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Early Days in the Hunt Laboratory at UVA, 1969-1980. 弗吉尼亚大学亨特实验室的早期生活,1969-1980 年。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-11-04 DOI: 10.1016/j.mcpro.2024.100874
P Jane Gale, George C Stafford, Howard R Morris, Charles N McEwen

Arriving at the University of Virginia in the autumn of 1969, Donald Hunt began his 50+ year career in academics with the study of organometallic chemistry, on which he had done his PhD thesis, and mass spectrometry, to which he was introduced while a postdoc in Klaus Biemann's laboratory at the Massachusetts Institute of Technology. In the 1970s, Hunt's lab pioneered the use of negative chemical ionization (CI) to enhance sensitivity for studying organic molecules, developed a system for simultaneously obtaining positive and negative CI spectra to augment structure elucidation, and built a prototype triple quadrupole instrument so effective at collisional dissociation that its commercial counterpart became the analytical instrument of choice for mixture analysis for the next decade and beyond. Foreseeing that the future lay in the analysis of biological molecules, by the end of the decade Hunt shifted his focus to peptides. The analysis of protein fragments had suddenly become more accessible thanks to the advent of the triple quadrupole and Barber's invention of fast atom bombardment. As the '80s began and Hunt and his team sought to pursue larger and larger pieces of proteins, his attention turned to the development of mass spectrometers with greater mass range. While recounting their memories of these events, several of Hunt's students and colleagues pay tribute to his support for them as individuals, as well as to his infectious enthusiasm for scientific endeavors that he so generously shared.

1969 年秋天,唐纳德-亨特来到弗吉尼亚大学,开始了他长达 50 多年的学术生涯。他的博士论文研究方向是有机金属化学,而他在麻省理工学院克劳斯-比曼 (Klaus Biemann) 实验室做博士后时开始接触质谱分析。20 世纪 70 年代,亨特的实验室率先使用负化学电离 (CI) 技术来提高有机分子研究的灵敏度,开发了一套同时获得正负 CI 光谱的系统来增强结构阐释能力,并建立了一个三重四极杆仪器原型,该仪器在碰撞解离方面非常有效,以至于其商业同类仪器在接下来的十年中成为混合物分析的首选。由于预见到生物分子分析是未来的发展趋势,亨特在这十年的末尾将工作重点转移到了肽的分析上。由于三重四极杆的出现和巴伯发明的快速原子轰击技术,蛋白质片段的分析突然变得更加容易。进入 80 年代后,亨特和他的团队开始寻求更大的蛋白质片段,他的注意力转向了质量范围更大的质谱仪的开发。在讲述他们对这些事件的回忆时,亨特的几位学生和同事对亨特作为个人对他们的支持以及他慷慨分享的对科学事业的感染力表示敬意。
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引用次数: 0
On the Hunt for the Histone Code. 寻找组蛋白密码
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-11-01 DOI: 10.1016/j.mcpro.2024.100873
Beatrix M Ueberheide, Sahana Mollah, Benjamin A Garcia

Our genome is not made of naked DNA, but a fiber (chromatin) composed of DNA and proteins packaged into our chromosomes. The basic building block of chromatin is the nucleosome, which has two copies of each of the proteins called histones (H2A, H2B, H3, and H4) wrapped by 146 base pairs of DNA. Regions of our genetic material are found between the more open (euchromatin) and more compact (heterochromatin) regions of the genome that can be variably accessible to the underlying genes. Furthermore, post-translational modifications (PTMs) on histones, such as on H3, are critical for regulating chromatin accessibility and gene expression. While site specific antibodies were the tool of choice for histone PTM analysis in the early days (pre-2000s), enter Don Hunt changing the histone PTM field forever. Don's clever thinking brought new innovative mass spectrometry-based approaches to the epigenetics field. His lab's effort led to the discovery of many new histone modifications and methods to facilitate the detection and quantification of histone PTMs, which are still considered state of the art in the proteomics field today. Due to Don's pioneering work in this area, many labs have been able to jump into the epigenetics field and "Hunt" down their own histone targets. A walkthrough of those early histone years in the Hunt Lab are described by three of us who were fortunate enough to be at the right place, at the right time.

