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Phosphoproteomic analysis reveals the mechanisms of human umbilical cord mesenchymal stem cell-derived exosomes attenuate renal aging 磷蛋白组分析揭示了人脐带间充质干细胞外泌体减轻肾脏衰老的机制
IF 2.8 2区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-20 DOI: 10.1016/j.jprot.2024.105335
Wenzhuo Yu , Xu Jia , Han Qiao , Di Liu , Yan Sun , Rong Yan , Chenglong Zhang , Na Yu , Yiping Song , Mingying Ling , Zhen Zhang , Xuehui Li , Chuanli Zhao , Yanqiu Xing
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.

Significance

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.
衰老是一个关键的生物过程,对肾脏的影响尤为显著。从人脐带间充质干细胞(hUC-MSCs)中提取的外泌体能够转移各种生物活性分子,对肾脏疾病具有有益的治疗作用。本研究证明,从人脐带间充质干细胞中提取的外泌体可改善自然衰老小鼠肾脏的细胞衰老。这些外泌体减少了衰老标志物和衰老相关分泌表型(SASP)的蛋白表达,导致DNA损伤灶减少,增殖指标Ki67的表达增加。在衰老过程中,许多蛋白质会发生磷酸化修饰。我们利用数据独立获取(DIA)磷酸化蛋白质组学研究了自然衰老小鼠的肾脏和用源自hUC-间充质干细胞的外泌体治疗的小鼠的肾脏。我们观察到不同磷酸化蛋白 Lamin A/C 在 Ser390 和 Ser392 位点的磷酸化水平升高,随后通过 Western 印迹进行了验证。总之,这项研究为 hUC 间充质干细胞衍生的外泌体在减轻肾脏细胞衰老方面提供了新的分子特征。意义:研究人员采用 DIA 磷酸化蛋白质组学研究了经 hUCMSC 外泌体处理的自然衰老小鼠肾脏组织中的磷酸化蛋白质。结果表明,DIA 技术能检测到更高丰度的磷酸化蛋白。我们发现了 24 种明显不同的磷酸化蛋白,并发现特定 Lamin A/C 位点的磷酸化对防止细胞衰老至关重要。这项研究将有助于更好地揭示参与 hUCMSC-exos 对自然衰老小鼠肾脏干预的相关磷酸化蛋白,为今后研究特定磷酸化位点蛋白作为肾脏衰老相关疾病的潜在治疗靶点奠定基础。
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引用次数: 0
In-depth proteomics and Phosphoproteomics reveal biomarkers and molecular pathways of chronic intermittent hypoxia in mice. 深度蛋白质组学和磷蛋白组学揭示了小鼠慢性间歇性缺氧的生物标记物和分子通路。
IF 2.8 2区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-19 DOI: 10.1016/j.jprot.2024.105334
Huanhuan Fang, Ye Zhang, Liangming Zhu, Jinzhao Lyu, Qiang Li

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.

阻塞性睡眠呼吸暂停(OSA)综合征的特点是慢性间歇性缺氧(CIH)。在这项研究中,我们采用了数据独立采集(DIA)质谱法,对我们之前建立的慢性间歇性缺氧(CIH)小鼠模型进行了全面的蛋白质组和磷酸蛋白质组分析。利用三份 CIH 和三份正常对照组的舌根样本,我们收集到了与 CIH 相关的分子变化的宝贵信息。我们的分析共鉴定出 4576 个蛋白质组和 13,867 个磷酸位点。蛋白质组数据的差异分析显示,CIH 样本中的 Ras 信号转导(Egf、Ngf 和 Fyb1)和钙信号转导(Tnn、Thbs4 和 Ppp2r2d)显著上调,而氧化磷酸化(Atp5mf、Atp5me 和 Atp5mg)则明显下降。此外,我们还观察到 CIH 样本中 PI3K-AKT 信号(Ptk2_Y861、Mapk3_T203 和 Eif4b_S230)和 HIF-1 信号(Gapdh_S208、Eno3_T229 和 Camk2b_T382)的磷酸化显著增加。这些发现促使人们深入研究这些特征蛋白和磷酸化蛋白与阻塞性睡眠呼吸暂停(OSA)的关联。综合分析揭示的分子特征可能对慢性间歇性缺氧的病理生理学及其与阻塞性睡眠呼吸暂停的联系有宝贵的见解。我们的观察结果为今后的研究工作奠定了基础,并为促进我们对这些病症的理解和治疗策略提供了潜在的途径。意义:本研究的意义在于全面探索了慢性间歇性缺氧(CIH)的分子机制,这是阻塞性睡眠呼吸暂停(OSA)的一个主要特征。通过采用数据独立采集(DIA)质谱技术,这项研究提供了深入的蛋白质组和磷酸化蛋白质组分析,揭示了与CIH相关的关键信号通路和分子改变。Ras和钙信号通路的重大变化,以及PI3K-AKT和HIF-1信号通路磷酸化的增加,为我们了解CIH和OSA的病理生理过程提供了新的视角。这些发现不仅加深了我们对 OSA 分子基础的理解,还为开发有针对性的治疗策略铺平了道路,最终有助于更好地管理和治疗 OSA 及相关疾病。
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引用次数: 0
TMT-based quantitative proteomics unveils the protective mechanism of Polygonatum sibiricum polysaccharides on septic acute liver injury 基于TMT的定量蛋白质组学揭示何首乌多糖对脓毒性急性肝损伤的保护机制
IF 2.8 2区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-18 DOI: 10.1016/j.jprot.2024.105331
Linxia Xiao , Yinuo Ping , Shangshang Sun , Ran Xu , Xinru Zhou , Hongyan Wu , Liang Qi
Polygonatum sibiricum 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.
Significance: PSP, recognized as a key component of Polygonatum sibiricum, 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.

