获取精准质量数读数，可快速为分子离子及其衍生的所有碎片离子分配元素组成。所采用的实验方案可生成可重复的多级质谱碎裂谱图树，最高可达MS5级。所得结果在5个月的周期内、100倍浓度变化以及归一化碰撞能量小幅波动的条件下均保持稳定——这一点对代谢物注释至关重要，同时有助于化合物结构与亚结构的解析。研究发现，多酚类母核结构的羟基化与甲氧基化模式差异，可通过整个分子的差异化碎裂得到体现；而糖基化位点的变化，则会导致同一苷元（aglycone）碎片离子衍生的碎片离子相对强度出现可重复的差异。因此，基于精准多级质谱的谱图树数据，是区分元素组成相似的代谢物的有力工具，可助力复杂生物样本（如植物粗提物）中的化合物鉴定工作。