Université Paris-Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SPI, 91191 Gif-sur-Yvette, France.
Laboratory for Food Safety, French Agency for Food, Environmental and Occupational Health & Safety (ANSES), Université Paris-Est, 94700 Maisons-Alfort, France.
J Proteome Res. 2022 Feb 4;21(2):547-556. doi: 10.1021/acs.jproteome.1c00886. Epub 2021 Dec 30.
We addressed here the need for improved sensitivity of top-down mass spectrometry for identification, differentiation, and absolute quantification of sequence variants of SEA, a bacterial toxin produced by and regularly involved in food poisoning outbreaks (FPO). We combined immunoaffinity enrichment, a protein internal standard, and optimized acquisition conditions, either by full-scan high-resolution mass spectrometry (HRMS) or multiplex parallel reaction monitoring (PRM) mode. Deconvolution of full-scan HRMS signal and PRM detection of variant-specific fragment ions allowed confident identification of each SEA variant. Summing the PRM signal of variant-common fragment ions was most efficient for absolute quantification, illustrated by a sensitivity down to 2.5 ng/mL and an assay variability below 15%. Additionally, we showed that relative PRM fragment ion abundances constituted a supplementary specificity criterion in top-down quantification. The top-down method was successfully evaluated on a panel of enterotoxin-producing strains isolated during FPO, in parallel to the conventional whole genome sequencing, ELISA, and bottom-up mass spectrometry methods. Top-down provided at the same time correct identification of the SEA variants produced and precise determination of the toxin level. The raw files generated in this study can be found on PASSEL (Peptide Atlas) under data set identifier PASS01710.
我们在这里探讨了提高自上而下的质谱法在鉴定、区分和绝对定量 SEA 序列变异体方面的灵敏度的需求，SEA 是一种由 产生的细菌毒素，经常与食物中毒爆发（FPO）有关。我们结合免疫亲和富集、蛋白质内标和优化的采集条件，无论是采用全扫描高分辨率质谱（HRMS）还是多路并行反应监测（PRM）模式。全扫描 HRMS 信号的解卷积和变异体特异性片段离子的 PRM 检测允许对每个 SEA 变异体进行有信心的鉴定。通过将变异体常见片段离子的 PRM 信号求和，对于绝对定量最为有效，灵敏度可低至 2.5ng/mL，检测变异性低于 15%。此外，我们表明，相对 PRM 片段离子丰度构成了自上而下定量的补充特异性标准。该自上而下的方法与传统的全基因组测序、ELISA 和自下而上的质谱法一起，成功地对 FPO 期间分离的产肠毒素菌株进行了评估。自上而下的方法同时提供了产生的 SEA 变异体的正确鉴定和毒素水平的精确测定。本研究中生成的原始文件可以在 PASSEL（肽图集）上找到，数据集标识符为 PASS01710。
