Pub Date : 2024-05-01Epub Date: 2024-04-02DOI: 10.1016/j.mcpro.2024.100758
John J M Bergeron
The acceleration of advances in proteomics has enabled integration with imaging at the EM and light microscopy levels, cryo-EM of protein structures, and artificial intelligence with proteins comprehensively and accurately resolved for cell structures at nanometer to subnanometer resolution. Proteomics continues to outpace experimentally based structural imaging, but their ultimate integration is a path toward the goal of a compendium of all proteins to understand mechanistically cell structure and function.
{"title":"Proteomics Impact on Cell Biology to Resolve Cell Structure and Function.","authors":"John J M Bergeron","doi":"10.1016/j.mcpro.2024.100758","DOIUrl":"10.1016/j.mcpro.2024.100758","url":null,"abstract":"<p><p>The acceleration of advances in proteomics has enabled integration with imaging at the EM and light microscopy levels, cryo-EM of protein structures, and artificial intelligence with proteins comprehensively and accurately resolved for cell structures at nanometer to subnanometer resolution. Proteomics continues to outpace experimentally based structural imaging, but their ultimate integration is a path toward the goal of a compendium of all proteins to understand mechanistically cell structure and function.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":null,"pages":null},"PeriodicalIF":7.0,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11070594/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140868247","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01Epub Date: 2024-05-11DOI: 10.1016/j.mcpro.2024.100774
Thais Pereira-Veiga, Susana Bravo, Antonio Gómez-Tato, Celso Yáñez-Gómez, Carmen Abuín, Vanesa Varela, Juan Cueva, Patricia Palacios, Ana B Dávila-Ibáñez, Roberto Piñeiro, Ana Vilar, María Del Pilar Chantada-Vázquez, Rafael López-López, Clotilde Costa
{"title":"Corrigendum to \"Red Blood Cells Protein Profile Is Modified in Breast Cancer Patients\".","authors":"Thais Pereira-Veiga, Susana Bravo, Antonio Gómez-Tato, Celso Yáñez-Gómez, Carmen Abuín, Vanesa Varela, Juan Cueva, Patricia Palacios, Ana B Dávila-Ibáñez, Roberto Piñeiro, Ana Vilar, María Del Pilar Chantada-Vázquez, Rafael López-López, Clotilde Costa","doi":"10.1016/j.mcpro.2024.100774","DOIUrl":"10.1016/j.mcpro.2024.100774","url":null,"abstract":"","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":null,"pages":null},"PeriodicalIF":7.0,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11103567/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140912447","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01Epub Date: 2024-03-13DOI: 10.1016/j.mcpro.2024.100747
Hau-Ming Jan, Shang-Chuen Wu, Carter J Stowell, Mary L Vallecillo-Zúniga, Anu Paul, Kashyap R Patel, Sasikala Muthusamy, Hsien-Ya Lin, Diyoly Ayona, Ryan Philip Jajosky, Samata P Varadkar, Hirotomo Nakahara, Rita Chan, Devika Bhave, William J Lane, Melissa Y Yeung, Marie A Hollenhorst, Seth Rakoff-Nahoum, Richard D Cummings, Connie M Arthur, Sean R Stowell
Although immune tolerance evolved to reduce reactivity with self, it creates a gap in the adaptive immune response against microbes that decorate themselves in self-like antigens. This is particularly apparent with carbohydrate-based blood group antigens, wherein microbes can envelope themselves in blood group structures similar to human cells. In this study, we demonstrate that the innate immune lectin, galectin-4 (Gal-4), exhibits strain-specific binding and killing behavior towards microbes that display blood group-like antigens. Examination of binding preferences using a combination of microarrays populated with ABO(H) glycans and a variety of microbial strains, including those that express blood group-like antigens, demonstrated that Gal-4 binds mammalian and microbial antigens that have features of blood group and mammalian-like structures. Although Gal-4 was thought to exist as a monomer that achieves functional bivalency through its two linked carbohydrate recognition domains, our data demonstrate that Gal-4 forms dimers and that differences in the intrinsic ability of each domain to dimerize likely influences binding affinity. While each Gal-4 domain exhibited blood group-binding activity, the C-terminal domain (Gal-4C) exhibited dimeric properties, while the N-terminal domain (Gal-4N) failed to similarly display dimeric activity. Gal-4C not only exhibited the ability to dimerize but also possessed higher affinity toward ABO(H) blood group antigens and microbes expressing glycans with blood group-like features. Furthermore, when compared to Gal-4N, Gal-4C exhibited more potent antimicrobial activity. Even in the context of the full-length protein, where Gal-4N is functionally bivalent by virtue of Gal-4C dimerization, Gal-4C continued to display higher antimicrobial activity. These results demonstrate that Gal-4 exists as a dimer and exhibits its antimicrobial activity primarily through its C-terminal domain. In doing so, these data provide important insight into key features of Gal-4 responsible for its innate immune activity against molecular mimicry.
