Journal of proteomics最新文献

{"title":"DIA proteomic and PRM validation through human granulose cells profiles screen suitable biomarkers for polycystic ovary syndrome patients","authors":"Faying Liu ,&nbsp;Lifeng Tian ,&nbsp;Ying Zhang ,&nbsp;Wei Deng ,&nbsp;Xiaoyun Xu ,&nbsp;Yang Zou ,&nbsp;Ruifang An","doi":"10.1016/j.jprot.2024.105332","DOIUrl":"10.1016/j.jprot.2024.105332","url":null,"abstract":"<div><div>The aim of this study is to identify differentially expressed proteins (DEPs) in granulose cells (GCs) from women with or withoutpolycystic ovary syndrome (PCOS) via data independent acquisition (DIA) proteomic analysis.A total of 63 women were recruited for this study, 34 PCOS patients as experimental group (P), and 29 women without PCOS as Normal group (NP). DIA-based proteomic analysis was performed to identify DEPs in GCs between the P and NP samples. Certain typical DEPs were further validated by Parallel reaction monitoring (PRM), and correlation analysis was performed between these DEPs and the clinical characteristics.Cell vitality was measured by CCK-8 assay. DIA analysis revealed 174 significantly DEPs, of which 7 were upregulated and 167 downregulated. Bioinformatics analysis was performed to analysis the significantly DEPs. The PRM experiment confirmed TOP2A and SPHKAP were upregulated significantly in P by comparing to NP, while GM2A, MRPS16, APOA2 and FGF2 were downregulated significantly. Most notably, Correlation analysis revealed that TOP2A, SPHKAP, MRPS16 and FGF2were positively correlated with TG, AMH and Age, but negatively correlated with Menarche age, DBIL, FT3, Basal serum FSH and LH.Meanwhile, CCK-8 assay has shown that downregulation of FGF2 could weaken cell viability. Finally, a panel of DEPs were identified in the GCs of patients with PCOS, of which certain significant DEPs might play essential roles in the pathogenesis of PCOS, could be regarded as candidate biomarkers for PCOS.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142468381","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}

{"title":"Untargeted metabolomics of 3xTg-AD neurotoxic astrocytes","authors":"Diego Carvalho ,&nbsp;Pablo Diaz-Amarilla ,&nbsp;Mathew R. Smith ,&nbsp;María Daniela Santi ,&nbsp;Marcela Martinez-Busi ,&nbsp;Young-Mi Go ,&nbsp;Dean P. Jones ,&nbsp;Pablo Duarte ,&nbsp;Eduardo Savio ,&nbsp;Juan A. Abin-Carriquiry ,&nbsp;Florencia Arredondo","doi":"10.1016/j.jprot.2024.105336","DOIUrl":"10.1016/j.jprot.2024.105336","url":null,"abstract":"<div><div>Alzheimer's disease (AD) is the most common form of dementia, affecting approximately 47 M people worldwide. Histological features and genetic risk factors, among other evidence, supported the amyloid hypothesis of the disease. This neuronocentric paradigm is currently undergoing a shift, considering evidence of the role of other cell types, such as microglia and astrocytes, in disease progression. Previously, we described a particular astrocyte subtype obtained from the 3xTg-AD murine model that displays neurotoxic properties in vitro. We continue here our exploratory analysis through the lens of metabolomics to identify potentially altered metabolites and biological pathways.</div><div>Cell extracts from neurotoxic and control astrocytes were compared using high-resolution mass spectrometry-based metabolomics. Around 12 % of metabolic features demonstrated significant differences between neurotoxic and control astrocytes, including alterations in the key metabolite glutamate. Consistent with our previous transcriptomic study, the present results illustrate many homeostatic and regulatory functions of metabolites, suggesting that neurotoxic 3xTg-AD astrocytes exhibit alterations in the Krebs cycle as well as the prostaglandin pathway.</div><div>This is the first metabolomic study performed in 3xTg-AD neurotoxic astrocytes. These results provide insight into metabolic alterations potentially associated with neurotoxicity and pathology progression in the 3xTg-AD mouse model and strengthen the therapeutic potential of astrocytes in AD.</div></div><div><h3>Biological significance</h3><div>Our study is the first high-resolution metabolomic characterization of the novel neurotoxic 3xTg-AD astrocytes. We propose key metabolites and pathway alterations, as well as possible associations with gene expression alterations in the model. Our results are in line with recent hypotheses beyond the amyloid cascade, considering the involvement of several stress response cascades during the development of Alzheimer's disease. This work could inspire other researchers to initiate similar studies in related models. Furthermore, this work illustrates a powerful workflow for metabolite annotation and selection that can be implemented in other studies.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142502655","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}