我们的基因组不是由裸露的 DNA 组成的,而是由 DNA 和蛋白质组成的纤维(染色质),包装在染色体中。染色质的基本组成部分是核小体,核小体由 146 个碱基对的 DNA 包裹着,每个核小体有两个被称为组蛋白(H2A、H2B、H3 和 H4)的蛋白质拷贝。我们的遗传物质区域位于基因组中较为开放(外染色质)和较为紧凑(异染色质)的区域之间,这些区域的潜在基因可以不同程度地进入。此外,组蛋白(如 H3)上的翻译后修饰(PTMs)对于调节染色质可及性和基因表达至关重要。虽然在早期(2000 年代以前),位点特异性抗体是组蛋白 PTM 分析的首选工具,但唐-亨特的出现彻底改变了组蛋白 PTM 领域。唐的聪明才智为表观遗传学领域带来了基于质谱的创新方法。在他的实验室的努力下,人们发现了许多新的组蛋白修饰,并找到了便于检测和量化组蛋白 PTM 的方法,这些方法至今仍被认为是蛋白质组学领域最先进的技术。由于唐在这一领域的开创性工作,许多实验室得以进入表观遗传学领域,"猎杀 "自己的组蛋白目标。我们三人有幸在正确的时间、正确的地点见证了亨特实验室早期组蛋白研究的历程。
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引用次数: 0
Screening of cancer-specific biomarkers for hepatitis B-related hepatocellular carcinoma based on a proteome microarray. 基于蛋白质组芯片筛选乙型肝炎相关肝细胞癌的癌症特异性生物标记物。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-11-01 DOI: 10.1016/j.mcpro.2024.100872
Wudi Hao, Danyang Zhao, Yuan Meng, Mei Yang, Meichen Ma, Jingwen Hu, Jianhua Liu, Xiaosong Qin

Hepatocellular carcinoma (HCC) is associated with one of the highest mortality rates among cancers, rendering its early diagnosis clinically invaluable. Serum biomarkers, specifically alpha-fetoprotein (AFP), represent the most promising and widely used diagnostic biomarkers for HCC. However, its detection rate is low in the early stages of HCC progression, and distinguishing specific false positives for other liver-related diseases, such as cirrhosis and acute hepatitis, remains challenging. Therefore, this study was conducted to identify biomarkers for hepatitis B (HBV)-related liver diseases by screening differentially expressed autoantibodies against tumor-associated antigens (TAAbs). We designed a large-scale multistage investigation, encompassing initial screening, HCC-focused, and ELISA validation cohorts to identify potential TAAbs in HBV-related liver diseases, spanning from healthy control (HC) individuals to patients with chronic hepatitis B (CHB), hepatitis B-related cirrhosis (HBC), and HCC, using protein microarray technology. The differential biological characteristics of TAAbs were analyzed using bioinformatics analysis. Validation of tumor-specific biomarkers for HCC was performed using ELISA. In the screening cohort, 547 candidate TAAbs were identified in the HCC group compared to those in the HC group. In the HCC-focused cohort, 64, 61, and 65 candidate TAAbs were identified in the CHB, HBC, and HCC groups, respectively, compared to those in the HC group. Thirty-four proteins exhibited continuously elevated expression from HCs to patients with CHB, HBC, and HCC. Among these, nine were identified as cancer-specific proteins. In the validation cohort, UBE2Z, CNOT3, and EID3 were correlated with liver function indicators in patients with hepatitis B-related HCC. Overall, UBE2Z, CNOT3, and EID3 emerged as cancer-specific biomarkers for HBV-related liver disease, providing a scientific basis for clinical application.