Significance

PSP have been identified as the most crucial components of Polygonatum sibiricum 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.
何首乌多糖(PSP)已被证明具有多种药理作用。我们之前的研究发现,何首乌多糖可通过抑制炎症反应来防止脓毒症急性肝损伤。然而,PSP减轻脓毒症急性肝损伤(SALI)的潜在分子机制仍然未知。在此,研究人员利用基于TMT的定量蛋白质组学探讨了PSP对脓毒性急性肝损伤的保护作用所涉及的重要通路和蛋白质。结果表明,在模型与对照和 PSP 与模型中分别发现了 632 和 176 个差异表达蛋白(DEPs)。GO注释显示出相似的趋势,表明这些差异表达蛋白主要参与细胞成分(Cellular Component)中的细胞解剖实体、生物过程(Biological Process)中的细胞过程和生物调控、分子功能(Molecular Function)中的结合和催化活性。同时,KEGG富集分析表明,在Model_vs_Control_up和PSP_vs_Model_down的前20条显著富集通路中,NF-κB信号通路、IL-17信号通路、TNF信号通路和Toll样受体信号通路等4条常见通路与败血症的发病机制密切相关。此外,TLR2、IKKi、JunB和CXCL9等几种常见DEPs的水平也得到了WB验证,这与蛋白质组学的结果一致。因此,PSP 对 SALI 的保护作用可能是通过阻断上述炎症通路实现的。意义:PSP 是何首乌的主要成分,具有多种药理作用。我们之前的研究发现,PSP 可预防 SALI,但未能阐明其作用机制。为了揭示PSP对SALI的保护作用所涉及的分子机制,本研究采用基于TMT的定量蛋白质组学分析方法,检测和分析对照组、模型组和PSP组肝脏组织中的DEPs。研究结果为探索药物的作用机制提供了理论参考,有助于PSP的综合利用。意义:PSP 是何首乌中最重要的成分,具有多种药理作用。我们之前的研究发现,PSP 可预防 SALI,但其作用机制尚不清楚。为揭示PSP对SALI的保护作用所涉及的分子机制,本研究采用基于TMT的定量蛋白质组学分析方法,检测和分析对照组、模型组和PSP组肝脏组织中的DEPs。研究结果为探索药物的作用机制提供了理论参考,有利于PSP的综合利用。
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引用次数: 0
4-plex quantitative glycoproteomics using glycan/protein-stable isotope labeling in cell culture 利用细胞培养中的聚糖/蛋白质稳定同位素标记技术进行四重定量糖蛋白组学研究。
IF 2.8 2区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-18 DOI: 10.1016/j.jprot.2024.105333
Peilin Jiang , Md Abdul Hakim , Arvin Saffarian Delkhosh , Parisa Ahmadi , Yunxiang Li , Yehia Mechref
<div><div>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.</div></div><div><h3>Significance</h3><div>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