虽然免疫耐受的进化是为了降低与自身的反应性,但它却造成了适应性免疫反应的缺失,无法抵御用类似自身的抗原装饰自身的微生物。这一点在以碳水化合物为基础的血型抗原上表现得尤为明显,微生物可将自身包裹在与人体细胞相似的血型结构中。在这项研究中,我们证明了先天性免疫凝集素--galectin-4(Gal-4)对显示血型类抗原的微生物具有菌株特异性结合和杀伤行为。通过结合使用 ABO(H)聚糖的微阵列和多种微生物菌株(包括表达血型样抗原的微生物菌株)对其结合偏好的研究表明,Gal-4 能结合具有血型和哺乳动物样结构特征的哺乳动物和微生物抗原。尽管人们认为 Gal-4 是作为单体存在的,通过其两个相连的碳水化合物识别结构域(CRDs)实现功能上的双价性,但我们的数据表明 Gal-4 形成了二聚体,而且每个结构域二聚化的内在能力差异可能会影响结合亲和力。虽然每个 Gal-4 结构域都表现出血型结合活性,但 C 端结构域(Gal-4C)表现出二聚体特性,而 N 端结构域(Gal-4N)未能表现出类似的二聚体活性。Gal-4C 不仅具有二聚化能力,而且对 ABO(H)血型抗原和表达具有血型相似特征的聚糖的微生物具有更高的亲和力。此外,与 Gal-4N 相比,Gal-4C 表现出更强的抗菌活性。即使在全长蛋白质中,Gal-4N 通过 Gal-4C 二聚化而具有二价功能,Gal-4C 仍然显示出更高的抗菌活性。这些结果表明,Gal-4 是以二聚体形式存在的,主要通过其 C 端结构域表现出抗菌活性。因此,这些数据为我们深入了解 Gal-4 对抗分子拟态的先天免疫活性的关键特征提供了重要依据。
{"title":"Galectin-4 Antimicrobial Activity Primarily Occurs Through its C-Terminal Domain.","authors":"Hau-Ming Jan, Shang-Chuen Wu, Carter J Stowell, Mary L Vallecillo-Zúniga, Anu Paul, Kashyap R Patel, Sasikala Muthusamy, Hsien-Ya Lin, Diyoly Ayona, Ryan Philip Jajosky, Samata P Varadkar, Hirotomo Nakahara, Rita Chan, Devika Bhave, William J Lane, Melissa Y Yeung, Marie A Hollenhorst, Seth Rakoff-Nahoum, Richard D Cummings, Connie M Arthur, Sean R Stowell","doi":"10.1016/j.mcpro.2024.100747","DOIUrl":"10.1016/j.mcpro.2024.100747","url":null,"abstract":"<p><p>Although immune tolerance evolved to reduce reactivity with self, it creates a gap in the adaptive immune response against microbes that decorate themselves in self-like antigens. This is particularly apparent with carbohydrate-based blood group antigens, wherein microbes can envelope themselves in blood group structures similar to human cells. In this study, we demonstrate that the innate immune lectin, galectin-4 (Gal-4), exhibits strain-specific binding and killing behavior towards microbes that display blood group-like antigens. Examination of binding preferences using a combination of microarrays populated with ABO(H) glycans and a variety of microbial strains, including those that express blood group-like antigens, demonstrated that Gal-4 binds mammalian and microbial antigens that have features of blood group and mammalian-like structures. Although Gal-4 was thought to exist as a monomer that achieves functional bivalency through its two linked carbohydrate recognition domains, our data demonstrate that Gal-4 forms dimers and that differences in the intrinsic ability of each domain to dimerize likely influences binding affinity. While each Gal-4 domain exhibited blood group-binding activity, the C-terminal domain (Gal-4C) exhibited dimeric properties, while the N-terminal domain (Gal-4N) failed to similarly display dimeric activity. Gal-4C not only exhibited the ability to dimerize but also possessed higher affinity toward ABO(H) blood group antigens and microbes expressing glycans with blood group-like features. Furthermore, when compared to Gal-4N, Gal-4C exhibited more potent antimicrobial activity. Even in the context of the full-length protein, where Gal-4N is functionally bivalent by virtue of Gal-4C dimerization, Gal-4C continued to display higher antimicrobial activity. These results demonstrate that Gal-4 exists as a dimer and exhibits its antimicrobial activity primarily through its C-terminal domain. In doing so, these data provide important insight into key features of Gal-4 responsible for its innate immune activity against molecular mimicry.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":null,"pages":null},"PeriodicalIF":6.1,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11097083/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140137028","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01Epub Date: 2024-04-03DOI: 10.1016/j.mcpro.2024.100760
Lia R Serrano, Trenton M Peters-Clarke, Tabiwang N Arrey, Eugen Damoc, Margaret Lea Robinson, Noah M Lancaster, Evgenia Shishkova, Corinne Moss, Anna Pashkova, Pavel Sinitcyn, Dain R Brademan, Scott T Quarmby, Amelia C Peterson, Martin Zeller, Daniel Hermanson, Hamish Stewart, Christian Hock, Alexander Makarov, Vlad Zabrouskov, Joshua J Coon
We describe deep analysis of the human proteome in less than 1 h. We achieve this expedited proteome characterization by leveraging state-of-the-art sample preparation, chromatographic separations, and data analysis tools, and by using the new Orbitrap Astral mass spectrometer equipped with a quadrupole mass filter, a high-field Orbitrap mass analyzer, and an asymmetric track lossless (Astral) mass analyzer. The system offers high tandem mass spectrometry acquisition speed of 200 Hz and detects hundreds of peptide sequences per second within data-independent acquisition or data-dependent acquisition modes of operation. The fast-switching capabilities of the new quadrupole complement the sensitivity and fast ion scanning of the Astral analyzer to enable narrow-bin data-independent analysis methods. Over a 30-min active chromatographic method consuming a total analysis time of 56 min, the Q-Orbitrap-Astral hybrid MS collects an average of 4319 MS1 scans and 438,062 tandem mass spectrometry scans per run, producing 235,916 peptide sequences (1% false discovery rate). On average, each 30-min analysis achieved detection of 10,411 protein groups (1% false discovery rate). We conclude, with these results and alongside other recent reports, that the 1-h human proteome is within reach.