肝细胞癌(HCC)是死亡率最高的癌症之一,因此早期诊断在临床上非常重要。血清生物标志物,特别是甲胎蛋白(AFP),是最有希望且应用最广泛的 HCC 诊断生物标志物。然而,在 HCC 进展的早期阶段,甲胎蛋白的检出率较低,而且区分其他肝脏相关疾病(如肝硬化和急性肝炎)的特异性假阳性仍具有挑战性。因此,本研究通过筛选针对肿瘤相关抗原(TAAbs)的不同表达的自身抗体,来确定乙型肝炎(HBV)相关肝病的生物标记物。我们设计了一项大规模的多阶段调查,包括初步筛选、以 HCC 为重点的队列和 ELISA 验证队列,利用蛋白质微阵列技术从健康对照(HC)个体到慢性乙型肝炎(CHB)、乙型肝炎相关肝硬化(HBC)和 HCC 患者中识别潜在的乙型肝炎相关肝病 TAAbs。利用生物信息学分析方法分析了 TAAbs 的不同生物学特征。利用酶联免疫吸附法对 HCC 的肿瘤特异性生物标记物进行了验证。在筛选队列中,与HC组相比,HCC组发现了547个候选TAAbs。在以HCC为重点的队列中,与HC组相比,CHB组、HBC组和HCC组分别发现了64、61和65个候选TAAbs。从 HC 到 CHB、HBC 和 HCC 患者,有 34 种蛋白质的表达量持续升高。其中,9 个蛋白被确定为癌症特异性蛋白。在验证队列中,UBE2Z、CNOT3 和 EID3 与乙肝相关 HCC 患者的肝功能指标相关。总之,UBE2Z、CNOT3 和 EID3 成为 HBV 相关肝病的癌症特异性生物标志物,为临床应用提供了科学依据。
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引用次数: 0
Functional analysis of MS-based proteomics data: from protein groups to networks. 基于 MS 的蛋白质组学数据的功能分析:从蛋白质组到网络。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-30 DOI: 10.1016/j.mcpro.2024.100871
Marie Locard-Paulet, Nadezhda T Doncheva, John H Morris, Lars Juhl Jensen

Mass-spectrometry-based proteomics allows the quantification of thousands of proteins, protein variants, and their modifications, in many biological samples. These are derived from the measurement of peptide relative quantities, and it is not always possible to distinguish proteins with similar sequences due to the absence of protein-specific peptides. In such cases, peptide signals are reported in protein groups that can correspond to several genes. Here, we show that multi-gene protein groups have a limited impact on GO-term enrichment, but selecting only one gene per group affects network analysis. We thus present the Cytoscape app Proteo Visualizer (https://apps.cytoscape.org/apps/ProteoVisualizer) that is designed for retrieving protein interaction networks from STRING using protein groups as input and thus allows visualisation and network analysis of bottom-up MS-based proteomics data sets.

基于质谱的蛋白质组学可以对许多生物样本中的数千种蛋白质、蛋白质变体及其修饰进行定量。由于缺乏蛋白质特异性肽,并不总能区分具有相似序列的蛋白质。在这种情况下,蛋白质组中的肽信号可能对应多个基因。在这里,我们发现多基因蛋白质组对 GO 项富集的影响有限,但每组只选择一个基因会影响网络分析。因此,我们推出了 Cytoscape 应用程序 Proteo Visualizer (https://apps.cytoscape.org/apps/ProteoVisualizer),该程序旨在使用蛋白质组作为输入,从 STRING 中检索蛋白质相互作用网络,从而对基于 MS 的自下而上蛋白质组学数据集进行可视化和网络分析。
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引用次数: 0
Targeted dynamic phospho-proteogenomic analysis of gastric cancer cells suggests host immunity provides survival benefit. 胃癌细胞的靶向动态磷酸蛋白基因组学分析表明,宿主免疫可带来生存益处。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-24 DOI: 10.1016/j.mcpro.2024.100870
Kohei Kume, Midori Iida, Takeshi Iwaya, Akiko Yashima-Abo, Yuka Koizumi, Akari Endo, Kaitlin Wade, Hayato Hiraki, Valerie Calvert, Julia Wulfkuhle, Virginia Espina, Doris R Siwak, Yiling Lu, Kazuhiro Takemoto, Yutaka Suzuki, Yasushi Sasaki, Takashi Tokino, Emanuel Petricoin, Lance A Liotta, Gordon B Mills, Satoshi S Nishizuka