糖蛋白丰度和聚糖结构的变化与多种疾病密切相关。糖蛋白组学的定量探索对于生物标记物的发现至关重要,但由于丰度低、复杂性高以及缺乏通用技术,对生物样本进行全面分析仍具有挑战性。我们利用 LC-ESI-MS 平台开发了一种多重糖蛋白组学方法,用于直接比较糖蛋白组学定量。在细胞培养过程中对糖肽进行同位素标记,实现了糖和肽的高标记效率(≥ 95 %)。将同一细胞系按 1:1:1:1 的比例混合后进行定量验证,并应用数学校正对比例进行解旋。该方法证明是可靠的,并被应用于三个乳腺癌细胞系(HTB22、MDA-MB-231、MDA-MB-231BR)和一个脑癌细胞系(CRL-1620)的糖蛋白组学比较研究,对三个重复的糖肽进行定量。对糖肽的表达进行了相对量化,并研究了细胞系之间的上调/下调情况。这种方法有助于深入了解糖基化微异质性,对乳腺癌脑转移研究至关重要。这种方法的优点包括消除了纳米电喷雾离子化产生的波动,缩短了分析时间,一次进样可完成多达 4 个复合物的分析。代谢标记引入了 MS1 级别的质量差异,确保提高灵敏度和分辨率,从而实现精确定量。意义:糖蛋白丰度的变化、糖基化水平的变化以及聚糖结构的变化与多种疾病密切相关。糖蛋白组学的定量探索已成为生物标记物发现的热门研究领域。然而,由于丰度低、固有的复杂性以及缺乏通用的定量技术,对生物样本中的糖蛋白组进行全面的定量分析仍然具有挑战性。在此,我们开发了一种使用 LC-ESI-MS 平台的多重糖蛋白组方法,以促进糖蛋白组定量的直接比较并提高通量。这种方法具有消除纳米电喷雾离子化(ESI)引起的定量波动和缩短分析时间等优点,一次进样可完成多达 4 个复合物的糖蛋白组分析。糖肽在细胞培养过程中进行了稳定的同位素标记,附着在单糖、氨基酸或两者上。我们实现了聚糖和肽的高标记效率(≥ 95 %)。我们以 1:1:1:1 的比例混合同一细胞系,对糖肽进行了定量验证。由于同位素重叠,采用数学校正法对 4 倍糖肽的比例进行了解旋。该方法证明了定量的可靠性,并成功地应用于三个乳腺癌细胞(HTB22、MDA-MB-231 和 MDA-MB-231BR)和一个脑癌细胞(CRL-1620)的糖蛋白组学比较研究,从三个重复品中鉴定出共计 264 个糖肽。对这四种细胞中糖肽的表达进行了相对量化,并对两种细胞系之间的上调/下调进行了研究。通过糖肽定量探索糖基化微异质性可能为进一步研究乳腺癌脑转移提供有价值的见解。结论我们所做工作的主要优势在于结合了 SILAC 和 IDAWG 这两种成熟的标记技术,从而实现了多重化,这两种技术在科学界都得到了有效的应用和广泛的引用。这种结合提高了我们方法的适用性和准确性,这一点从这两种技术的广泛引用和成功独立使用中可见一斑。我们相信,尽管该方法目前仅限于细胞系统,但这种多路复用方法将极大地推动该领域的发展。
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引用次数: 0
DSSBU: A novel mass spectrometry-cleavable analogue of the BS3 cross-linker DSSBU:BS3 交联剂的新型质谱可裂解类似物。
IF 2.8 2区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-18 DOI: 10.1016/j.jprot.2024.105330
Banerjee Swati , Sýs Jakub , Machara Aleš , Junková Petra , Hubálek Martin
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 N-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 PXD055284.

