Journal of Proteome Research最新文献

Clean and Complete Protein Digestion with an Autolysis Resistant Trypsin for Peptide Mapping. 使用抗自溶的胰蛋白酶对蛋白质进行干净彻底的消化，以绘制肽图。

IF 3.8 2区生物学

Journal of Proteome Research Pub Date : 2024-10-11 DOI: 10.1021/acs.jproteome.4c00598

Beatrice Muriithi, Samantha Ippoliti, Abraham Finny, Balasubrahmanyam Addepalli, Matthew Lauber

{"title":"Clean and Complete Protein Digestion with an Autolysis Resistant Trypsin for Peptide Mapping.","authors":"Beatrice Muriithi, Samantha Ippoliti, Abraham Finny, Balasubrahmanyam Addepalli, Matthew Lauber","doi":"10.1021/acs.jproteome.4c00598","DOIUrl":"https://doi.org/10.1021/acs.jproteome.4c00598","url":null,"abstract":"Peptide mapping requires cleavage of proteins in a predictable fashion so that target protein-specific peptides can be reliably identified and quantified. Trypsin, a commonly used protease in this process, can also undergo self-cleavage or autolysis, thereby reducing the effectivity and even cleavage specificity at lysine and arginine residues. Here, we report highly efficient and reproducible peptide mapping of biotherapeutic monoclonal antibodies. We highlight the properties of a homogeneous chemically modified trypsin on thermal stability, a 54% increase in melting temperature with an 84% increase in energy required for unfolding, an indication of more thermally stable trypsin, >90% retained intact mass peak area after exposure to digestion conditions confirming autolysis resistance, 10× more intensity for intact enzyme compared to trypsin of similar source and narrower molecular weight distribution with LC-MS indicative of low degradation compared to 3 other types of trypsin. Finally, we show the utility of this autolysis-resistant trypsin in characterizing biotherapeutic monoclonal antibodies consistently and reliably showing a >30% reduction in missed cleavage for a short-duration protein digestion time of 30 min compared to heterogeneously modified trypsin of a similar source.","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142398630","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}

引用次数: 0

Proteomic Characterization of Urinary Exosomes with Pancreatic Cancer by Phosphatidylserine Imprinted Polymer Enrichment and Mass Spectrometry Analysis. 通过磷脂酰丝氨酸印迹聚合物富集和质谱分析确定胰腺癌患者尿液外泌体的蛋白质组特征

IF 3.8 2区生物学

Journal of Proteome Research Pub Date : 2024-10-11 DOI: 10.1021/acs.jproteome.4c00508

Xianhui Cheng, Wenjing Yu, Yuanyuan Liu, Shengnan Jia, Dongxue Wang, Lianghai Hu

{"title":"Proteomic Characterization of Urinary Exosomes with Pancreatic Cancer by Phosphatidylserine Imprinted Polymer Enrichment and Mass Spectrometry Analysis.","authors":"Xianhui Cheng, Wenjing Yu, Yuanyuan Liu, Shengnan Jia, Dongxue Wang, Lianghai Hu","doi":"10.1021/acs.jproteome.4c00508","DOIUrl":"https://doi.org/10.1021/acs.jproteome.4c00508","url":null,"abstract":"Exosomes, as carriers of cell-to-cell communication, can serve as promising biomarkers for probing the early diagnosis of cancer. Pancreatic cancer is a common malignant tumor of the pancreas with an insidious onset and difficult early diagnosis. The aim of this study was to capture exosomes in urine samples by phosphatidylserine-molecularly imprinted polymers (PS-MIPs). Transmission electron microscopy and nanoparticle tracking analysis as well as Western blot showed that our molecularly imprinted material can effectively capture urinary exosomes. Three parallel tests verified the reproducibility of the mass spectrometry assay and the stability of the material capture efficiency. Mass Spectrometry with nontargeted proteomics was combined to show differentially expressed proteins in exosomes between 5 pancreatic cancer patients and 5 healthy controls. The most significant changes in the proteomic profile in pancreatic cancer patients compared to healthy controls were the overexpression of SLC9A3R1, SPAG9, and ferritin light chain (FTL) These proteins may have an important role in diagnosis and prognostic assessment, supporting further scientific and clinical studies on pancreatic cancer.","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142398632","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}