{"title":"The One Hour Human Proteome.","authors":"Lia R Serrano, Trenton M Peters-Clarke, Tabiwang N Arrey, Eugen Damoc, Margaret Lea Robinson, Noah M Lancaster, Evgenia Shishkova, Corinne Moss, Anna Pashkova, Pavel Sinitcyn, Dain R Brademan, Scott T Quarmby, Amelia C Peterson, Martin Zeller, Daniel Hermanson, Hamish Stewart, Christian Hock, Alexander Makarov, Vlad Zabrouskov, Joshua J Coon","doi":"10.1016/j.mcpro.2024.100760","DOIUrl":"10.1016/j.mcpro.2024.100760","url":null,"abstract":"<p><p>We describe deep analysis of the human proteome in less than 1 h. We achieve this expedited proteome characterization by leveraging state-of-the-art sample preparation, chromatographic separations, and data analysis tools, and by using the new Orbitrap Astral mass spectrometer equipped with a quadrupole mass filter, a high-field Orbitrap mass analyzer, and an asymmetric track lossless (Astral) mass analyzer. The system offers high tandem mass spectrometry acquisition speed of 200 Hz and detects hundreds of peptide sequences per second within data-independent acquisition or data-dependent acquisition modes of operation. The fast-switching capabilities of the new quadrupole complement the sensitivity and fast ion scanning of the Astral analyzer to enable narrow-bin data-independent analysis methods. Over a 30-min active chromatographic method consuming a total analysis time of 56 min, the Q-Orbitrap-Astral hybrid MS collects an average of 4319 MS<sup>1</sup> scans and 438,062 tandem mass spectrometry scans per run, producing 235,916 peptide sequences (1% false discovery rate). On average, each 30-min analysis achieved detection of 10,411 protein groups (1% false discovery rate). We conclude, with these results and alongside other recent reports, that the 1-h human proteome is within reach.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":null,"pages":null},"PeriodicalIF":7.0,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11103439/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140849312","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01Epub Date: 2024-04-10DOI: 10.1016/j.mcpro.2024.100766
Jiaying Liu, Xiaona Chang, Liujia Qian, Shuo Chen, Zhangzhi Xue, Junhua Wu, Danju Luo, Bo Huang, Jun Fan, Tiannan Guo, Xiu Nie
The diagnosis of primary lung adenocarcinomas with intestinal or mucinous differentiation (PAIM) remains challenging due to the overlapping histomorphological, immunohistochemical (IHC), and genetic characteristics with lung metastatic colorectal cancer (lmCRC). This study aimed to explore the protein biomarkers that could distinguish between PAIM and lmCRC. To uncover differences between the two diseases, we used tandem mass tagging-based shotgun proteomics to characterize proteomes of formalin-fixed, paraffin-embedded tumor samples of PAIM (n = 22) and lmCRC (n = 17).Then three machine learning algorithms, namely support vector machine (SVM), random forest, and the Least Absolute Shrinkage and Selection Operator, were utilized to select protein features with diagnostic significance. These candidate proteins were further validated in an independent cohort (PAIM, n = 11; lmCRC, n = 19) by IHC to confirm their diagnostic performance. In total, 105 proteins out of 7871 proteins were significantly dysregulated between PAIM and lmCRC samples and well-separated two groups by Uniform Manifold Approximation and Projection. The upregulated proteins in PAIM were involved in actin cytoskeleton organization, platelet degranulation, and regulation of leukocyte chemotaxis, while downregulated ones were involved in mitochondrial transmembrane transport, vasculature development, and stem cell proliferation. A set of ten candidate proteins (high-level expression in lmCRC: CDH17, ATP1B3, GLB1, OXNAD1, LYST, FABP1; high-level expression in PAIM: CK7 (an established marker), NARR, MLPH, S100A14) was ultimately selected to distinguish PAIM from lmCRC by machine learning algorithms. We further confirmed using IHC that the five protein biomarkers including CDH17, CK7, MLPH, FABP1 and NARR were effective biomarkers for distinguishing PAIM from lmCRC. Our study depicts PAIM-specific proteomic characteristics and demonstrates the potential utility of new protein biomarkers for the differential diagnosis of PAIM and lmCRC. These findings may contribute to improving the diagnostic accuracy and guide appropriate treatments for these patients.
