Pub Date : 2024-10-30DOI: 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。
{"title":"Functional analysis of MS-based proteomics data: from protein groups to networks.","authors":"Marie Locard-Paulet, Nadezhda T Doncheva, John H Morris, Lars Juhl Jensen","doi":"10.1016/j.mcpro.2024.100871","DOIUrl":"https://doi.org/10.1016/j.mcpro.2024.100871","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100871"},"PeriodicalIF":6.1,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564690","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-24DOI: 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.
{"title":"Targeted dynamic phospho-proteogenomic analysis of gastric cancer cells suggests host immunity provides survival benefit.","authors":"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","doi":"10.1016/j.mcpro.2024.100870","DOIUrl":"https://doi.org/10.1016/j.mcpro.2024.100870","url":null,"abstract":"<p><p>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<sup>+</sup> 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.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100870"},"PeriodicalIF":6.1,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504286","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-23DOI: 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.
{"title":"A Donald F. Hunt Story (John's Version).","authors":"John R Yates","doi":"10.1016/j.mcpro.2024.100869","DOIUrl":"https://doi.org/10.1016/j.mcpro.2024.100869","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100869"},"PeriodicalIF":6.1,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504282","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-22DOI: 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 SureQuant 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. 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 and interleukin 1-β, 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。
{"title":"High-Throughput and High-Sensitivity Biomarker Monitoring in Body Fluid by Fast LC SureQuant IS-Targeted Quantitation.","authors":"Konstantinos Kalogeropoulos, Simonas Savickas, Aleksander M Haack, Cathrine A Larsen, Jacek Mikosiński, Erwin M Schoof, Hans Smola, Louise Bundgaard, Ulrich Auf dem Keller","doi":"10.1016/j.mcpro.2024.100868","DOIUrl":"10.1016/j.mcpro.2024.100868","url":null,"abstract":"<p><p>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 SureQuant 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. 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 and interleukin 1-β, 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.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100868"},"PeriodicalIF":6.1,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504284","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-21DOI: 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。
{"title":"Mass Spectrometry-based proteomics for the masses: Peptide and protein identification in the Hunt laboratory during the 2000's.","authors":"Jessica R Chapman","doi":"10.1016/j.mcpro.2024.100866","DOIUrl":"https://doi.org/10.1016/j.mcpro.2024.100866","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100866"},"PeriodicalIF":6.1,"publicationDate":"2024-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504285","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-21DOI: 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 subpopulation of phage retained by each LEAP ortholog. 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) revealed a subpopulation of the proteome constituting the CtP repertoire in common between the two dehydrin orthologs (LEA14 and GmPm12) compared to bovine serum albumin (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 analysis. Moreover, LEAP:CtP interactions for four other CtPs were confirmed in planta using bimolecular fluorescence complementation assays. The results provide insights into the involvement of the dehydrin Y-segments and K-domains in protein binding.
{"title":"Dehydrin Client Proteins Identified Using Phage Display Affinity Selected Libraries Processed With Paired-End Phage Sequencing.","authors":"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","doi":"10.1016/j.mcpro.2024.100867","DOIUrl":"10.1016/j.mcpro.2024.100867","url":null,"abstract":"<p><p>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 subpopulation of phage retained by each LEAP ortholog. 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) revealed a subpopulation of the proteome constituting the CtP repertoire in common between the two dehydrin orthologs (LEA14 and GmPm12) compared to bovine serum albumin (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 analysis. Moreover, LEAP:CtP interactions for four other CtPs were confirmed in planta using bimolecular fluorescence complementation assays. The results provide insights into the involvement of the dehydrin Y-segments and K-domains in protein binding.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100867"},"PeriodicalIF":5.3,"publicationDate":"2024-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504283","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-19DOI: 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, for example, 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。
{"title":"Nanobodies: From High-Throughput Identification to Therapeutic Development.","authors":"Peter C Fridy, Michael P Rout, Natalia E Ketaren","doi":"10.1016/j.mcpro.2024.100865","DOIUrl":"10.1016/j.mcpro.2024.100865","url":null,"abstract":"<p><p>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, for example, 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.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100865"},"PeriodicalIF":6.1,"publicationDate":"2024-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142470074","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-16DOI: 10.1016/j.mcpro.2024.100861
Kasandra Buchholtz, Rosa Jersie-Christensen, Karen Angeliki Krogfelt, Biljana Mojsoska
Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic human pathogen, causing serious chronic infections. P. aeruginosa can adapt efficiently to antibiotic stressors via different genotypic or phenotypic strategies such as resistance and tolerance. The adaptation regulatory system is not always very well understood. In this study, we use shotgun proteomics to investigate the system-level response to tobramycin in two clinical wound P. aeruginosa isolates and PAO1. We profiled each strain for its antibiotic drug-tolerant phenotype using supra-minimum inhibitory concentrations (supra-MIC) of tobramycin and applied proteomics to investigate the protein expression profiles. The MIC revealed that all isolates were susceptible to tobramycin but at supra-MIC concentrations at stationary growth, a degree of tolerance was observed for the isolates. We identified around 40 % of the total proteins encoded by the P. aeruginosa genome and highlighted shared and unique protein signatures for all isolates. Comparative proteome profiling in the absence of antibiotic treatment showed divergent fingerprints, despite similarities in the growth behavior of the isolates. In the presence of tobramycin, the isolates shared a common response in the downregulation of proteins involved in the two-component system, whereas stress response proteins were present at higher levels. Our findings provide insight into the use of proteomic tools to dissect the system-level response in clinical isolates in the absence and presence of antibiotic stress.