{"title":"Serum proteomic analysis identified ITIH4 as a potential novel biomarker for feline infectious peritonitis","authors":"Yuzhou Jiao ,&nbsp;Mengfang Yang ,&nbsp;Lingying Fang ,&nbsp;Yuanyuan Yan ,&nbsp;Zhen Fu ,&nbsp;Mengxia Li ,&nbsp;Lisha Li ,&nbsp;Zirui Liu ,&nbsp;Xiaoshuai Hu ,&nbsp;Benyuan Wu ,&nbsp;Yuejun Shi ,&nbsp;Chao Kang ,&nbsp;Zhou Shen ,&nbsp;Guiqing Peng","doi":"10.1016/j.jprot.2024.105338","DOIUrl":"10.1016/j.jprot.2024.105338","url":null,"abstract":"<div><div>Feline infectious peritonitis (FIP) is a fatal feline disease. At present, the reference standard for FIP diagnosis is immunohistochemistry (IHC) of organs, but this method involves high time-related costs, invasive sampling procedures and professional requirements. Serological detection is a common auxiliary method for diagnosing diseases. As a result, we assessed the changes in the serum proteome of FIP patients with the aim of identifying novel specific serum biomarkers that could aid in the clinical diagnosis of FIP. Pre- and postinfection groups were compared and 92 differentially expressed proteins (DEPs) were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the DEPs revealed that the enriched GO terms and KEGG pathways among the DEPs were immune activation, peptidase regulator activity and the complement and coagulation cascade pathways. The level of peptidase regulator interalpha-trypsin inhibitor heavy chain 4 (ITIH4) in cat serum was significantly correlated with FIP. The areas under the ROC curve (AUCs) of full-length ITIH4 (f-ITIH4) and cleaved ITIH4 (c-ITIH4) expression were 0.922 and 1.000, respectively, which allowed the discrimination of FIP cats from healthy cats. These results suggest that ITIH4 may be a potential serum biomarker for detecting early FIP.</div></div><div><h3>Significance</h3><div>FIP causes fatal disease in cats of almost all ages, and there is currently no effective vaccine or treatment for FIP. Therefore, early diagnosis is extremely important for disease prevention and control. The results of the model and clinical samples revealed that ITIH4 was significantly increased in the serum of FIP cats. This study is the first to propose ITIH4 as a diagnostic biomarker in cats with FIP and our results suggest that serum ITIH4 levels might identify cats with FIP during the early stage.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142502654","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}

{"title":"Proteomic analysis of the human amniotic mesenchymal stromal cell secretome by integrated approaches via filter-aided sample preparation","authors":"Alexandra Muntiu ,&nbsp;Andrea Papait ,&nbsp;Federica Vincenzoni ,&nbsp;Diana Valeria Rossetti ,&nbsp;Pietro Romele ,&nbsp;Anna Cargnoni ,&nbsp;Antonietta Silini ,&nbsp;Ornella Parolini ,&nbsp;Claudia Desiderio","doi":"10.1016/j.jprot.2024.105339","DOIUrl":"10.1016/j.jprot.2024.105339","url":null,"abstract":"<div><div>The immunomodulatory, anti-inflammatory and regenerative properties of the human amniotic mesenchymal stromal cells (hAMSCs) secretome are acknowledged but the understanding of the specific bioactive components remains incomplete. To address these limitations, the present investigation aimed to profile the proteins and peptides content of the hAMSC secretome through sample pretreatment and fractionation on 10 kDa molecular cut-off FASP (Filter Aided Sample Preparation) device and LC-MS analysis. The filter retained protein fraction underwent trypsin digestion, while the unretained was collected unchanged for intact small proteins and peptides analysis. This combined approach (C-FASP) collects in a single step two complementary fractions, advantageously saving sample volume and time of analysis. The bottom-up analysis of the C-FASP proteins fraction &gt;10 kDa confirmed our previous findings, establishing a set of proteins consistently characterizing the hAMSC secretome. The analysis of the fraction &lt;10 kDa, never been investigated to our knowledge, identified peptide fragments of thymosin beta 4 and beta 10, collagen alpha 1 chains I and III, alpha-enolase, and glyceraldehyde-3-phosphate dehydrogenase, involved in wound healing, anti-inflammatory response, tissue repair and regeneration, key biological activities of the secretome. C-FASP provided a comprehensive molecular profile of the hAMSC secretome offering new insights for enhanced therapeutic applications in regenerative medicine.