{"title":"Proteomics-Derived Biomarker Panel Facilitates Distinguishing Primary Lung Adenocarcinomas With Intestinal or Mucinous Differentiation From Lung Metastatic Colorectal Cancer.","authors":"Jiaying Liu, Xiaona Chang, Liujia Qian, Shuo Chen, Zhangzhi Xue, Junhua Wu, Danju Luo, Bo Huang, Jun Fan, Tiannan Guo, Xiu Nie","doi":"10.1016/j.mcpro.2024.100766","DOIUrl":"10.1016/j.mcpro.2024.100766","url":null,"abstract":"<p><p>The diagnosis of primary lung adenocarcinomas with intestinal or mucinous differentiation (PAIM) remains challenging due to the overlapping histomorphological, immunohistochemical (IHC), and genetic characteristics with lung metastatic colorectal cancer (lmCRC). This study aimed to explore the protein biomarkers that could distinguish between PAIM and lmCRC. To uncover differences between the two diseases, we used tandem mass tagging-based shotgun proteomics to characterize proteomes of formalin-fixed, paraffin-embedded tumor samples of PAIM (n = 22) and lmCRC (n = 17).Then three machine learning algorithms, namely support vector machine (SVM), random forest, and the Least Absolute Shrinkage and Selection Operator, were utilized to select protein features with diagnostic significance. These candidate proteins were further validated in an independent cohort (PAIM, n = 11; lmCRC, n = 19) by IHC to confirm their diagnostic performance. In total, 105 proteins out of 7871 proteins were significantly dysregulated between PAIM and lmCRC samples and well-separated two groups by Uniform Manifold Approximation and Projection. The upregulated proteins in PAIM were involved in actin cytoskeleton organization, platelet degranulation, and regulation of leukocyte chemotaxis, while downregulated ones were involved in mitochondrial transmembrane transport, vasculature development, and stem cell proliferation. A set of ten candidate proteins (high-level expression in lmCRC: CDH17, ATP1B3, GLB1, OXNAD1, LYST, FABP1; high-level expression in PAIM: CK7 (an established marker), NARR, MLPH, S100A14) was ultimately selected to distinguish PAIM from lmCRC by machine learning algorithms. We further confirmed using IHC that the five protein biomarkers including CDH17, CK7, MLPH, FABP1 and NARR were effective biomarkers for distinguishing PAIM from lmCRC. Our study depicts PAIM-specific proteomic characteristics and demonstrates the potential utility of new protein biomarkers for the differential diagnosis of PAIM and lmCRC. These findings may contribute to improving the diagnostic accuracy and guide appropriate treatments for these patients.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":null,"pages":null},"PeriodicalIF":7.0,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11092395/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140850640","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01Epub Date: 2024-04-07DOI: 10.1016/j.mcpro.2024.100761
Xing Xu, Kejun Yin, Ronghu Wu
Glycoproteins located on the cell surface play a pivotal role in nearly every extracellular activity. N-glycosylation is one of the most common and important protein modifications in eukaryotic cells, and it often regulates protein folding and trafficking. Glycosylation of cell-surface proteins undergoes meticulous regulation by various enzymes in the endoplasmic reticulum (ER) and the Golgi, ensuring their proper folding and trafficking to the cell surface. However, the impacts of protein N-glycosylation, N-glycan maturity, and protein folding status on the trafficking of cell-surface glycoproteins remain to be explored. In this work, we comprehensively and site-specifically studied the trafficking of cell-surface glycoproteins in human cells. Integrating metabolic labeling, bioorthogonal chemistry, and multiplexed proteomics, we investigated 706 N-glycosylation sites on 396 cell-surface glycoproteins in monocytes, either by inhibiting protein N-glycosylation, disturbing N-glycan maturation, or perturbing protein folding in the ER. The current results reveal their distinct impacts on the trafficking of surface glycoproteins. The inhibition of protein N-glycosylation dramatically suppresses the trafficking of many cell-surface glycoproteins. The N-glycan immaturity has more substantial effects on proteins with high N-glycosylation site densities, while the perturbation of protein folding in the ER exerts a more pronounced impact on surface glycoproteins with larger sizes. Furthermore, for N-glycosylated proteins, their trafficking to the cell surface is related to the secondary structures and adjacent amino acid residues of glycosylation sites. Systematic analysis of surface glycoprotein trafficking advances our understanding of the mechanisms underlying protein secretion and surface presentation.