{"title":"Analysis of antibiotic response in Clinical Wound Pseudomonas aeruginosa isolates: Unveiling Proteome Dynamics of tobramycin tolerant phenotype.","authors":"Kasandra Buchholtz, Rosa Jersie-Christensen, Karen Angeliki Krogfelt, Biljana Mojsoska","doi":"10.1016/j.mcpro.2024.100861","DOIUrl":"https://doi.org/10.1016/j.mcpro.2024.100861","url":null,"abstract":"<p><p>Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic human pathogen, causing serious chronic infections. P. aeruginosa can adapt efficiently to antibiotic stressors via different genotypic or phenotypic strategies such as resistance and tolerance. The adaptation regulatory system is not always very well understood. In this study, we use shotgun proteomics to investigate the system-level response to tobramycin in two clinical wound P. aeruginosa isolates and PAO1. We profiled each strain for its antibiotic drug-tolerant phenotype using supra-minimum inhibitory concentrations (supra-MIC) of tobramycin and applied proteomics to investigate the protein expression profiles. The MIC revealed that all isolates were susceptible to tobramycin but at supra-MIC concentrations at stationary growth, a degree of tolerance was observed for the isolates. We identified around 40 % of the total proteins encoded by the P. aeruginosa genome and highlighted shared and unique protein signatures for all isolates. Comparative proteome profiling in the absence of antibiotic treatment showed divergent fingerprints, despite similarities in the growth behavior of the isolates. In the presence of tobramycin, the isolates shared a common response in the downregulation of proteins involved in the two-component system, whereas stress response proteins were present at higher levels. Our findings provide insight into the use of proteomic tools to dissect the system-level response in clinical isolates in the absence and presence of antibiotic stress.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100861"},"PeriodicalIF":6.1,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142470060","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-16DOI: 10.1016/j.mcpro.2024.100860
Mingbo Peng, Tianjing Wang, Yujie Li, Zheng Zhang, Cuihong Wan
sORF-encoded peptides (SEPs) refer to proteins encoded by small open reading frames (sORFs) with a length of less than 100 amino acids, which play an important role in various life activities. Analysis of known SEPs showed that using non-canonical initiation codons of SEPs was more common. However, the current analysis of SEP sequences mainly relies on bioinformatics prediction, and most of them use AUG as the start site, which may not be completely correct for SEPs. Chemical labeling was used to systematically analyze the N-terminal sequences of SEPs to accurately define the start sites of SEPs. By comparison, we found that dimethylation and guanidinylation are more efficient than acetylation. The ACN precipitation and heating precipitation performed better in SEP enrichment. As an N-terminal peptide enrichment material, Hexadhexaldehyde was superior to CNBr-activated agarose and NHS-activated agarose. Combining these methods, we identified 128 SEPs with 131 N-terminal sequences. Among them, two-thirds are novel N-terminal sequences, and most of them start from the 11-31st amino acids of the original sequence. Partial novel N-termini were produced by proteolysis or signal peptide removal. Some SEPs' transcription start sites were corrected to be non-AUG start codons. One novel start codon was validated using GFP-tag vectors. These results demonstrated that the chemical labeling approaches would be beneficial for identifying the start codons of sORFs and the real N-terminal of their encoded peptides, which helps better understand the characterization of SEPs.