</div></div><div><h3>Significance</h3><div>In this investigation we originally present the comprehensive proteomic investigation of the human amniotic mesenchymal stromal cell secretome by combining the analysis of the proteome and of the peptidome following sample pretreatment and fractionation by Filter Aided Sample Preparation (FASP) with 10 kDa molecular cut-off in coupling with LC-MS analysis. The proteome fraction retained by FASP filter was analyzed after enzymatic digestion, while the unretained fraction, below 10 kDa molecular mass, was analyzed unchanged in its intact form. This dual approach provides novel insights, previously unexplored, into the molecular components potentially responsible for the immunomodulatory and anti-inflammatory properties of the hAMSC secretome. These findings could significantly enhance the therapeutic potential of hAMSCs in regenerative medicine.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142502653","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}

{"title":"Intraspecific venom variation in the Iberian asp viper (Vipera aspis zinnikeri) across natural and intensive agricultural habitats","authors":"Jon Buldain ,&nbsp;Rui Vitorino ,&nbsp;Tânia Lima ,&nbsp;Ignazio Avella ,&nbsp;Óscar Zuazo ,&nbsp;Fernando Martínez-Freiría","doi":"10.1016/j.jprot.2024.105337","DOIUrl":"10.1016/j.jprot.2024.105337","url":null,"abstract":"<div><div>Snake venom composition varies at different levels. To date, comparative venom studies have seldom taken into account the role of habitat type in the occurrence of snake venom variation. Here we investigated the presence of venom variation across different populations of the Iberian asp viper (<em>Vipera aspis zinnikeri</em>) inhabiting two contrasting habitats: natural vs. intensive agricultural. We used shotgun proteomics to describe the protein composition of the venoms of six adults from two distinct localities. Furthermore, to test whether local conditions and habitat can alter venom composition in this taxon, we compared the SDS-PAGE profiles of 40 adult venoms from six populations, three in natural habitats and three in intensive agricultural environments. The venoms were composed of 21 toxin families, of which five (CTL, PLA₂, VEGF, svSP, and svMP) comprised 69–82 % of each proteome. The relative abundances of toxin families varied considerably at inter- and intra-population levels. Linear regression performed on non-metric multidimensional scaling values showed a significant effect of locality of origin and habitat type on the differences detected between individual SDS-PAGE venom profiles. Our results suggest the presence of regional variation in <em>V. a. zinnikeri</em> venom, potentially reinforcing the role of local pressures in shaping snake venom composition.</div></div><div><h3>Significance</h3><div>This work provides the first proteomic characterization of the venom of the Iberian asp viper, <em>Vipera aspis zinnikeri</em>, obtained by means of shotgun proteomics. The statistical analysis of 40 individual SDS-PAGE venom profiles highlights that venom variation in this taxon can be associated with geographical origin and habitat type of the area where each viper was collected. Our results suggest the presence of regional variation in <em>V. a. zinnikeri</em> venom, reinforcing the role that local pressures may play as drivers of snake venom variation.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142502652","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}

{"title":"Phosphoproteomic analysis reveals the mechanisms of human umbilical cord mesenchymal stem cell-derived exosomes attenuate renal aging","authors":"Wenzhuo Yu ,&nbsp;Xu Jia ,&nbsp;Han Qiao ,&nbsp;Di Liu ,&nbsp;Yan Sun ,&nbsp;Rong Yan ,&nbsp;Chenglong Zhang ,&nbsp;Na Yu ,&nbsp;Yiping Song ,&nbsp;Mingying Ling ,&nbsp;Zhen Zhang ,&nbsp;Xuehui Li ,&nbsp;Chuanli Zhao ,&nbsp;Yanqiu Xing","doi":"10.1016/j.jprot.2024.105335","DOIUrl":"10.1016/j.jprot.2024.105335","url":null,"abstract":"<div><div>Aging is a critical biological process, with particularly notable impacts on the kidneys. Exosomes derived from human umbilical cord mesenchymal stem cells (hUC-MSCs) are capable of transferring various bioactive molecules, which exhibit beneficial therapeutic effects on kidney diseases. This study demonstrates that exosomes derived from hUC-MSCs ameliorate cellular senescence in the kidneys of naturally aging mice. These exosomes reduce the protein expression of senescence markers and senescence-associated secretory phenotypes (SASP) leading to fewer DNA damage foci and increased expression of the proliferation indicator Ki67. During the aging process, many proteins undergo phosphorylation modifications. We utilized data-independent acquisition (DIA) phosphoproteomics to study kidneys of naturally aging mice and those treated with hUC-MSC-derived exosomes. We observed elevated phosphorylation levels of the differentially phosphorylated proteins, Lamin A/C, at Ser390 and Ser392 sites, which were subsequently verified by western blotting. Overall, this study provides a new molecular characterization of hUC-MSC-derived exosomes in mitigating cellular senescence in the kidneys.