Despite of massive emergence of molecular targeting drugs, the mainstay of advanced gastric cancer (GC) therapy is DNA-damaging drugs. Using a reverse-phase protein array-based proteogenomic analysis of a panel of eight GC cell lines, we identified genetic alterations and signaling pathways, potentially associated with resistance to DNA-damaging drugs, including 5-fluorouracil (5FU), cisplatin, and etoposide. Resistance to cisplatin and etoposide, but not 5FU, was negatively associated with global copy number loss, vimentin expression, and caspase activity, which are considered hallmarks of previously established EMT subtype. The segregation of 19,392 protein expression time courses by sensitive and resistant cell lines for the drugs tested revealed that 5FU-resistant cell lines had lower changes in global protein dynamics, suggesting their robust protein level regulation, compared to their sensitive counterparts, whereas the cell lines that are resistant to other drugs showed increased protein dynamics in response to each drug. Despite faint global protein dynamics, 5FU-resistant cell lines showed increased STAT1 phosphorylation and PD-L1 expression in response to 5FU. In publicly available cohort data, expression of STAT1 and NFκB target genes induced by proinflammatory cytokines was associated with prolonged survival in GC. In our validation cohort, total lymphocyte count (TLC), rather than PD-L1 positivity, predicted a better relapse-free survival rate in GC patients with 5FU-based adjuvant chemotherapy than those with surgery alone. Moreover, TLC+ patients who had no survival benefit from adjuvant chemotherapy were discriminated by expression of IκBα, a potent negative regulator of NFκB. Collectively, our results suggest that 5FU resistance observed in cell lines may be overcome by host immunity or by combination therapy with immune checkpoint blockade.

尽管出现了大量分子靶向药物,但晚期胃癌(GC)的主要治疗手段仍是DNA损伤药物。通过对八种胃癌细胞系进行基于反相蛋白质阵列的蛋白质基因组分析,我们发现了可能与对DNA损伤药物(包括5-氟尿嘧啶(5FU)、顺铂和依托泊苷)的耐药性有关的基因改变和信号通路。对顺铂和依托泊苷(而非 5FU )的耐药性与全局拷贝数丢失、波形蛋白表达和 Caspase 活性呈负相关,而这些因素被认为是先前确定的 EMT 亚型的标志。对测试药物敏感和耐药细胞系的19,392个蛋白质表达时间过程的分离显示,与敏感细胞系相比,对5FU耐药的细胞系的全局蛋白质动态变化较小,这表明它们的蛋白质水平调节能力较强,而对其他药物耐药的细胞系对每种药物的反应都显示出蛋白质动态变化增加。尽管全局蛋白动态变化微弱,但对5FU耐药的细胞系对5FU的反应显示STAT1磷酸化和PD-L1表达增加。在公开的队列数据中,促炎细胞因子诱导的 STAT1 和 NFκB 靶基因的表达与 GC 存活时间的延长有关。在我们的验证队列中,总淋巴细胞计数(TLC)而非 PD-L1 阳性预示着接受 5FU 辅助化疗的 GC 患者的无复发生存率优于仅接受手术治疗的患者。此外,TLC+患者无法从辅助化疗中获益,而IκBα是NFκB的一种有效负调控因子,通过IκBα的表达可以区分TLC+患者。总之,我们的研究结果表明,在细胞系中观察到的5FU耐药性可以通过宿主免疫或与免疫检查点阻断剂联合治疗来克服。
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引用次数: 0
A Donald F. Hunt Story (John's Version). 唐纳德-F-亨特的故事(约翰版本)。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-23 DOI: 10.1016/j.mcpro.2024.100869
John R Yates

A personal narative of my time in the Hunt laboraotry and beyond is provided. The impact of the Hunt laboratory on the analysis of peptides and proteins by tandem mass spectrometry is described in the context of the time.