Significance

We have synthesized the novel protein cross-linker DSSBU, which combines sulfo-NHS ester chemistry with a mass spectrometry-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 N-hydroxysuccinimide ester cross-linkers.
蛋白质交联在结构蛋白质组学中发挥着不可替代的作用。最近,人们大力开发新型质谱(MS)可溶解试剂。目前,市场上只有不溶于水的 MS 可溶解交联剂。然而,要全面分析构成蛋白质的各种化学和结构基团,就必须针对亲水性程度不同的蛋白质位点。在此,我们介绍了内部为此开发的新型 MS 可裂解交联剂二磺酸二琥珀酰亚胺基二丁基脲(DSSBU)。DSSBU 含有一个 N-羟基磺基琥珀酰亚胺(sulfo-NHS)反应基团,因此可以作为广泛使用的交联剂二琥珀酰亚胺基二丁基脲(DSBU)的水溶性对应物。为了研究 DSSBU 的适用性,我们比较了四种类似交联剂:双[磺基琥珀酰亚胺基]辛二酸酯(BS3)、双琥珀酰亚胺基辛二酸酯(DSS)、DSBU 和 DSSBU 与牛血清白蛋白的功效。此外,我们还比较了 DSBU 和 DSSBU 与人类血红蛋白的功效。我们的研究结果表明,磺基-NHS 基团可确保 DSSBU 具有优异的水溶性,因此无需使用二甲亚砜等有机溶剂,同时还能保持 DSBU 等脲基 MS 可裂解交联剂的功效。此外,这种方法还能瞄准蛋白质的极性区域。收集到的数据可通过蛋白质组交换(ProteomeXchange)获取,标识符为 PXD055284。意义:我们合成了新型蛋白质交联剂 DSSBU,它结合了磺基-NHS 酯化学和质谱可裂解脲基。这使得 DSSBU 成为一种可与氨基反应的水溶性、质谱可裂解交联剂。据我们所知,这是第一种兼具上述三种特性的交联剂。我们在牛血清白蛋白(交联质谱界广泛使用的模型)和人类血红蛋白上测试了新型交联剂的性能。我们全面评估了 DSSBU 的性能,并将其与目前使用的其他三种交联剂(BS3、DSS 和 DSBU)的功效进行了比较。我们的结论是,我们的新型交联剂在性能上超过了其 MS-非可溶解类似物 BS3,其水溶性使其无需使用有机溶剂,而其亲水性则使其可以靶向蛋白质中的极性区域。因此,它很可能成为 N-羟基琥珀酰亚胺酯交联剂产品组合的重要补充。
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引用次数: 0
Proteo-transcriptomic profiles reveal genetic mechanisms underlying primary hair follicle development in coarse sheep fetal skin 蛋白质转录组图谱揭示粗毛绵羊胎儿皮肤初级毛囊发育的遗传机制
IF 2.8 2区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-11 DOI: 10.1016/j.jprot.2024.105327
Dehong Tian , Wenkui Zhang , Lei Wang , Junying Qi , Teng Xu , Mingxing Zuo , Buying Han , Xue Li , Kai Zhao
Long hair trait represents a valuable genetic asset in Qinghai Tibetan sheep, with its quality and yield being contingent upon the characteristics of hair follicles (HFs). This study aims to elucidate the genetic mechanism underlying primary hair follicles (PFs) formation through an integrated analysis of proteomics and transcriptomics. Samples were collected at key stages of fetal HF formation (E65 and E85) for histological observation, revealing significant alterations in the microstructure of PF (E65) during the developmental process. In this study, a comprehensive analysis revealed a total of 217 overlapping genes that exhibited concordant expression patterns at both the proteomic and transcriptomic levels. Furthermore, to ensure the reliability of our findings, we employed parallel response monitoring (PRM) to validate the obtained proteomic data. The protein-protein interaction (PPI) network diagram highlights five hub core proteins (TTN, IGTA2, F2, EGFR, and MYH14). These differentially expressed proteins (DEPs) play crucial roles in metabolic processes, cell adhesion, and diverse biological processes. The potential synergy between transcriptional regulation and post-translational modifications plays a pivotal role in governing the initiation PF development. The findings presented in this study offer innovative insights into the molecular mechanisms underlying HFs generation and establish a robust foundation for targeted breeding strategies aimed at augmenting wool traits in sheep.