</div></div><div><h3>Significance</h3><div>DIA phosphoproteomics was employed to investigate phosphorylated proteins in the kidney tissues of naturally aging mice with hUCMSC-exos treated. The results demonstrated that the DIA technique detected a higher abundance of phosphorylated proteins. We identified 24 significantly differentially phosphorylated proteins, and found that the phosphorylation of specific Lamin A/C sites is crucial for preventing cellular senescence. This study will help to better reveal the related phosphorylated proteins involved in hUCMSC-exos intervention in the kidneys of naturally aging mice, providing a foundation for future research on specific phosphorylation sites of proteins as potential therapeutic targets for renal aging-related diseases.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142468379","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}

{"title":"In-depth proteomics and Phosphoproteomics reveal biomarkers and molecular pathways of chronic intermittent hypoxia in mice.","authors":"Huanhuan Fang, Ye Zhang, Liangming Zhu, Jinzhao Lyu, Qiang Li","doi":"10.1016/j.jprot.2024.105334","DOIUrl":"https://doi.org/10.1016/j.jprot.2024.105334","url":null,"abstract":"<p><p>Obstructive sleep apnea (OSA) syndrome is characterized by Chronic Intermittent Hypoxia (CIH). In this study, we employed Data-independent acquisition (DIA) Mass Spectrometry to conduct comprehensive proteomic and phosphoproteomic profiling of a murine model subjected to Chronic Intermittent Hypoxia (CIH), a model we had previously established. Utilizing three CIH and three normal control genioglossus samples, we gathered valuable insights into the molecular alterations associated with CIH. Our analyses identified a total of 4576 protein groups and 13,867 phosphosites. Differential analysis of the proteomic data highlighted a significant upregulation of Ras signaling (Egf, Ngf, and Fyb1) and calcium signaling (Tnn, Thbs4, and Ppp2r2d) in CIH samples, contrasting with a notable decrease in oxidative phosphorylation (Atp5mf, Atp5me, and Atp5mg). Additionally, we observed a substantial increase in the phosphorylation of PI3K-AKT signaling (Ptk2_Y861, Mapk3_T203, and Eif4b_S230) and HIF-1 signaling (Gapdh_S208, Eno3_T229, and Camk2b_T382) in CIH samples. These findings prompted a deeper investigation into the association of the characterized proteins and phosphoproteins with Obstructive Sleep Apnea (OSA). The comprehensive profiling revealed molecular signatures that may serve as valuable insights into the pathophysiology of chronic intermittent hypoxia and its link to obstructive sleep apnea. Our observations provide a foundation for future research endeavors, offering potential avenues for advancing our understanding and treatment strategies for these conditions. SIGNIFICANCE: The significance of this study lies in its comprehensive exploration of the molecular mechanisms underpinning Chronic Intermittent Hypoxia (CIH), a key feature of Obstructive Sleep Apnea (OSA). By employing Data-independent acquisition (DIA) Mass Spectrometry, this research provides an in-depth proteomic and phosphoproteomic analysis, uncovering critical signaling pathways and molecular alterations associated with CIH. The identification of significant changes in Ras and calcium signaling pathways, along with increased phosphorylation in PI3K-AKT and HIF-1 signaling, offers novel insights into the pathophysiological processes involved in CIH and OSA. These findings not only enhance our understanding of the molecular basis of OSA but also pave the way for the development of targeted therapeutic strategies, ultimately contributing to better management and treatment of OSA and related conditions.</p>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142468378","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}