引用次数: 0

Longitudinal Assessment of Nasopharyngeal Biomarkers Post-COVID-19: Unveiling Persistent Markers and Severity Correlations. COVID-19后鼻咽生物标志物的纵向评估：揭示持续性标记物和严重程度相关性

IF 3.8 2区生物学

Journal of Proteome Research Pub Date : 2024-10-11 DOI: 10.1021/acs.jproteome.4c00536

Francisco Javier Redondo-Calvo, Yoana Rabanal-Ruiz, Gema Verdugo-Moreno, Natalia Bejarano-Ramírez, Raquel Bodoque-Villar, Mario Durán-Prado, Soledad Illescas, Eduardo Chicano-Galvez, Francisco Javier Gómez-Romero, José Martinez-Alarcón, Javier Arias-Pardilla, Pilar Lopez-Juarez, Juan Fernando Padin, Juan Ramón Peinado, Leticia Serrano-Oviedo

{"title":"Longitudinal Assessment of Nasopharyngeal Biomarkers Post-COVID-19: Unveiling Persistent Markers and Severity Correlations.","authors":"Francisco Javier Redondo-Calvo, Yoana Rabanal-Ruiz, Gema Verdugo-Moreno, Natalia Bejarano-Ramírez, Raquel Bodoque-Villar, Mario Durán-Prado, Soledad Illescas, Eduardo Chicano-Galvez, Francisco Javier Gómez-Romero, José Martinez-Alarcón, Javier Arias-Pardilla, Pilar Lopez-Juarez, Juan Fernando Padin, Juan Ramón Peinado, Leticia Serrano-Oviedo","doi":"10.1021/acs.jproteome.4c00536","DOIUrl":"https://doi.org/10.1021/acs.jproteome.4c00536","url":null,"abstract":"SARS-CoV-19 infection provokes a variety of symptoms; most patients present mild/moderate symptoms, whereas a small proportion of patients progress to severe illness with multiorgan failure accompanied by metabolic disturbances requiring ICU-level care. Given the importance of the disease, researchers focused on identifying severity-associated biomarkers in infected patients as well as markers associated with patients suffering long-COVID. However, little is known about the presence of biomarkers that remain a few years after SARS-CoV-2 infection once the patients fully recover of the symptoms. In this study, we evaluated the presence of persistent biomarkers in the nasopharyngeal tract two years after SARS-Cov-2 infection in fully asymptomatic patients, taking into account the severity of their infection (mild/moderate and severe infections). In addition to the direct identification of several components of the Coronavirus Infection Pathway in those individuals that suffered severe infections, we describe herein 371 proteins and their associated canonical pathways that define the different adverse effects of SARS-CoV-2 infections. The persistence of these biomarkers for up to two years after infection, along with their ability to distinguish the severity of the infection endured, highlights the surprising presence of persistent nasopharyngeal exudate changes in fully recovered patients.","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142405715","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}

引用次数: 0

Proteomic Analysis Reveals Major Proteins and Pathways That Mediate the Effect of 17-β-Estradiol in Cell Division and Apoptosis in Breast Cancer MCF7 Cells. 蛋白质组分析揭示了介导 17-β 雌二醇对乳腺癌 MCF7 细胞分裂和凋亡影响的主要蛋白质和途径

IF 3.8 2区生物学

Journal of Proteome Research Pub Date : 2024-10-11 DOI: 10.1021/acs.jproteome.4c00102