Significance

The composition of coarse hair primarily consists of long, myelinated fibers originating from primary hair follicles. Sheep fetal skin initiates the formation of primary hair follicles around E65, followed by the development of secondary hair follicles around E85. Conducting differential proteomic and transcriptomic analyses during these developmental stages enhances our understanding of the molecular mechanisms underlying primary hair follicle development and offers valuable insights for sustainable utilization of high-quality germplasm resources.
长毛性状是青海藏羊的宝贵遗传资产,其质量和产量取决于毛囊(HFs)的特性。本研究旨在通过蛋白质组学和转录组学的综合分析,阐明初级毛囊(PFs)形成的遗传机制。研究人员在胎儿毛囊形成的关键阶段(E65 和 E85)采集样本进行组织学观察,结果发现在发育过程中,胎儿毛囊(E65)的微观结构发生了显著变化。在这项研究中,通过综合分析发现共有 217 个重叠基因在蛋白质组和转录组水平上表现出一致的表达模式。此外,为了确保研究结果的可靠性,我们采用了平行响应监测(PRM)来验证所获得的蛋白质组数据。蛋白-蛋白相互作用(PPI)网络图突出显示了五个中心核心蛋白(TTN、IGTA2、F2、表皮生长因子受体和 MYH14)。这些差异表达蛋白(DEPs)在代谢过程、细胞粘附和多种生物过程中发挥着关键作用。转录调控和翻译后修饰之间的潜在协同作用在调控 PF 发育的启动过程中发挥着关键作用。本研究的发现为高频毛发的分子机制提供了创新性的见解,并为旨在增强绵羊羊毛性状的定向育种策略奠定了坚实的基础。绵羊胎儿皮肤在 E65 左右开始形成初级毛囊,随后在 E85 左右形成次级毛囊。在这些发育阶段进行不同的蛋白质组和转录组分析,可加深我们对初级毛囊发育的分子机制的了解,并为可持续利用优质种质资源提供宝贵的见解。
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引用次数: 0
Protein degradation patterns as biomarkers for post-mortem interval estimation: A comprehensive review of proteomic approaches in forensic science 蛋白质降解模式作为死后间隔估计的生物标志物:法医学蛋白质组学方法综述。
IF 2.8 2区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-09 DOI: 10.1016/j.jprot.2024.105326
Anjali Chhikara, Pallavi Kumari, Jyoti Dalal, Kiran Kumari
The determination of post-mortem interval (PMI) is a critical process for forensic medical-legal investigations. Proteomic techniques are gaining prominence in analysing forensic biological samples. After death, studying the proteins present in human bodies could be critical in discovering important new biomarkers that can serve as reliable indicators of various factors. A literature review is conducted on estimating PMI through protein degradation analysis using PubMed, NCBI, SCOPUS, Research Gate, Science Direct, and Google Scholar. A total of 32 studies were identified and studied. It is found that the most commonly studied tissue type is the skeleton muscle (15 studies), followed by others. The kinetics of several proteins and proteases were particularly correlated with PMI. Different proteins degrade differently after death: alpha-actinin, GAPDH, and alpha-tubulin breakdown slowly, but meta-vinculin breaks down early. Tropomyosin does not change for a long time after death, up to 10 days. Certain markers had a positive correlation with PMI, meaning that their amount increased as PMI hours increased, while other markers showed a negative correlation, suggesting that their number decreased with time. The level of several biological markers, such as SERBP1, COX7B, and SOD2, changed gradually and consistently as the PMI increased. The information gathered from this analysis provides new opportunities for precise PMI measurements in legal contexts by expanding the research area's use in human skeletal tissue.