本文介绍了我在亨特实验室工作期间及之后的个人经历。在当时的背景下,介绍了亨特实验室对通过串联质谱分析肽和蛋白质的影响。
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引用次数: 0
Mass Spectrometry-based proteomics for the masses: Peptide and protein identification in the Hunt laboratory during the 2000's. 基于质谱的大众蛋白质组学:2000 年代亨特实验室的肽和蛋白质鉴定。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-21 DOI: 10.1016/j.mcpro.2024.100866
Jessica R Chapman

There has been a rapid increase in the number of individuals utilizing mass spectrometry (MS)-based proteomics to study complex biological systems and questions since the start of the 2000's. Building off the advancements in ionization and liquid chromatography scientists continued to push towards technology that would enable in-depth analysis of biological specimen. Donald F Hunt and the Hunt laboratory were major contributors to this effort with their work on improving upon existing Fourier Transform MS, development of electron transfer dissociation, and continued work on ion-ion reactions to improve intact protein analysis. Collaboration with other instrumentation laboratories and instrument companies led to the sharing of technology and eventual commercialization providing greater access. Additionally, the Hunt laboratory spread the gospel of mass spectrometry-based proteomics through collaborations that lasted decades with other scientists who were experts in immunology, cellular signaling, epigenetics, and other fascinating fields. This article attempts to highlight the many contributions of Don and the Hunt laboratory to peptide and protein identification since the year 2000.

自 2000 年代初以来,利用基于质谱(MS)的蛋白质组学研究复杂生物系统和问题的人数迅速增加。在电离和液相色谱技术取得进步的基础上,科学家们继续推动能够对生物样本进行深入分析的技术。唐纳德-亨特(Donald F Hunt)和亨特实验室在这方面做出了重大贡献,他们改进了现有的傅立叶变换质谱,开发了电子转移解离技术,并继续研究离子-离子反应以改进完整蛋白质分析。与其他仪器实验室和仪器公司的合作促进了技术共享,并最终实现了商业化,提供了更多的机会。此外,亨特实验室还通过与免疫学、细胞信号、表观遗传学和其他精彩领域的其他科学家进行长达数十年的合作,传播了基于质谱的蛋白质组学的福音。本文试图重点介绍唐和亨特实验室自 2000 年以来在多肽和蛋白质鉴定方面做出的诸多贡献。
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引用次数: 0
High-throughput and high-sensitivity biomarker monitoring in body fluid by fast LC SureQuant™ IS targeted quantitation. 利用快速液相色谱 SureQuant™ IS 靶向定量技术对体液中的生物标记物进行高通量、高灵敏度监测。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-21 DOI: 10.1016/j.mcpro.2024.100868
Konstantinos Kalogeropoulos, Simonas Savickas, Aleksander M Haack, Cathrine A Larsen, Jacek Mikosiński, Erwin M Schoof, Hans Smola, Louise Bundgaard, Ulrich Auf dem Keller

Targeted proteomics methods have been greatly improved and refined over the last decade and are becoming increasingly the method of choice in protein and peptide quantitative assays. Despite the tremendous progress, targeted proteomics assays still suffer from inadequate sensitivity for lower abundant proteins and throughput, especially in complex biological samples. These attributes are essential for establishing targeted proteomics methods at the forefront of clinical use. Here, we report an assay utilizing the SureQuantTM internal standard triggered targeted method on a latest generation mass spectrometer coupled with an EvoSep One liquid chromatography platform, which displays high sensitivity and a high throughput of 100 samples per day (SPD). We demonstrate the robustness of this method by quantifying proteins spanning six orders of magnitude in human wound fluid exudates, a biological fluid that exhibits sample complexity and composition similar to plasma. Among the targets quantified were low-abundance proteins such at tumor necrosis factor A (TNFA) and interleukin 1-β (IL1B), highlighting the value of this method in the quantification of trace amounts of invaluable biomarkers that were until recently hardly accessible by targeted proteomics methods. Taken together, this method extends the toolkit of targeted proteomics assays and will help to drive forward mass spectrometry-based proteomics biomarker quantification.

