Alice Nevone, Francesca Lattarulo, Monica Russo, Pasquale Cascino, Giampaolo Merlini, Giovanni Palladini, Mario Nuvolone
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To address these limitations, we have recently developed a novel method, termed Single Molecule Real-Time Sequencing of the M protein (SMaRT M-Seq), which combines the unbiased amplification of expressed immunoglobulin genes via inverse PCR from circularized cDNA with long-read DNA sequencing, followed by bioinformatic and immunogenetic analyses. This approach enables the unambiguous identification of full-length variable sequences of M protein genes across diverse patient cohorts, including those with low tumor burden. Our protocol has undergone technical validation and has been successfully applied to large patient cohorts with monoclonal gammopathies. Here we present the step-by-step protocol for SMaRT M-Seq. By enabling the parallel sequencing of M proteins from a large number of samples in a cost-effective and time-efficient manner, SMaRT M-Seq facilitates access to patient-specific M protein sequences, advancing personalized medicine approaches and enabling deeper mechanistic studies in monoclonal gammopathies.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.5000,"publicationDate":"2024-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11520399/pdf/","citationCount":"0","resultStr":"{\"title\":\"SMaRT M-Seq: an optimized step-by-step protocol for M protein sequencing in monoclonal gammopathies.\",\"authors\":\"Alice Nevone, Francesca Lattarulo, Monica Russo, Pasquale Cascino, Giampaolo Merlini, Giovanni Palladini, Mario Nuvolone\",\"doi\":\"10.1093/biomethods/bpae074\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>In patients with monoclonal gammopathies, tumor B cells or plasma cells secrete a monoclonal antibody (M protein) that not only serves as a biomarker for tumor tracking but can also cause potentially life-threatening organ damage. 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Our protocol has undergone technical validation and has been successfully applied to large patient cohorts with monoclonal gammopathies. Here we present the step-by-step protocol for SMaRT M-Seq. 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引用次数: 0
摘要
在单克隆丙种球蛋白病患者中,肿瘤 B 细胞或浆细胞会分泌一种单克隆抗体(M 蛋白),这种抗体不仅是追踪肿瘤的生物标记物,还可能造成潜在的危及生命的器官损伤。这种损害是由患者特异性的 M 蛋白序列驱动的。对 M 蛋白进行测序的方法一直受限于其高昂的成本和耗时的性质,虽然最近使用下一代测序或质谱法的方法取得了进步,但它们可能需要对肿瘤细胞进行分选,局限于免疫球蛋白基因或患者的子集,和/或缺乏广泛的验证。为了解决这些局限性,我们最近开发了一种新方法,称为 M 蛋白单分子实时测序(SMaRT M-Seq),它将通过环化 cDNA 反 PCR 无偏扩增表达的免疫球蛋白基因与长线程 DNA 测序相结合,然后进行生物信息学和免疫遗传学分析。这种方法能在不同的患者队列(包括肿瘤负荷较低的患者)中明确鉴定 M 蛋白基因的全长可变序列。我们的方案已经过技术验证,并已成功应用于大型单克隆丙种球蛋白病患者队列。我们在此介绍 SMaRT M-Seq 的分步方案。SMaRT M-Seq能以低成本、高效率的方式对大量样本中的M蛋白进行平行测序,有助于获得患者特异性的M蛋白序列,从而推动个性化医疗方法的发展,并能对单克隆丙种球蛋白病进行更深入的机理研究。
SMaRT M-Seq: an optimized step-by-step protocol for M protein sequencing in monoclonal gammopathies.
In patients with monoclonal gammopathies, tumor B cells or plasma cells secrete a monoclonal antibody (M protein) that not only serves as a biomarker for tumor tracking but can also cause potentially life-threatening organ damage. This damage is driven by the patient-specific sequence of the M protein. Methods for sequencing M proteins have been limited by their high cost and time-consuming nature, and while recent approaches using next-generation sequencing or mass spectrometry offer advancements, they may require tumor cell sorting, are limited to subsets of immunoglobulin genes or patients, and/or lack extensive validation. To address these limitations, we have recently developed a novel method, termed Single Molecule Real-Time Sequencing of the M protein (SMaRT M-Seq), which combines the unbiased amplification of expressed immunoglobulin genes via inverse PCR from circularized cDNA with long-read DNA sequencing, followed by bioinformatic and immunogenetic analyses. This approach enables the unambiguous identification of full-length variable sequences of M protein genes across diverse patient cohorts, including those with low tumor burden. Our protocol has undergone technical validation and has been successfully applied to large patient cohorts with monoclonal gammopathies. Here we present the step-by-step protocol for SMaRT M-Seq. By enabling the parallel sequencing of M proteins from a large number of samples in a cost-effective and time-efficient manner, SMaRT M-Seq facilitates access to patient-specific M protein sequences, advancing personalized medicine approaches and enabling deeper mechanistic studies in monoclonal gammopathies.