确定死后间隔(PMI)是法医法律调查的一个关键过程。蛋白质组技术在分析法医生物样本方面的作用日益突出。人死后,研究人体内的蛋白质对于发现重要的新生物标记物至关重要,这些标记物可作为各种因素的可靠指标。本研究利用 PubMed、NCBI、SCOPUS、Research Gate、Science Direct 和 Google Scholar 对通过蛋白质降解分析估计 PMI 进行了文献综述。共发现并研究了 32 项研究。研究发现,最常研究的组织类型是骨骼肌(15 项研究),其次是其他组织。几种蛋白质和蛋白酶的动力学与 PMI 尤为相关。不同的蛋白质在死亡后的降解方式不同:α-肌动蛋白、GAPDH 和α-微管蛋白的分解速度较慢,而元肌球蛋白的分解速度较快。肌球蛋白在死亡后很长一段时间(长达 10 天)都不会发生变化。某些标记物与 PMI 呈正相关,即随着 PMI 小时数的增加,其数量也会增加,而其他标记物则呈负相关,表明其数量会随着时间的推移而减少。随着 PMI 的增加,SERBP1、COX7B 和 SOD2 等几种生物标记物的水平逐渐发生持续变化。本分析收集的信息为在法律背景下精确测量 PMI 提供了新的机会,扩大了该研究领域在人体骨骼组织中的应用。
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引用次数: 0
Proteomic integrity of tape-stripped stratum corneum samples endures higher temperatures 用胶带剥离的角质层样本在较高温度下仍能保持蛋白质组的完整性。
IF 2.8 2区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-05 DOI: 10.1016/j.jprot.2024.105329
Ali Azimi , Lauren Faul , Petrina Chand , Pablo Fernandez-Penas
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引用次数: 0
Dehydration priming remodels protein abundance and phosphorylation level regulating tolerance to subsequent dehydration or salt stress in creeping bentgrass 脱水启动可重塑蛋白质丰度和磷酸化水平,从而调节匍匐翦股颖对后续脱水或盐胁迫的耐受性。
IF 2.8 2区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-05 DOI: 10.1016/j.jprot.2024.105325
Huizhen Yang , Yan Yuan , Zhou Li
Dehydration priming (DP) induces stress memory which plays a positive role in plant adaptability, but it is not well understood how DP differentially regulates subsequent dehydration (cis priming) or salt (trans priming) tolerance at the post-translational level. Purpose of this study was to identify proteins, phosphorylation levels and sites, and relevant metabolic pathways for DP-induced dehydration or salt tolerance in Agrostis stolonifera. DP-induced differentially regulated proteins (DRPs) were mostly located in the cytoplasm, chloroplast, and cell membrane, and differentially regulated phosphoproteins (DRPPs) were mostly nuclear proteins and cytoplasmic proteins. DP regulated common phosphorylation sites ([SP] and [RxxS]) under dehydration and salt conditions and also individually affected 8 or 11 phosphorylation sites under dehydration or salt stress. DP-regulated DRPPs were mainly rich in glycolysis and glutathione metabolism pathways, RNA splicing, and dynamin family proteins under dehydration stress, whereas DP-regulated salt tolerance was mainly related to chlorophyll metabolism, photosynthesis, MAPK signaling cascade, and ABC transporter I family at the phosphorylation level. In addition, the DP also significantly up-regulated phosphorylation of histones (ATXR3 and SETD1A) in response to subsequent dehydration and salt stress as well as abundances of antioxidant enzymes, dynamin family protein, and KCS6 under dehydration stress or abundances of PETE, HMGA, XTH, and ABCI6 under salt stress, respectively. Transcriptomics analysis further indicated that DP-regulated dehydration or salt tolerance was also related to transcriptional regulation in the early stage. Current results provided better understanding of the role of stress memory in plant adaptability to repeated or crossed stress via post-translational modifications (PTMs).