{"title":"TMT-based quantitative proteomics unveils the protective mechanism of Polygonatum sibiricum polysaccharides on septic acute liver injury","authors":"Linxia Xiao ,&nbsp;Yinuo Ping ,&nbsp;Shangshang Sun ,&nbsp;Ran Xu ,&nbsp;Xinru Zhou ,&nbsp;Hongyan Wu ,&nbsp;Liang Qi","doi":"10.1016/j.jprot.2024.105331","DOIUrl":"10.1016/j.jprot.2024.105331","url":null,"abstract":"<div><div><em>Polygonatum sibiricum</em> polysaccharides (PSP) has been shown to possess multiple pharmacological functions. Our previous study found that PSP could protect against acute liver injury during sepsis via inhibiting inflammatory response. However, the underlying molecular mechanism by which PSP alleviates septic acute liver injury (SALI) remains unknown. Herein, TMT-based quantitative proteomics was utilized to explore the essential pathways and proteins involved in the protective effects of PSP on SALI. The results revealed that 632 and 176 differentially expressed proteins (DEPs) were identified in Model_vs_Control and PSP_vs_Model, respectively. GO annotation showed similar trends, suggesting that these DEPs were primarily involved in the cellular anatomical entity in Cellular Component, the cellular processe and the biological regulation in Biological Process, the binding and the catalytic activity in Molecular Function. Meanwhile, KEGG enrichment analysis implied that four common pathways, including the NF-κB signaling pathway, the IL-17 signaling pathway, the TNF signaling pathway and the Toll-like receptor signaling pathway, were closely associated with the pathogenesis of sepsis among the top 20 remarkably enriched pathways in Model_vs_Control_up and PSP_vs_Model_down. Moreover, the levels of several common DEPs, including TLR2, IKKi, JunB and CXCL9, were validated by WB, which was in line with the results of proteomics. Therefore, the protective effects of PSP on SALI might exert via blocking the above-mentioned inflammation pathways.</div><div>Significance: PSP, recognized as a key component of <em>Polygonatum sibiricum</em>, exhibits a range of pharmacological functions. Our previous study found that PSP could protect against SALI, yet failing to clarify the mechanism of action. To reveal the underlying molecular mechanism involved in the protective effects of PSP on SALI, a TMT-based quantitative proteomic analysis was performed to detect and analyse the DEPs in liver tissue among the control group, the model group and the PSP group in this study. The results provide theoretical references for exploring the action mechanism of drugs and facilitate the comprehensive utilization of PSP.</div></div><div><h3>Significance</h3><div>PSP have been identified as the most crucial components of <em>Polygonatum sibiricum</em> with various pharmacological functions. Our previous study found that PSP could protect against SALI, but the mechanism of action remains unknown. To reveal the underlying molecular mechanism involved in the protective effects of PSP on SALI, a TMT-based quantitative proteomic analysis was performed to detect and analyse the DEPs in liver tissue among the control group, the model group and the PSP group in this study. The results provide theoretical references for exploring the action mechanism of drugs and facilitate the comprehensive utilization of PSP.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142468380","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}

{"title":"4-plex quantitative glycoproteomics using glycan/protein-stable isotope labeling in cell culture","authors":"Peilin Jiang ,&nbsp;Md Abdul Hakim ,&nbsp;Arvin Saffarian Delkhosh ,&nbsp;Parisa Ahmadi ,&nbsp;Yunxiang Li ,&nbsp;Yehia Mechref","doi":"10.1016/j.jprot.2024.105333","DOIUrl":"10.1016/j.jprot.2024.105333","url":null,"abstract":"&lt;div&gt;&lt;div&gt;Alterations in glycoprotein abundance and glycan structures are closely linked to numerous diseases. The quantitative exploration of glycoproteomics is pivotal for biomarker discovery, but comprehensive analysis within biological samples remains challenging due to low abundance, complexity, and lack of universal technology. We developed a multiplex glycoproteomic approach using an LC-ESI-MS platform for direct comparison of glycoproteomic quantitation. Glycopeptides were isotopically labeled during cell culture, achieving high labeling efficiency (≥ 95 %) for both glycans and peptides. Quantitation was validated by mixing the same cell line in a 1:1:1:1 ratio, with mathematical correction applied to deconvolute the ratios. This method proved reliable and was applied to a comparative glycoproteomic study of three breast cancer cell lines (HTB22, MDA-MB-231, MDA-MB-231BR) and one brain cancer cell line (CRL-1620), quantifying glycopeptides from three replicates. The expression of glycopeptides was relatively quantified, and up/down-regulation between cell lines was investigated. This approach provided insights into glycosylation microheterogeneity, crucial for breast cancer brain metastasis research. Benefits include eliminating fluctuations from nano electrospray ionization and reducing analysis time, enabling up to 4-plex profiling in a single injection. Metabolic labeling introduced mass differences at the MS1 level, ensuring increased sensitivity and higher resolution for accurate quantitation.