{"title":"Systematic Investigation of the Trafficking of Glycoproteins on the Cell Surface.","authors":"Xing Xu, Kejun Yin, Ronghu Wu","doi":"10.1016/j.mcpro.2024.100761","DOIUrl":"10.1016/j.mcpro.2024.100761","url":null,"abstract":"<p><p>Glycoproteins located on the cell surface play a pivotal role in nearly every extracellular activity. N-glycosylation is one of the most common and important protein modifications in eukaryotic cells, and it often regulates protein folding and trafficking. Glycosylation of cell-surface proteins undergoes meticulous regulation by various enzymes in the endoplasmic reticulum (ER) and the Golgi, ensuring their proper folding and trafficking to the cell surface. However, the impacts of protein N-glycosylation, N-glycan maturity, and protein folding status on the trafficking of cell-surface glycoproteins remain to be explored. In this work, we comprehensively and site-specifically studied the trafficking of cell-surface glycoproteins in human cells. Integrating metabolic labeling, bioorthogonal chemistry, and multiplexed proteomics, we investigated 706 N-glycosylation sites on 396 cell-surface glycoproteins in monocytes, either by inhibiting protein N-glycosylation, disturbing N-glycan maturation, or perturbing protein folding in the ER. The current results reveal their distinct impacts on the trafficking of surface glycoproteins. The inhibition of protein N-glycosylation dramatically suppresses the trafficking of many cell-surface glycoproteins. The N-glycan immaturity has more substantial effects on proteins with high N-glycosylation site densities, while the perturbation of protein folding in the ER exerts a more pronounced impact on surface glycoproteins with larger sizes. Furthermore, for N-glycosylated proteins, their trafficking to the cell surface is related to the secondary structures and adjacent amino acid residues of glycosylation sites. Systematic analysis of surface glycoprotein trafficking advances our understanding of the mechanisms underlying protein secretion and surface presentation.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":null,"pages":null},"PeriodicalIF":6.1,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11087972/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140857057","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01Epub Date: 2024-03-20DOI: 10.1016/j.mcpro.2024.100749
Liyuan Dai, Qiaoyun Tan, Lin Li, Ning Lou, Cuiling Zheng, Jianliang Yang, Liling Huang, Shasha Wang, Rongrong Luo, Guangyu Fan, Tongji Xie, Jiarui Yao, Zhishang Zhang, Le Tang, Yuankai Shi, Xiaohong Han
Chemoimmunotherapy has evolved as a standard treatment for advanced non-small cell lung cancer (aNSCLC). However, inevitable drug resistance has limited its efficacy, highlighting the urgent need for biomarkers of chemoimmunotherapy. A three-phase strategy to discover, verify, and validate longitudinal predictive autoantibodies (AAbs) for aNSCLC before and after chemoimmunotherapy was employed. A total of 528 plasma samples from 267 aNSCLC patients before and after anti-PD1 immunotherapy were collected, plus 30 independent formalin-fixed paraffin-embedded samples. Candidate AAbs were firstly selected using a HuProt high-density microarray containing 21,000 proteins in the discovery phase, followed by validation using an aNSCLC-focused microarray. Longitudinal predictive AAbs were chosen for ELISA based on responders versus non-responders comparison and progression-free survival (PFS) survival analysis. Prognostic markers were also validated using immunohistochemistry and publicly available immunotherapy datasets. We identified and validated a panel of two AAbs (MAX and DHX29) as pre-treatment biomarkers and another panel of two AAbs (MAX and TAPBP) as on-treatment predictive markers in aNSCLC patients undergoing chemoimmunotherapy. All three AAbs exhibited a positive correlation with early responses and PFS (p < 0.05). The kinetics of MAX AAb showed an increasing trend in responders (p < 0.05) and a tendency to initially increase and then decrease in non-responders (p < 0.05). Importantly, MAX protein and mRNA levels effectively discriminated PFS (p < 0.05) in aNSCLC patients treated with immunotherapy. Our results present a longitudinal analysis of changes in prognostic AAbs in aNSCLC patients undergoing chemoimmunotherapy.