Zhenqi Zhou, Brihget Sicairos, Jianhong Zhou, Yuchun Du

{"title":"Proteomic Analysis Reveals Major Proteins and Pathways That Mediate the Effect of 17-β-Estradiol in Cell Division and Apoptosis in Breast Cancer MCF7 Cells.","authors":"Zhenqi Zhou, Brihget Sicairos, Jianhong Zhou, Yuchun Du","doi":"10.1021/acs.jproteome.4c00102","DOIUrl":"https://doi.org/10.1021/acs.jproteome.4c00102","url":null,"abstract":"Despite extensive research, the genes/proteins and pathways responsible for the physiological effects of estrogen remain elusive. In this study, we determined the effect of estrogen on global protein expression in breast cancer MCF7 cells using a proteomic method. The expression of 77 cytosolic, 74 nuclear, and 81 membrane/organelle proteins was significantly altered by 17-β-estradiol (E2). Protein enrichment analyses suggest that E2 may stimulate cell division primarily by promoting the G1 to S phase transition and advancing the G2/M checkpoint. The effect of E2 on cell survival was complex, as it could simultaneously enhance and inhibit apoptosis. Bioinformatics analysis suggests that E2 may enhance apoptosis by promoting the accumulation of the pore-forming protein Bax in the mitochondria and inhibit apoptosis by activating the PI3K/AKT/mTOR signaling pathway. We verified the activation of the PI3K signaling and the accumulation of Bax in the membrane/organelle fraction in E2-treated cells using immunoblotting. Treatment of MCF7 cells with E2 and the PI3K inhibitor Ly294002 significantly enhanced apoptosis compared to those treated with E2 alone, suggesting that combining estrogen with a PI3K inhibitor could be a promising strategy for treating ERα-positive breast cancer. Interestingly, many of the E2-upregulated proteins contained the HEAT, KH, and RRM domains.","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142398631","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}

引用次数: 0

In-Depth Proteomic Analysis Reveals Phenotypic Diversity of Macrophages in Liver Fibrosis. 深入的蛋白质组分析揭示了肝纤维化中巨噬细胞的表型多样性

IF 3.8 2区生物学

Journal of Proteome Research Pub Date : 2024-10-09 DOI: 10.1021/acs.jproteome.4c00681

Wenting Yang, Liling Chen, Jian Zhang, Chenyi Qiu, Wenhao Hou, Xiangye Zhang, Bin Fu, Dianyuan Zhao, Huan Wang, Di Liu, Fang Yan, Wantao Ying, Li Tang

{"title":"In-Depth Proteomic Analysis Reveals Phenotypic Diversity of Macrophages in Liver Fibrosis.","authors":"Wenting Yang, Liling Chen, Jian Zhang, Chenyi Qiu, Wenhao Hou, Xiangye Zhang, Bin Fu, Dianyuan Zhao, Huan Wang, Di Liu, Fang Yan, Wantao Ying, Li Tang","doi":"10.1021/acs.jproteome.4c00681","DOIUrl":"https://doi.org/10.1021/acs.jproteome.4c00681","url":null,"abstract":"Macrophages make up a heterogeneous population of immune cells that exhibit diverse phenotypes and functions in health and disease. Although macrophage epigenomic and transcriptomic profiles have been reported, the proteomes of distinct macrophage populations under various pathological conditions remain largely elusive. Here, we employed a label-free proteomic approach to characterize the diversity of the hepatic macrophage pool in an experimental model of CCl4-induced liver fibrosis. We found a decrease in the proportion of liver resident embryo-derived KCs (EmKCs), and a drastic increase in the proportion of monocyte-derived KCs (MoKCs) and CLEC2-Macs. Proteomic profiling revealed that MoKCs largely resembled EmKCs, whereas CLEC2-Macs exhibited greater proteomic alternations compared with EmKCs, suggesting two distinct destinations for monocyte differentiation during liver fibrosis. Furthermore, CLEC2-Macs were characterized by increased expression of proteins associated with inflammatory response, antigen processing and presentation processes, which may be involved in the pathogenesis of liver fibrosis. Collectively, our study provides insights into the considerable heterogeneity within the hepatic macrophage pool during liver fibrosis.","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142386408","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}