在过去的十年中,靶向蛋白质组学方法得到了极大的改进和完善,并逐渐成为蛋白质和肽定量检测的首选方法。尽管取得了巨大进步,但靶向蛋白质组学检测仍存在对低丰度蛋白质的灵敏度和通量不足的问题,尤其是在复杂的生物样本中。这些特性对于将靶向蛋白质组学方法应用于临床至关重要。在此,我们报告了一种利用 SureQuantTM 内标触发靶向方法在最新一代质谱仪和 EvoSep One 液相色谱平台上进行的检测,该方法具有高灵敏度和每天 100 个样品 (SPD) 的高通量。我们通过量化人体伤口渗出液(一种样本复杂、成分类似血浆的生物液体)中跨越六个数量级的蛋白质,证明了这种方法的稳健性。定量的目标蛋白包括肿瘤坏死因子 A (TNFA) 和白细胞介素 1-β (IL1B)等低丰度蛋白,突出了这种方法在定量痕量宝贵生物标志物方面的价值,直到最近,靶向蛋白质组学方法还很难获得这些生物标志物。总之,这种方法扩展了靶向蛋白质组学测定的工具包,将有助于推动基于质谱的蛋白质组学生物标记物定量。
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引用次数: 0
Dehydrin client proteins identified using phage display affinity selected libraries processed with Paired-End PhAge Sequencing (PEPA-Seq). 利用噬菌体展示亲和选择文库和成对末端 PhAge 测序(PEPA-Seq)鉴定脱水蛋白客户蛋白。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-21 DOI: 10.1016/j.mcpro.2024.100867
Sandra Helena Unêda-Trevisoli, Lynnette M A Dirk, Francisco Elder Carlos Bezerra Pereira, Manohar Chakrabarti, Guijie Hao, James M Campbell, Sai Deepshikha Bassetti Nayakwadi, Ashley Morrison, Sanjay Joshi, Sharyn E Perry, Vijyesh Sharma, Caleb Mensah, Barbara Willard, Laura de Lorenzo, Baseerat Afroza, Arthur G Hunt, Tomokazu Kawashima, Lisa Vaillancourt, Daniel Guariz Pinheiro, A Bruce Downie

The LATE EMBRYOGENESIS ABUNDANT PROTEINs (LEAPs) are a class of noncatalytic, intrinsically disordered proteins with a malleable structure. Some LEAPs exhibit a protein and/or membrane binding capacity and LEAP binding to various targets has been positively correlated with abiotic stress tolerance. Regarding the LEAPs' presumptive role in protein protection, identifying client proteins (CtPs) to which LEAPs bind is one practicable means of revealing the mechanism by which they exert their function. To this end, we used phage display affinity selection to screen libraries derived from Arabidopsis thaliana seed mRNA with recombinant orthologous LEAPs from Arabidopsis and soybean (Glycine max). Subsequent high throughput sequencing of DNA from affinity-purified phage was performed to characterize the entire sub-population of phage retained by each LEAP orthologue. This entailed cataloging in-frame fusions, elimination of false positives, and aligning the hits on the CtP scaffold to reveal domains of respective CtPs that bound to orthologous LEAPs. This approach (Paired-end PhAge Sequencing, or PEPA-Seq) revealed a subpopulation of the proteome constituting the CtP repertoire in common between the two DHNs orthologues (LEA14 and GmPm12) compared to BSA (unrelated binding control). The veracity of LEAP:CtP binding for one of the CtPs (LEA14 and GmPM12 self-association) was independently assessed using temperature related intensity change (TRIC) analysis. Moreover, LEAP:CtP interactions for four other CtPs were confirmed in planta using bimolecular fluorescence complementation (BiFC) assays. The results provide insights into the involvement of the DHN Y-segments and K-domains in protein binding.

