Data for: Oligonucleotide mapping via mass spectrometry to enable comprehensive primary structure characterization of an mRNA vaccine against SARS-CoV-2

Oligonucleotide mapping via liquid chromatography mass spectrometry mass spectrometry (LC-MS/MS) was recently developed to support development of Comirnaty®, the world’s first commercial mRNA vaccine which immunizes against the SARS-CoV-2 virus. Analogous to peptide mapping of therapeutic protein modalities, oligonucleotide mapping described here provides direct primary structure characterization of mRNA, through enzymatic digestion, accurate mass determinations, and optimized collisionally-induced fragmentation. Sample preparation for oligonucleotide mapping is a rapid, one-pot, one-enzyme digestion. The digest is analyzed via LC-MS/MS with an extended gradient and resulting data analysis employs semi-automated software. In a single method, oligonucleotide mapping readouts include a highly reproducible and completely annotated UV chromatogram with >98% sequence coverage and a microheterogeneity assessment of 5´ terminus capping and 3´ terminus poly(A) tail length. Oligonucleotide ma..., Oligonucleotide mapping was developed with a representative batch of Comirnaty® BNT162b2 Original DS (i.e., the original Pfizer-BioNTech COVID-19 vaccine that encodes for the spike glycoprotein (S) of the SARS-CoV-2 virus, the Wuhan-Hu-1 isolate: GenBank: QHD43416.1), and it has been applied to subsequent Comirnaty® BNT162b2 constructs (BNT162b2s04 [Delta] and BNT162b2s05 [Omicron]) and other portfolio mRNA molecules. Fifty micrograms of mRNA DS was digested with 2500 U of RNase T1 in a 50 mM Tris(hydroxymethyl)aminomethane (Tris) pH 7.5 buffer with 20 mM Ethylenediaminetetraacetic acid (EDTA) 1 h at 37°C. The resulting enzymatic fragment solution was spiked with 10× triethylamine (TEA) and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) emulsion to give a final v/v concentration of 0.1% TEA 1% HFIP. A 4 µg load was injected and fragments were separated by ion-pair reversed-phase ultrahigh performance liquid chromatography (IP RP-UHPLC) with UV detection at 260 nm using a 1290 Infinity II Bio ..., The Thermo mass spectrometer .raw files can be opened by Xcalibur Qual Browser (Thermo Fisher Scientific), which is (proprietary) commercial software. An open-source alternative is ThermoRawFileParser (https://doi.org/10.1021%2Facs.jproteome.9b00328).
Automated identification of oligonucleotides was accomplished using BioPharma Finder v5.0 (Thermo Fisher Scientific), which is (propriety) commercial software. An open-source alternative is attached in this dataset: three Excel VBA spreadsheets that may be used to in the identification of any MS feature. As Excel VBA spreadsheets, they are fully compiled and do not require any library code extensions or external links.
The \"Oligonucleotide MS Peak ID Given Sequence v8.xlsm\" spreadsheet may be used to generate a list of theoretical RNaseT1 digest oligonucleotides and match observed precursor masses to candidate theoretical RNaseT1 digest oligonucleotides. Inputs are a single or list of observed masses and a target mRNA construct sequence.
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基于液相色谱-串联质谱法（LC-MS/MS）的寡核苷酸图谱分析（Oligonucleotide mapping），近日被开发用于支持复必泰®（Comirnaty®）的研发——该疫苗是全球首款商用新型冠状病毒（SARS-CoV-2）信使核糖核酸（mRNA）疫苗。与治疗性蛋白制剂的肽图谱分析类似，本文所述的寡核苷酸图谱分析可通过酶解、精准质量测定及优化的碰撞诱导解离，直接实现mRNA的一级结构表征。寡核苷酸图谱分析的样品前处理采用快速的单管单酶解流程，酶解产物通过延长梯度的LC-MS/MS进行分析，后续数据分析采用半自动化软件完成。在单一分析方法中，寡核苷酸图谱分析的输出结果包含：重现性极佳且完整注释的紫外色谱图（序列覆盖率＞98%），以及对5'端加帽修饰和3'端poly(A)尾长的微观异质性评估。寡核苷酸图谱分析最初基于一批代表性的复必泰® BNT162b2原始原料药（DS），即首款辉瑞-生物泰克新冠疫苗，其编码新型冠状病毒Wuhan-Hu-1毒株的刺突糖蛋白（S蛋白），该序列的GenBank登录号为QHD43416.1；该方法后续已被应用于复必泰® BNT162b2的衍生构建株（BNT162b2s04 [德尔塔（Delta）]及BNT162b2s05 [奥密克戎（Omicron）]）以及其他管线内mRNA分子。取50 μg mRNA原料药，于37℃下在含20 mM乙二胺四乙酸（EDTA）的50 mM三(羟甲基)氨基甲烷（Tris）缓冲液（pH 7.5）中，用2500 U核糖核酸酶T1（RNase T1）酶解1小时。向酶解得到的片段溶液中添加10×三乙胺（TEA）与1,1,1,3,3,3-六氟-2-丙醇（HFIP）乳化液，最终体系的体积分数为0.1% TEA与1% HFIP。进样4 μg酶解产物，采用1290 Infinity II Bio系列液相色谱系统，通过离子对反相超高效液相色谱（IP RP-UHPLC）分离片段，并以260 nm波长进行紫外检测。赛默飞质谱仪生成的.raw格式文件可通过赛默飞世尔科技（Thermo Fisher Scientific）的Xcalibur定性浏览器（Xcalibur Qual Browser）打开，该软件为商用专有软件；其开源替代工具为ThermoRawFileParser，相关DOI为10.1021/acs.jproteome.9b00328。寡核苷酸的自动化鉴定可通过赛默飞世尔科技的BioPharma Finder v5.0软件完成，该软件为商用专有软件；本数据集附带一款开源替代工具：可用于鉴定任意质谱特征的3份Excel VBA宏工作表。作为Excel VBA工作表，这些文件已完全编译，无需任何库代码扩展或外部链接。其中名为"Oligonucleotide MS Peak ID Given Sequence v8.xlsm"的工作表可用于生成理论核糖核酸酶T1酶解寡核苷酸列表，并将观测到的前体质量与候选理论酶解寡核苷酸进行匹配；其输入项为单个或一组观测质量，以及目标mRNA构建体的序列。……