Significance

Recurrent moderate drought may buffer drought legacies in many plant species. When plants were exposed to repeated drought stress, their adaptability to subsequent stress could be enhanced, which is known as “stress memory”. Dehydration priming has been found to be an important approach to induce stress memory. Current results provided better understanding of the role of stress memory in plant adaptability to repeated or crossed stress via post-translational modifications.
脱水诱导(DP)可诱导胁迫记忆,对植物的适应性起积极作用,但目前还不太清楚脱水诱导如何在翻译后水平上对随后的脱水(顺式诱导)或耐盐(反式诱导)进行差异调控。本研究的目的是鉴定 Agrostis stolonifera 中 DP 诱导的脱水或耐盐性的蛋白质、磷酸化水平和位点以及相关代谢途径。DP诱导的差异调控蛋白(DEPs)主要位于细胞质、叶绿体和细胞膜,差异调控磷酸化蛋白(DRPPs)主要是核蛋白和细胞质蛋白。在脱水和盐胁迫条件下,DP调控共同的磷酸化位点([SP]和[RxxS]),在脱水或盐胁迫条件下,DP还单独影响8或11个磷酸化位点。在脱水胁迫下,DP调控的DRPPs主要富集于糖酵解和谷胱甘肽代谢途径、RNA剪接和dynamin家族蛋白;而在磷酸化水平上,DP调控的耐盐性主要与叶绿素代谢、光合作用、MAPK信号级联和ABC转运体I家族有关。此外,DP还能显著上调组蛋白(ATXR3和SETD1A)的磷酸化水平,以应对随后的脱水和盐胁迫;在脱水胁迫下,还能显著上调抗氧化酶、dynamin家族蛋白和KCS6的丰度;在盐胁迫下,还能显著上调PETE、HMGA、XTH和ABCI6的丰度。转录组学分析还进一步表明,DP调控的脱水或耐盐性在早期阶段也与转录调控有关。目前的研究结果使人们更好地理解了胁迫记忆通过翻译后修饰(PTMs)在植物适应重复或交叉胁迫中的作用。意义:反复出现的中度干旱可能会缓冲许多植物物种的干旱遗产。当植物受到反复干旱胁迫时,它们对后续胁迫的适应性会增强,这就是所谓的 "胁迫记忆"。脱水引物被认为是诱导胁迫记忆的一种重要方法。目前的研究结果让人们更好地了解了胁迫记忆通过翻译后修饰在植物适应重复或交叉胁迫中的作用。
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引用次数: 0
Integrated transcriptomic and proteomic analysis revealed the regulatory role of 5-azacytidine in kenaf salt stress alleviation 转录组和蛋白质组的综合分析揭示了 5-azacytidine 在减轻剑麻盐胁迫中的调控作用。
IF 2.8 2区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-04 DOI: 10.1016/j.jprot.2024.105328
Dengjie Luo , Zengqiang Li , Samavia Mubeen , Muzammal Rehman , Shan Cao , Caijin Wang , Jiao Yue , Jiao Pan , Gang Jin , Ru Li , Tao Chen , Peng Chen
Salinity stress limits agricultural production. The DNA methyltransferase inhibitor, 5-azacitidine (5-azaC), plays a role in plant abiotic stress regulation, but its molecular basis in mediating salinity tolerance in kenaf remains unclear. To investigate the effects on 5-azaC on alleviating salt stress, kenaf seedlings were pre-treated with 0, 50, 100, 150, and 200 μM 5-azaC and then exposed to 150 mM NaCl in a nutrient solution. Physiological, transcriptomic, and proteomic analyses were conducted on the root system to understand the regulatory mechanism of 5-azaC (comparing 5-azaC150 and control group 5-azaC0) under salt stress. The results indicated that 5-azaC significantly mitigated salt stress in kenaf by activating the antioxidant system, reducing reactive oxygen species (ROS), and increasing starch, soluble sugars, and adenosine triphosphate (ATP) content. A total of 14,348 differentially expressed genes (DEGs) and 313 differentially abundant proteins (DAPs) were identified. Combined proteomic and transcriptomic analysis revealed 27 DEGs/DAPs, with jointly up-regulated proteins (genes) including HcTHI1, HcBGLU11, and HcCBL1, and jointly down-regulated proteins (genes) including HcGAPDH, HcSS, and HcPP2C52. Overexpression and virus-induced gene silencing (VIGS) of HcPP2C52 demonstrated its role as a negative regulator of salt tolerance. These findings provide insights into the regulatory role of 5-azaC in plant responses to abiotic stresses.

Significance

The specific molecular mechanism by which 5-azaC affects gene expression and protein activity of kenaf has been revealed, leading to enhanced salt tolerance.
盐分胁迫限制了农业生产。DNA 甲基转移酶抑制剂--5-氮杂胞苷(5-azaC)在植物非生物胁迫调控中发挥作用,但其介导剑麻耐盐性的分子基础仍不清楚。为了研究 5-azaC 对缓解盐胁迫的影响,我们用 0、50、100、150 和 200 μM 5-azaC 预处理剑麻幼苗,然后将其置于 150 mM NaCl 营养液中。对根系进行生理、转录组和蛋白质组分析,以了解盐胁迫下 5-azaC 的调控机制(5-azaC150 与对照组 5-azaC0比较)。结果表明,5-azaC 能激活抗氧化系统,减少活性氧(ROS),增加淀粉、可溶性糖和三磷酸腺苷(ATP)的含量,从而显著缓解剑麻的盐胁迫。共鉴定出 14 348 个差异表达基因(DEG)和 313 个差异丰富蛋白(DAP)。结合蛋白质组和转录组分析发现了 27 个 DEGs/DAPs,共同上调的蛋白质(基因)包括 HcTHI1、HcBGLU11 和 HcCBL1,共同下调的蛋白质(基因)包括 HcGAPDH、HcSS 和 HcPP2C52。HcPP2C52的过表达和病毒诱导基因沉默(VIGS)证明了其作为耐盐性负调控因子的作用。这些发现深入揭示了 5-azaC 在植物对非生物胁迫反应中的调控作用。意义:揭示了 5-azaC 影响剑麻基因表达和蛋白质活性的具体分子机制,从而增强了剑麻的耐盐性。
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引用次数: 0
期刊
Journal of proteomics
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