引用次数: 0

Deep Proteome Analysis of Cerebrospinal Fluid from Pediatric Patients with Central Nervous System Cancer. 中枢神经系统癌症小儿患者脑脊液的深度蛋白质组分析

IF 3.8 2区生物学

Journal of Proteome Research Pub Date : 2024-10-09 DOI: 10.1021/acs.jproteome.4c00471

Christian Mirian, Ole Østergaard, Maria Thastrup, Signe Modvig, Jon Foss-Skiftesvik, Jane Skjøth-Rasmussen, Marianne Berntsen, Josefine Britze, Alex Christian Yde Nielsen, René Mathiasen, Kjeld Schmiegelow, Jesper Velgaard Olsen

{"title":"Deep Proteome Analysis of Cerebrospinal Fluid from Pediatric Patients with Central Nervous System Cancer.","authors":"Christian Mirian, Ole Østergaard, Maria Thastrup, Signe Modvig, Jon Foss-Skiftesvik, Jane Skjøth-Rasmussen, Marianne Berntsen, Josefine Britze, Alex Christian Yde Nielsen, René Mathiasen, Kjeld Schmiegelow, Jesper Velgaard Olsen","doi":"10.1021/acs.jproteome.4c00471","DOIUrl":"https://doi.org/10.1021/acs.jproteome.4c00471","url":null,"abstract":"The cerebrospinal fluid (CSF) is a key matrix for discovery of biomarkers relevant for prognosis and the development of therapeutic targets in pediatric central nervous system malignancies. However, the wide range of protein concentrations and age-related differences in children makes such discoveries challenging. In addition, pediatric CSF samples are often sparse and first prioritized for clinical purposes. The present work focused on optimizing each step of the proteome analysis workflow to extract the most detailed proteome information possible from the limited CSF resources available for research purposes. The strategy included applying sequential ultracentrifugation to enrich for extracellular vesicles (EV) in addition to analysis of a small volume of raw CSF, which allowed quantification of 1351 proteins (+55% relative to raw CSF) from 400 μL CSF. When including a spectral library, a total of 2103 proteins (+240%) could be quantified. The workflow was optimized for CSF input volume, tryptic digestion method, gradient length, mass spectrometry data acquisition method and database search strategy to quantify as many proteins a possible. The fully optimized workflow included protein aggregation capture (PAC) digestion, paired with data-independent acquisition (DIA, 21 min gradient) and allowed 2989 unique proteins to be quantified from only 400 μL CSF, which is a 340% increase in proteins compared to analysis of a tryptic digest of raw CSF.","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142386405","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}

引用次数: 0

Carrier-Guided Proteome Analysis in a High Protein Background: An Improved Approach to Host Cell Protein Identification. 高蛋白背景下的载体引导蛋白质组分析：宿主细胞蛋白质鉴定的改进方法。

IF 3.8 2区生物学

Journal of Proteome Research Pub Date : 2024-10-09 DOI: 10.1021/acs.jproteome.4c00158

Divyanshi Karmani, Niloofar Seifihesar, Mukhayyo Sultonova, Beau Blackmore, Joao A Paulo, Matthew Harty, J Patrick Murphy