{"title":"High-Throughput Antigen Microarray Identifies Longitudinal Prognostic Autoantibody for Chemoimmunotherapy in Advanced Non-Small Cell Lung Cancer.","authors":"Liyuan Dai, Qiaoyun Tan, Lin Li, Ning Lou, Cuiling Zheng, Jianliang Yang, Liling Huang, Shasha Wang, Rongrong Luo, Guangyu Fan, Tongji Xie, Jiarui Yao, Zhishang Zhang, Le Tang, Yuankai Shi, Xiaohong Han","doi":"10.1016/j.mcpro.2024.100749","DOIUrl":"10.1016/j.mcpro.2024.100749","url":null,"abstract":"<p><p>Chemoimmunotherapy has evolved as a standard treatment for advanced non-small cell lung cancer (aNSCLC). However, inevitable drug resistance has limited its efficacy, highlighting the urgent need for biomarkers of chemoimmunotherapy. A three-phase strategy to discover, verify, and validate longitudinal predictive autoantibodies (AAbs) for aNSCLC before and after chemoimmunotherapy was employed. A total of 528 plasma samples from 267 aNSCLC patients before and after anti-PD1 immunotherapy were collected, plus 30 independent formalin-fixed paraffin-embedded samples. Candidate AAbs were firstly selected using a HuProt high-density microarray containing 21,000 proteins in the discovery phase, followed by validation using an aNSCLC-focused microarray. Longitudinal predictive AAbs were chosen for ELISA based on responders versus non-responders comparison and progression-free survival (PFS) survival analysis. Prognostic markers were also validated using immunohistochemistry and publicly available immunotherapy datasets. We identified and validated a panel of two AAbs (MAX and DHX29) as pre-treatment biomarkers and another panel of two AAbs (MAX and TAPBP) as on-treatment predictive markers in aNSCLC patients undergoing chemoimmunotherapy. All three AAbs exhibited a positive correlation with early responses and PFS (p < 0.05). The kinetics of MAX AAb showed an increasing trend in responders (p < 0.05) and a tendency to initially increase and then decrease in non-responders (p < 0.05). Importantly, MAX protein and mRNA levels effectively discriminated PFS (p < 0.05) in aNSCLC patients treated with immunotherapy. Our results present a longitudinal analysis of changes in prognostic AAbs in aNSCLC patients undergoing chemoimmunotherapy.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":null,"pages":null},"PeriodicalIF":7.0,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11070596/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140184856","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01Epub Date: 2024-04-02DOI: 10.1016/j.mcpro.2024.100759
Søren Heissel, Yi He, Andris Jankevics, Yuqi Shi, Henrik Molina, Rosa Viner, Richard A Scheltema
Recombinant expression of proteins, propelled by therapeutic antibodies, has evolved into a multibillion dollar industry. Essential here is the quality control assessment of critical attributes, such as sequence fidelity, proper folding, and posttranslational modifications. Errors can lead to diminished bioactivity and, in the context of therapeutic proteins, an elevated risk for immunogenicity. Over the years, many techniques were developed and applied to validate proteins in a standardized and high-throughput fashion. One parameter has, however, so far been challenging to assess. Disulfide bridges, covalent bonds linking two cysteine residues, assist in the correct folding and stability of proteins and thus have a major influence on their efficacy. Mass spectrometry promises to be an optimal technique to uncover them in a fast and accurate fashion. In this work, we present a unique combination of sample preparation, data acquisition, and analysis facilitating the rapid and accurate assessment of disulfide bridges in purified proteins. Through microwave-assisted acid hydrolysis, the proteins are digested rapidly and artifact-free into peptides, with a substantial degree of overlap over the sequence. The nonspecific nature of this procedure, however, introduces chemical background, which is efficiently removed by integrating ion mobility preceding the mass spectrometric measurement. The nonspecific nature of the digestion step additionally necessitates new developments in data analysis, for which we extended the XlinkX node in Proteome Discoverer to efficiently process the data and ensure correctness through effective false discovery rate correction. The entire workflow can be completed within 1 h, allowing for high-throughput, high-accuracy disulfide mapping.