&lt;/div&gt;&lt;/div&gt;&lt;div&gt;&lt;h3&gt;Significance&lt;/h3&gt;&lt;div&gt;Alternations in glycoprotein abundance, changes in glycosylation levels, and variations in glycan structures are closely linked to numerous diseases. The quantitative exploration of glycoproteomics has emerged as a popular area of research for biomarker discovery. However, conducting a comprehensive quantitative analysis of the glycoproteome within biological samples remains challenging due to low abundance, inherent complexities, and the absence of universal quantitative technology. Here, we developed a multiplex glycoproteomic approach using an LC-ESI-MS platform to facilitate direct comparison of glycoproteomic quantitation and enhance throughput. This approach offers benefits such as eliminating quantitative fluctuations arising from nano electrospray ionization (ESI) and reducing analysis time, enabling up to 4-plex glycoproteomic profiling in a single injection. Glycopeptides were stable isotopic labeled during cell culture procedure, attaching to monosaccharides, amino acids, or both. We achieved a high labeling efficiency (≥ 95 %) for both glycans and peptides. Quantitation validation was tested on glycopeptides by mixing the same cell line with 1:1:1:1 ratio. Due to the overlapped isotopes, a mathematical correction was applied to deconvolute the ratio of 4-plex glycopeptides. This method demonstrated quantitative reliability and was successfully applied to a comparative glycoproteomic study of","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142468370","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}

{"title":"DSSBU: A novel mass spectrometry-cleavable analogue of the BS3 cross-linker","authors":"Banerjee Swati ,&nbsp;Sýs Jakub ,&nbsp;Machara Aleš ,&nbsp;Junková Petra ,&nbsp;Hubálek Martin","doi":"10.1016/j.jprot.2024.105330","DOIUrl":"10.1016/j.jprot.2024.105330","url":null,"abstract":"<div><div>Protein cross-linking has assumed an irreplaceable role in structural proteomics. Recently, significant efforts have been made to develop novel mass spectrometry (MS)-cleavable reagents. At present, only water-insoluble MS-cleavable cross-linkers are commercially available. However, to comprehensively analyse the various chemical and structural motifs making up proteins, it is necessary to target different protein sites with varying degrees of hydrophilicity. Here we introduce the new MS-cleavable cross-linker disulfodisuccinimidyl dibutyric urea (DSSBU), which we have developed in-house for this purpose. DSSBU contains an <em>N</em>-hydroxysulfosuccinimide (sulfo-NHS) reactive group, so it can serve as a water-soluble counterpart to the widely used cross-linker disuccinimidyl dibutyric urea (DSBU). To investigate the applicability of DSSBU, we compared the efficacy of four similar cross-linkers: bis[sulfosuccinimidyl] suberate (BS³), disuccinimidyl suberate (DSS), DSBU and DSSBU with bovine serum albumin. In addition, we compared the efficacy of DSBU and DSSBU with human haemoglobin. Our results demonstrate that the sulfo-NHS group ensures the superior water solubility of DSSBU and thus negates the need for organic solvents such as dimethyl sulfoxide while preserving the effectivity of urea-based MS-cleavable crosslinkers such as DSBU. Additionally, it makes it possible to target polar regions in proteins. The data gathered are available via ProteomeXchange under identifier <span><span>PXD055284</span><svg><path></path></svg></span>.</div></div><div><h3>Significance</h3><div>We have synthesized the novel protein cross-linker DSSBU, which combines sulfo-NHS ester chemistry with a mass <em>spectrometry-</em>cleavable urea group. This makes DSSBU a water-soluble, MS-cleavable cross-linker that reacts with amino groups. To our knowledge, it is the first cross-linker which combines all three of these characteristics. We have tested the performance of our novel cross-linker on bovine serum albumin, a model widely used by the cross-linking mass spectrometry community, and on human haemoglobin. We have comprehensively assessed the performance of DSSBU and compared its efficacy with that of three other cross-linkers in current use (BS³, DSS and DSBU). We conclude that our novel cross-linker surpasses its MS-non-cleavable analogue BS³ in performance and that its water solubility eliminates the need for organic solvents while its hydrophilicity allows for the targetting of polar regions in proteins. Therefore, it will likely become a significant addition to the portfolio of <em>N</em>-hydroxysuccinimide ester cross-linkers.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142468377","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}