{"title":"Carrier-Guided Proteome Analysis in a High Protein Background: An Improved Approach to Host Cell Protein Identification.","authors":"Divyanshi Karmani, Niloofar Seifihesar, Mukhayyo Sultonova, Beau Blackmore, Joao A Paulo, Matthew Harty, J Patrick Murphy","doi":"10.1021/acs.jproteome.4c00158","DOIUrl":"https://doi.org/10.1021/acs.jproteome.4c00158","url":null,"abstract":"Many shotgun proteomics experiments are negatively influenced by highly abundant proteins, such as those measuring residual host cell proteins (HCP) amidst highly abundant recombinant biotherapeutic or plasma proteins amidst albumin and immunoglobulins. While western blotting and ELISAs can reveal the presence of specific low abundance proteins from highly abundant background proteins, mass spectrometry approaches are required to define the low abundance protein composition in these scenarios. The challenge in detecting low abundance proteins in a high protein background by standard shotgun approaches is that spectra are often not triggered on their peptides in data dependent acquisition methods but rather on the highly abundant background peptides. Here, we use tandem mass tags (TMT) to introduce a carrier proteome approach to enhance the detection of proteins, such as from residual host cell proteomes amidst a highly abundant background. Using a mixture of bovine serum albumin (BSA) and E. coli as a mock high background/low abundance target protein formulation, we demonstrate proof-of-principle experiments allowing the improved detection of target proteins amidst a high protein background. While we observed significant coisolation interference, we mitigated it by using a spike-in interference detection TMT channel. Finally, we use the approach to identify 300 residual E. coli proteins from a protein A pulldown of a human IgG antibody, demonstrating that it may be applicable to analysis of HCPs in biotherapeutic protein formulations.","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142386404","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}

引用次数: 0

Achieving a 35-Plex Tandem Mass Tag Reagent Set through Deuterium Incorporation. 通过氘结合实现 35 种串联质量标签试剂组合

IF 3.8 2区生物学

Journal of Proteome Research Pub Date : 2024-10-08 DOI: 10.1021/acs.jproteome.4c00668

Nathan R Zuniga, Dustin C Frost, Karsten Kuhn, Myungsun Shin, Rebecca L Whitehouse, Ting-Yu Wei, Yuchen He, Shane L Dawson, Ian Pike, Ryan D Bomgarden, Steven P Gygi, Joao A Paulo

{"title":"Achieving a 35-Plex Tandem Mass Tag Reagent Set through Deuterium Incorporation.","authors":"Nathan R Zuniga, Dustin C Frost, Karsten Kuhn, Myungsun Shin, Rebecca L Whitehouse, Ting-Yu Wei, Yuchen He, Shane L Dawson, Ian Pike, Ryan D Bomgarden, Steven P Gygi, Joao A Paulo","doi":"10.1021/acs.jproteome.4c00668","DOIUrl":"https://doi.org/10.1021/acs.jproteome.4c00668","url":null,"abstract":"Mass spectrometry-based sample multiplexing with isobaric tags permits the development of high-throughput and precise quantitative biological assays with proteome-wide coverage and minimal missing values. Here, we nearly doubled the multiplexing capability of the TMTpro reagent set to a 35-plex through the incorporation of one deuterium isotope into the reporter group. Substituting deuterium frequently results in suboptimal peak coelution, which can compromise the accuracy of reporter ion-based quantification. To counteract the deuterium effect on quantitation, we implemented a strategy that necessitated the segregation of nondeuterium and deuterium-containing channels into distinct subplexes during normalization procedures, with reassembly through a common bridge channel. This multiplexing strategy of \"design independent sub-plexes but acquire together\" (DISAT) was used to compare protein expression differences between human cell lines and in a cysteine-profiling (i.e., chemoproteomics) experiment to identify compounds binding to cysteine-113 of Pin1.","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142386403","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}

引用次数: 0

DiDBiT-TMT: A Novel Method to Quantify Changes in the Proteomic Landscape Induced by Neural Plasticity. DiDBiT-TMT：量化神经可塑性引起的蛋白质组景观变化的新方法。

IF 3.8 2区生物学

Journal of Proteome Research Pub Date : 2024-10-07 DOI: 10.1021/acs.jproteome.4c00180

Mariam Gamaleldin, Nam-Kyung Yu, Jolene K Diedrich, Yuanhui Ma, Anne Wienand, Daniel B McClatchy, Anders Nykjaer, Sadegh Nabavi, John R Yates