{"title":"Fast and Accurate Disulfide Bridge Detection.","authors":"Søren Heissel, Yi He, Andris Jankevics, Yuqi Shi, Henrik Molina, Rosa Viner, Richard A Scheltema","doi":"10.1016/j.mcpro.2024.100759","DOIUrl":"10.1016/j.mcpro.2024.100759","url":null,"abstract":"<p><p>Recombinant expression of proteins, propelled by therapeutic antibodies, has evolved into a multibillion dollar industry. Essential here is the quality control assessment of critical attributes, such as sequence fidelity, proper folding, and posttranslational modifications. Errors can lead to diminished bioactivity and, in the context of therapeutic proteins, an elevated risk for immunogenicity. Over the years, many techniques were developed and applied to validate proteins in a standardized and high-throughput fashion. One parameter has, however, so far been challenging to assess. Disulfide bridges, covalent bonds linking two cysteine residues, assist in the correct folding and stability of proteins and thus have a major influence on their efficacy. Mass spectrometry promises to be an optimal technique to uncover them in a fast and accurate fashion. In this work, we present a unique combination of sample preparation, data acquisition, and analysis facilitating the rapid and accurate assessment of disulfide bridges in purified proteins. Through microwave-assisted acid hydrolysis, the proteins are digested rapidly and artifact-free into peptides, with a substantial degree of overlap over the sequence. The nonspecific nature of this procedure, however, introduces chemical background, which is efficiently removed by integrating ion mobility preceding the mass spectrometric measurement. The nonspecific nature of the digestion step additionally necessitates new developments in data analysis, for which we extended the XlinkX node in Proteome Discoverer to efficiently process the data and ensure correctness through effective false discovery rate correction. The entire workflow can be completed within 1 h, allowing for high-throughput, high-accuracy disulfide mapping.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":null,"pages":null},"PeriodicalIF":7.0,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11067345/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140850527","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01Epub Date: 2024-04-10DOI: 10.1016/j.mcpro.2024.100763
Zhongzhi Sun, Zhibin Ning, Daniel Figeys
The human gut microbiome is closely associated with human health and diseases. Metaproteomics has emerged as a valuable tool for studying the functionality of the gut microbiome by analyzing the entire proteins present in microbial communities. Recent advancements in liquid chromatography and tandem mass spectrometry (LC-MS/MS) techniques have expanded the detection range of metaproteomics. However, the overall coverage of the proteome in metaproteomics is still limited. While metagenomics studies have revealed substantial microbial diversity and functional potential of the human gut microbiome, few studies have summarized and studied the human gut microbiome landscape revealed with metaproteomics. In this article, we present the current landscape of human gut metaproteomics studies by re-analyzing the identification results from 15 published studies. We quantified the limited proteome coverage in metaproteomics and revealed a high proportion of annotation coverage of metaproteomics-identified proteins. We conducted a preliminary comparison between the metaproteomics view and the metagenomics view of the human gut microbiome, identifying key areas of consistency and divergence. Based on the current landscape of human gut metaproteomics, we discuss the feasibility of using metaproteomics to study functionally unknown proteins and propose a whole workflow peptide-centric analysis. Additionally, we suggest enhancing metaproteomics analysis by refining taxonomic classification and calculating confidence scores, as well as developing tools for analyzing the interaction between taxonomy and function.