晚幼粒细胞增殖蛋白(LEAPs)是一类非催化性的、具有可塑性结构的内在紊乱蛋白。一些 LEAPs 具有与蛋白质和/或膜结合的能力,LEAP 与不同靶标的结合与非生物胁迫耐受性呈正相关。关于 LEAPs 在蛋白质保护中的推测作用,识别与 LEAPs 结合的客户蛋白(CtPs)是揭示 LEAPs 发挥其功能的机制的一种可行方法。为此,我们使用噬菌体展示亲和选择技术筛选拟南芥种子 mRNA 与拟南芥和大豆(Glycine max)的重组同源 LEAPs 的文库。随后对亲合纯化噬菌体的 DNA 进行了高通量测序,以确定每个 LEAP 同源物保留的整个噬菌体亚群的特征。这就需要对框架内融合进行编目,消除假阳性,并对 CtP 支架上的命中进行对齐,以揭示与同源 LEAP 结合的各自 CtP 的结构域。这种方法(成对端 PhAge 测序或 PEPA-Seq)揭示了与 BSA(非相关结合对照)相比,构成两个 DHNs 同源物(LEA14 和 GmPm12)共同的 CtP 复合物的蛋白质组亚群。使用温度相关强度变化(TRIC)分析法独立评估了 LEAP:CtP 与其中一个 CtP 结合(LEA14 和 GmPM12 自结合)的真实性。此外,还利用双分子荧光互补(BiFC)试验在植物体内证实了 LEAP:CtP 与其他四个 CtP 的相互作用。这些结果有助于深入了解 DHN Y 段和 K 域参与蛋白质结合的情况。
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引用次数: 0
Nanobodies: from high-throughput identification to therapeutic development. 纳米抗体:从高通量鉴定到治疗开发。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-19 DOI: 10.1016/j.mcpro.2024.100865
Peter C Fridy, Michael P Rout, Natalia E Ketaren

The camelid single-domain antibody fragment, commonly referred to as a nanobody, achieves the targeting power of conventional monoclonal antibodies (mAbs) at only a fraction of their size. Isolated from camelid species (including llamas, alpacas, and camels), their small size at ∼15 kDa, low structural complexity and high stability compared with conventional antibodies have propelled nanobody technology into the limelight of biologic development. Nanobodies are proving themselves to be a potent complement to traditional mAb therapies, showing success in the treatment of e.g. autoimmune diseases and cancer, and more recently as therapeutic options to treat infectious diseases caused by rapidly evolving biological targets such as the SARS-CoV-2 virus. This review highlights the benefits of applying a proteomic approach to identify diverse nanobody sequences against a single antigen. This proteomic approach coupled with conventional yeast/phage display methods enables the production of highly diverse repertoires of nanobodies able to bind the vast epitope landscape of an antigen, with epitope sampling surpassing that of mAbs. Additionally, we aim to highlight recent findings illuminating the structural attributes of nanobodies that make them particularly amenable to comprehensive antigen sampling and to synergistic activity - underscoring the powerful advantage of acquiring a large, diverse nanobody repertoire against a single antigen. Lastly, we highlight the efforts being made in the clinical development of nanobodies, which have great potential as powerful diagnostic reagents and treatment options, especially when targeting infectious disease agents.

驼科动物单域抗体片段通常被称为纳米抗体,其靶向能力仅为传统单克隆抗体(mAbs)的一小部分。纳米抗体是从驼科动物(包括美洲驼、羊驼和骆驼)身上分离出来的,与传统抗体相比,它的体积小(15 kDa)、结构复杂度低、稳定性高,因此纳米抗体技术成为生物技术发展的焦点。纳米抗体被证明是对传统 mAb 疗法的有力补充,在治疗自身免疫性疾病和癌症等方面取得了成功,最近还成为治疗由快速发展的生物靶标(如 SARS-CoV-2 病毒)引起的传染性疾病的治疗选择。本综述强调了应用蛋白质组学方法识别针对单一抗原的多种纳米抗体序列的好处。这种蛋白质组学方法与传统的酵母/噬菌体展示方法相结合,能生产出高度多样化的纳米抗体,这些抗体能结合抗原的大量表位,其表位采样率超过了 mAbs。此外,我们还将重点介绍纳米抗体的最新研究成果,这些研究成果揭示了纳米抗体的结构特性,这些特性使纳米抗体特别适合于全面的抗原取样和协同活性--强调了获得针对单一抗原的大量、多样化纳米抗体库的强大优势。最后,我们重点介绍了纳米抗体的临床开发工作,纳米抗体作为强大的诊断试剂和治疗方案具有巨大的潜力,尤其是在针对传染病病原体时。
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Molecular & Cellular Proteomics
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