{"title":"DiDBiT-TMT: A Novel Method to Quantify Changes in the Proteomic Landscape Induced by Neural Plasticity.","authors":"Mariam Gamaleldin, Nam-Kyung Yu, Jolene K Diedrich, Yuanhui Ma, Anne Wienand, Daniel B McClatchy, Anders Nykjaer, Sadegh Nabavi, John R Yates","doi":"10.1021/acs.jproteome.4c00180","DOIUrl":"https://doi.org/10.1021/acs.jproteome.4c00180","url":null,"abstract":"Direct detection of biotinylated proteins (DiDBiT) is a proteomic method that can enrich and detect newly synthesized proteins (NSPs) labeled with bio-orthogonal amino acids with 20-fold improved detectability compared to conventional methods. However, DiDBiT has currently been used to compare only two conditions per experiment. Here, we present DiDBiT-TMT, a method that can be used to quantify NSPs across many conditions and replicates in the same experiment by combining isobaric tandem mass tagging (TMT) with DiDBiT. We applied DiDBiT-TMT to brain slices to determine changes in the de novo proteome that occur after inducing chemical long-term potentiation (cLTP) or treatment with the neuromodulator norepinephrine. We successfully demonstrated DiDBiT-TMT's capacity to quantitatively compare up to 9 samples in parallel. We showed that there is a minimal overlap among NSPs that are differentially expressed in cLTP-treated organotypic brain slices, norepinephrine-treated organotypic brain slices, and organotypic slices undergoing combinatorial treatment with norepinephrine and cLTP. Our results point to the possible divergence of the molecular mechanisms underlying these treatments and showcase the applicability of DiDBiT-TMT for studying neurobiology.","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142386406","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}

引用次数: 0

End-to-End Throughput Chemical Proteomics for Photoaffinity Labeling Target Engagement and Deconvolution. 用于光亲和标记目标参与和解旋的端到端通量化学蛋白质组学。

IF 3.8 2区生物学

Journal of Proteome Research Pub Date : 2024-10-07 DOI: 10.1021/acs.jproteome.4c00442

Sheldon T Cheung, Yongkang Kim, Ji-Hoon Cho, Kristoffer R Brandvold, Brahma Ghosh, Amanda M Del Rosario, Harris Bell-Temin

{"title":"End-to-End Throughput Chemical Proteomics for Photoaffinity Labeling Target Engagement and Deconvolution.","authors":"Sheldon T Cheung, Yongkang Kim, Ji-Hoon Cho, Kristoffer R Brandvold, Brahma Ghosh, Amanda M Del Rosario, Harris Bell-Temin","doi":"10.1021/acs.jproteome.4c00442","DOIUrl":"https://doi.org/10.1021/acs.jproteome.4c00442","url":null,"abstract":"Photoaffinity labeling (PAL) methodologies have proven to be instrumental for the unbiased deconvolution of protein-ligand binding events in physiologically relevant systems. However, like other chemical proteomic workflows, they are limited in many ways by time-intensive sample manipulations and data acquisition techniques. Here, we describe an approach to address this challenge through the innovation of a carboxylate bead-based protein cleanup procedure to remove excess small-molecule contaminants and couple it to plate-based, proteomic sample processing as a semiautomated solution. The analysis of samples via label-free, data-independent acquisition (DIA) techniques led to significant improvements on a workflow time per sample basis over current standard practices. Experiments utilizing three established PAL ligands with known targets, (+)-JQ-1, lenalidomide, and dasatinib, demonstrated the utility of having the flexibility to design experiments with a myriad of variables. Data revealed that this workflow can enable the confident identification and rank ordering of known and putative targets with outstanding protein signal-to-background enrichment sensitivity. This unified end-to-end throughput strategy for processing and analyzing these complex samples could greatly facilitate efficient drug discovery efforts and open up new opportunities in the chemical proteomics field.","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142386407","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}

引用次数: 0