van der Laan Tom, Elfrink Hyung, Azadi-Chegeni Fatemeh, Dubbelman Anne-Charlotte, Harms Amy C, Jacobs Doris M, Braumann Ulrich, Velders Aldrik H, van Duynhoven John, Hankemeier Thomas
Analytical Biosciences and Metabolomics, Division of Systems Biomedicine and Pharmacology, Leiden Academic Center for Drug Research, Leiden University, 2333 CC, Leiden, the Netherlands.
Agrotechnology and Food Sciences, Laboratory of BioNanoTechnology, Wageningen University, 6700 EK, Wageningen, the Netherlands.
Anal Chim Acta. 2021 Jan 15;1142:28-37. doi: 10.1016/j.aca.2020.10.054. Epub 2020 Oct 30.
The unambiguous identification of unknown compounds is of utmost importance in the field of metabolomics. However, current identification workflows often suffer from error-sensitive methodologies, which may lead to incorrect structure annotations of small molecules. Therefore, we have developed a comprehensive identification workflow including two highly complementary techniques, i.e. liquid chromatography (LC) combined with mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR), and used it to identify five taste-related retention time and m/z features in soy sauce. An off-line directed two-dimensional separation was performed in order to purify the features prior to the identification. Fractions collected during the first dimension separation (reversed phase low pH) were evaluated for the presence of remaining impurities next to the features of interest. Based on the separation between the feature and impurities, the most orthogonal second dimension chromatography (hydrophilic interaction chromatography or reversed phase high pH) was selected for further purification. Unknown compounds down to tens of micromolar concentrations were tentatively annotated by MS and structurally confirmed by MS and NMR. The mass (0.4-4.2 μg) and purity of the isolated compounds were sufficient for the acquisition of one and two-dimensional NMR spectra. The use of a directed two-dimensional chromatography allowed for a fractionation that was tailored to each feature and remaining impurities. This makes the fractionation more widely applicable to different sample matrices than one-dimensional or fixed two-dimensional chromatography. Five proline-based 2,5-diketopiperazines were successfully identified in soy sauce. These cyclic dipeptides might contribute to taste by giving a bitter flavour or indirectly enhancing umami flavour.
在代谢组学领域，明确鉴定未知化合物至关重要。然而，当前的鉴定流程往往受误差敏感方法的影响，这可能导致小分子结构注释错误。因此，我们开发了一种综合鉴定流程，包括两种高度互补的技术，即液相色谱（LC）与质谱（MS）联用以及核磁共振波谱（NMR），并将其用于鉴定酱油中五个与风味相关的保留时间和质荷比特征。为了在鉴定前纯化这些特征，进行了离线定向二维分离。对在第一维分离（反相低pH）过程中收集的馏分进行评估，以确定除目标特征外是否存在残留杂质。根据特征与杂质之间的分离情况，选择最正交的第二维色谱（亲水相互作用色谱或反相高pH）进行进一步纯化。低至数十微摩尔浓度的未知化合物通过质谱初步注释，并通过质谱和核磁共振进行结构确认。分离得到的化合物的质量（0.4 - 4.2μg）和纯度足以获取一维和二维核磁共振谱。使用定向二维色谱可以针对每个特征和残留杂质进行定制分馏。这使得分馏比一维或固定二维色谱更广泛地适用于不同的样品基质。在酱油中成功鉴定出五种基于脯氨酸的2,5 - 二酮哌嗪。这些环二肽可能通过产生苦味或间接增强鲜味来影响风味。