sORF编码肽(SEPs)是指由长度小于100个氨基酸的小开放阅读框(sORFs)编码的蛋白质,它们在各种生命活动中发挥着重要作用。对已知 SEP 的分析表明,使用 SEP 的非规范起始密码子较为常见。然而,目前对SEP序列的分析主要依赖于生物信息学预测,且大多使用AUG作为起始位点,这对于SEP来说可能并不完全正确。我们采用化学标记法系统分析了SEPs的N端序列,以准确界定SEPs的起始位点。通过比较,我们发现二甲基化和鸟苷酸化比乙酰化更有效。ACN 沉淀和加热沉淀的 SEP 富集效果更好。作为 N 端多肽富集材料,六甲醛优于 CNBr 活化的琼脂糖和 NHS 活化的琼脂糖。结合这些方法,我们共鉴定出 128 个 SEPs,131 个 N 端序列。其中,三分之二是新的 N 端序列,它们大多从原始序列的第 11-31 个氨基酸开始。部分新型 N 端是通过蛋白水解或信号肽去除产生的。一些 SEP 的转录起始位点被修正为非 AUG 起始密码子。使用 GFP 标记载体对一个新的起始密码子进行了验证。这些结果表明,化学标记方法有助于鉴定 sORFs 的起始密码子及其编码肽的真正 N-末端,从而有助于更好地理解 SEPs 的特征。
{"title":"Mapping Start Codons of Small Open Reading Frames by N-Terminomics Approach.","authors":"Mingbo Peng, Tianjing Wang, Yujie Li, Zheng Zhang, Cuihong Wan","doi":"10.1016/j.mcpro.2024.100860","DOIUrl":"10.1016/j.mcpro.2024.100860","url":null,"abstract":"<p><p>sORF-encoded peptides (SEPs) refer to proteins encoded by small open reading frames (sORFs) with a length of less than 100 amino acids, which play an important role in various life activities. Analysis of known SEPs showed that using non-canonical initiation codons of SEPs was more common. However, the current analysis of SEP sequences mainly relies on bioinformatics prediction, and most of them use AUG as the start site, which may not be completely correct for SEPs. Chemical labeling was used to systematically analyze the N-terminal sequences of SEPs to accurately define the start sites of SEPs. By comparison, we found that dimethylation and guanidinylation are more efficient than acetylation. The ACN precipitation and heating precipitation performed better in SEP enrichment. As an N-terminal peptide enrichment material, Hexadhexaldehyde was superior to CNBr-activated agarose and NHS-activated agarose. Combining these methods, we identified 128 SEPs with 131 N-terminal sequences. Among them, two-thirds are novel N-terminal sequences, and most of them start from the 11-31st amino acids of the original sequence. Partial novel N-termini were produced by proteolysis or signal peptide removal. Some SEPs' transcription start sites were corrected to be non-AUG start codons. One novel start codon was validated using GFP-tag vectors. These results demonstrated that the chemical labeling approaches would be beneficial for identifying the start codons of sORFs and the real N-terminal of their encoded peptides, which helps better understand the characterization of SEPs.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100860"},"PeriodicalIF":6.1,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142470073","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-15DOI: 10.1016/j.mcpro.2024.100862
Xiang Zhang, Juan Ge, Yue Wang, Minjian Chen, Xuejiang Guo, Shuai Zhu, Hui Wang, Qiang Wang
Well-controlled metabolism is associated with high-quality oocytes and optimal development of a healthy embryo. However, the metabolic framework that controls mammalian oocyte growth remains unknown. In the present study, we comprehensively depict the temporal metabolic dynamics of mouse oocytes during in vivo growth through the integrated analysis of metabolomics and proteomics. Many novel metabolic features are discovered during this process. Of note, glycolysis is enhanced, and oxidative phosphorylation capacity is reduced in the growing oocytes, presenting a Warburg-like metabolic program. For nucleotide biosynthesis, the salvage pathway is markedly activated during oocyte growth, whereas the de novo pathway is evidently suppressed. Fatty acid synthesis and channeling into phosphoinositides are specifically elevated in oocytes accompanying primordial follicle activation; nevertheless, fatty acid oxidation is reduced in these oocytes simultaneously. Our data establish the metabolic landscape during in vivo oocyte growth and serve as a broad resource for probing mammalian oocyte metabolism.
{"title":"Integrative Omics Reveals the Metabolic Patterns During Oocyte Growth.","authors":"Xiang Zhang, Juan Ge, Yue Wang, Minjian Chen, Xuejiang Guo, Shuai Zhu, Hui Wang, Qiang Wang","doi":"10.1016/j.mcpro.2024.100862","DOIUrl":"10.1016/j.mcpro.2024.100862","url":null,"abstract":"<p><p>Well-controlled metabolism is associated with high-quality oocytes and optimal development of a healthy embryo. However, the metabolic framework that controls mammalian oocyte growth remains unknown. In the present study, we comprehensively depict the temporal metabolic dynamics of mouse oocytes during in vivo growth through the integrated analysis of metabolomics and proteomics. Many novel metabolic features are discovered during this process. Of note, glycolysis is enhanced, and oxidative phosphorylation capacity is reduced in the growing oocytes, presenting a Warburg-like metabolic program. For nucleotide biosynthesis, the salvage pathway is markedly activated during oocyte growth, whereas the de novo pathway is evidently suppressed. Fatty acid synthesis and channeling into phosphoinositides are specifically elevated in oocytes accompanying primordial follicle activation; nevertheless, fatty acid oxidation is reduced in these oocytes simultaneously. Our data establish the metabolic landscape during in vivo oocyte growth and serve as a broad resource for probing mammalian oocyte metabolism.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100862"},"PeriodicalIF":6.1,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142470072","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}