{"title":"The Landscape and Perspectives of the Human Gut Metaproteomics.","authors":"Zhongzhi Sun, Zhibin Ning, Daniel Figeys","doi":"10.1016/j.mcpro.2024.100763","DOIUrl":"10.1016/j.mcpro.2024.100763","url":null,"abstract":"<p><p>The human gut microbiome is closely associated with human health and diseases. Metaproteomics has emerged as a valuable tool for studying the functionality of the gut microbiome by analyzing the entire proteins present in microbial communities. Recent advancements in liquid chromatography and tandem mass spectrometry (LC-MS/MS) techniques have expanded the detection range of metaproteomics. However, the overall coverage of the proteome in metaproteomics is still limited. While metagenomics studies have revealed substantial microbial diversity and functional potential of the human gut microbiome, few studies have summarized and studied the human gut microbiome landscape revealed with metaproteomics. In this article, we present the current landscape of human gut metaproteomics studies by re-analyzing the identification results from 15 published studies. We quantified the limited proteome coverage in metaproteomics and revealed a high proportion of annotation coverage of metaproteomics-identified proteins. We conducted a preliminary comparison between the metaproteomics view and the metagenomics view of the human gut microbiome, identifying key areas of consistency and divergence. Based on the current landscape of human gut metaproteomics, we discuss the feasibility of using metaproteomics to study functionally unknown proteins and propose a whole workflow peptide-centric analysis. Additionally, we suggest enhancing metaproteomics analysis by refining taxonomic classification and calculating confidence scores, as well as developing tools for analyzing the interaction between taxonomy and function.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":null,"pages":null},"PeriodicalIF":7.0,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11098955/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140853420","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01Epub Date: 2024-03-27DOI: 10.1016/j.mcpro.2024.100754
Patricia Bortel, Ilaria Piga, Claire Koenig, Christopher Gerner, Ana Martinez-Val, Jesper V Olsen
Improving coverage, robustness, and sensitivity is crucial for routine phosphoproteomics analysis by single-shot liquid chromatography-tandem mass spectrometry (LC-MS/MS) from minimal peptide inputs. Here, we systematically optimized key experimental parameters for automated on-bead phosphoproteomics sample preparation with a focus on low-input samples. Assessing the number of identified phosphopeptides, enrichment efficiency, site localization scores, and relative enrichment of multiply-phosphorylated peptides pinpointed critical variables influencing the resulting phosphoproteome. Optimizing glycolic acid concentration in the loading buffer, percentage of ammonium hydroxide in the elution buffer, peptide-to-beads ratio, binding time, sample, and loading buffer volumes allowed us to confidently identify >16,000 phosphopeptides in half-an-hour LC-MS/MS on an Orbitrap Exploris 480 using 30 μg of peptides as starting material. Furthermore, we evaluated how sequential enrichment can boost phosphoproteome coverage and showed that pooling fractions into a single LC-MS/MS analysis increased the depth. We also present an alternative phosphopeptide enrichment strategy based on stepwise addition of beads thereby boosting phosphoproteome coverage by 20%. Finally, we applied our optimized strategy to evaluate phosphoproteome depth with the Orbitrap Astral MS using a cell dilution series and were able to identify >32,000 phosphopeptides from 0.5 million HeLa cells in half-an-hour LC-MS/MS using narrow-window data-independent acquisition (nDIA).
{"title":"Systematic Optimization of Automated Phosphopeptide Enrichment for High-Sensitivity Phosphoproteomics.","authors":"Patricia Bortel, Ilaria Piga, Claire Koenig, Christopher Gerner, Ana Martinez-Val, Jesper V Olsen","doi":"10.1016/j.mcpro.2024.100754","DOIUrl":"10.1016/j.mcpro.2024.100754","url":null,"abstract":"<p><p>Improving coverage, robustness, and sensitivity is crucial for routine phosphoproteomics analysis by single-shot liquid chromatography-tandem mass spectrometry (LC-MS/MS) from minimal peptide inputs. Here, we systematically optimized key experimental parameters for automated on-bead phosphoproteomics sample preparation with a focus on low-input samples. Assessing the number of identified phosphopeptides, enrichment efficiency, site localization scores, and relative enrichment of multiply-phosphorylated peptides pinpointed critical variables influencing the resulting phosphoproteome. Optimizing glycolic acid concentration in the loading buffer, percentage of ammonium hydroxide in the elution buffer, peptide-to-beads ratio, binding time, sample, and loading buffer volumes allowed us to confidently identify >16,000 phosphopeptides in half-an-hour LC-MS/MS on an Orbitrap Exploris 480 using 30 μg of peptides as starting material. Furthermore, we evaluated how sequential enrichment can boost phosphoproteome coverage and showed that pooling fractions into a single LC-MS/MS analysis increased the depth. We also present an alternative phosphopeptide enrichment strategy based on stepwise addition of beads thereby boosting phosphoproteome coverage by 20%. Finally, we applied our optimized strategy to evaluate phosphoproteome depth with the Orbitrap Astral MS using a cell dilution series and were able to identify >32,000 phosphopeptides from 0.5 million HeLa cells in half-an-hour LC-MS/MS using narrow-window data-independent acquisition (nDIA).</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":null,"pages":null},"PeriodicalIF":7.0,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11087715/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140318716","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}