Tandem mass spectrometry (MS/MS) can provide direct and accurate sequence characterization of synthetic peptide drugs, and peptide drug products including side chain modifications in the Peptide drugs. This article explains a step-by-step guide to developing a high-throughput method using high resolution mass spectrometry for characterization of Calcitonin Salmon injection containing high proportion of UV-active excipients. The major challenge in the method development of Amino acid sequencing and Peptide mapping was presence of phenol in drug product. Phenol is a UV-active excipient and reacts with both Dithiothreitol (DTT) and Trypsin. Hence Calcitonin Salmon was extracted from the Calcitonin Salmon injection using solid phase extraction after the extraction, Amino acid sequencing and peptide mapping study was performed. Upon incubation of Calcitonin Salmon with Trypsin and DTT, digested fragments were generated which were separated by mass compatible reverse phase chromatography and the molecular mass of each fragment was determined using HRMS. A reverse phase chromatographic method was developed using UHPLC-HRMS for the determination of direct mass, peptide mapping and to determine the amino acid sequencing in the Calcitonin Salmon injection. The method was found Specific and fragments after trypsin digest are well resolved from each other and the molecular mass of each fragment was determined using HRMS. Sequencing was performed using automated identification of and ions annotation and identifications based on MS/MS spectra using Biopharma finder and Proteome discoverer software. Using this approach 100% protein coverage was obtained and protein was identified as Calcitonin Salmon and the observed masses of tryptic digest of peptide was found similar with theoretical masses. The method can be used for both UV and MS based Peptide mapping and whereas the UV based peptide mapping method can be used as identification test for Calcitonin Salmon drug substance and drug product in quality control.

中文翻译：

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使用超高效液相色谱-高分辨率质谱 (UHPLC-HRMS) 并应用生物信息学工具，对鲑鱼降钙素注射液中的降钙素鲑鱼进行肽图分析和氨基酸测序的高通量方法

串联质谱（MS/MS）可以提供合成肽药物和肽药物产品（包括肽药物中的侧链修饰）的直接、准确的序列表征。本文介绍了使用高分辨率质谱开发高通量方法的分步指南，用于表征含有高比例紫外线活性赋形剂的降钙素鲑鱼注射液。氨基酸测序和肽图分析方法开发的主要挑战是药品中苯酚的存在。苯酚是一种紫外线活性赋形剂，可与二硫苏糖醇 (DTT) 和胰蛋白酶发生反应。因此，从鲑鱼降钙素注射液中提取降钙素，提取后进行固相萃取，并进行氨基酸测序和肽图谱研究。将鲑鱼降钙素与胰蛋白酶和 DTT 一起温育后，生成消化片段，通过质量兼容的反相色谱分离这些片段，并使用 HRMS 测定每个片段的分子量。使用 UHPLC-HRMS 开发了一种反相色谱方法，用于测定降钙素鲑鱼注射液中的直接质量、肽图谱和氨基酸测序。该方法发现胰蛋白酶消化后的特异性片段彼此很好地分离，并且使用 HRMS 测定了每个片段的分子量。使用 Biopharma finder 和 Proteome discoveryr 软件基于 MS/MS 谱图的自动识别和离子注释和识别进行测序。使用这种方法获得了 100% 的蛋白质覆盖率，并且蛋白质被鉴定为鲑鱼降钙素，并且发现肽的胰蛋白酶消化物的观察质量与理论质量相似。该方法可用于基于 UV 和 MS 的肽图谱，而基于 UV 的肽图谱方法可用作质量控制中降钙素鲑鱼原料药和药品的鉴定测试。