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Proteomics Impact on Cell Biology to Resolve Cell Structure and Function. 蛋白质组学对细胞生物学的影响,以解析细胞结构和功能。
IF 7 2区 生物学 Q1 Chemistry Pub Date : 2024-05-01 Epub Date: 2024-04-02 DOI: 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.

蛋白质组学的加速发展使其能够与电磁显微镜和光学显微镜成像、蛋白质结构的冷冻电镜以及人工智能相结合,以纳米到亚纳米分辨率全面准确地解析细胞结构中的蛋白质。蛋白质组学的发展速度继续超过以实验为基础的结构成像,但它们的最终整合是实现所有蛋白质汇编目标的必经之路,从而从机理上了解细胞的结构和功能。
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引用次数: 0
Corrigendum to "Red Blood Cells Protein Profile Is Modified in Breast Cancer Patients". 乳腺癌患者的红细胞蛋白谱发生改变 "的更正。
IF 7 2区 生物学 Q1 Chemistry Pub Date : 2024-05-01 Epub Date: 2024-05-11 DOI: 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
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引用次数: 0
Galectin-4 Antimicrobial Activity Primarily Occurs Through its C-Terminal Domain. Galectin-4 的抗菌活性主要通过其 C 端结构域产生。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-01 Epub Date: 2024-03-13 DOI: 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。
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引用次数: 0
The One Hour Human Proteome. 一小时人类蛋白质组
IF 7 2区 生物学 Q1 Chemistry Pub Date : 2024-05-01 Epub Date: 2024-04-03 DOI: 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.

我们描述了在不到一小时内对人类蛋白质组的深入分析。我们利用最先进的样品制备、色谱分离和数据分析工具,并通过使用配备四极杆质量滤波器、高场 Orbitrap 质量分析器和非对称轨道无损(Astral)质量分析器的新型 Orbitrap Astral 质谱仪,实现了蛋白质组的快速表征。该系统的 MS/MS 采集速度高达 200 Hz,在数据独立或数据依赖采集操作模式下,每秒可检测数百个肽序列。新型四极杆的快速切换功能与 Astral 分析仪的灵敏度和快速离子扫描功能相得益彰,从而实现了窄仓数据独立分析 (DIA) 方法。在总分析时间为 56 分钟的 30 分钟活性色谱法中,Q-Orbitrap-Astral 混合质谱每次运行平均收集 4,319 次 MS1 扫描和 438,062 次 MS/MS 扫描,产生 235,916 个肽序列(误发现率为 1% (FDR))。平均而言,每次 30 分钟的分析可检测到 10,411 个蛋白质组(1% FDR)。我们根据这些结果和最近的其他报告得出结论,一小时人类蛋白质组的实现指日可待。
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引用次数: 0
Proteomics-Derived Biomarker Panel Facilitates Distinguishing Primary Lung Adenocarcinomas With Intestinal or Mucinous Differentiation From Lung Metastatic Colorectal Cancer. 蛋白质组学衍生生物标记物面板有助于区分肠道或粘液性分化的原发性肺腺癌(PAIM)和肺转移性结直肠癌(lmCRC)。
IF 7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-01 Epub Date: 2024-04-10 DOI: 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.

具有肠或粘液分化的原发性肺腺癌(PAIM)与肺转移性结直肠癌(lmCRC)在组织形态学、免疫组化和遗传学特征上存在重叠,因此诊断PAIM仍具有挑战性。本研究旨在探索能区分 PAIM 和 lmCRC 的蛋白质生物标志物。为了揭示这两种疾病之间的差异,我们使用了基于串联质量标记(TMT)的枪式蛋白质组学来表征福尔马林固定石蜡包埋(FFPE)肿瘤样本的蛋白质组,其中PAIM样本22个,lmCRC样本17个,然后利用三种机器学习算法,即支持向量机(SVM)、随机森林和最小绝对收缩和选择操作器(LASSO),来选择具有诊断意义的蛋白质特征。这些候选蛋白质在一个独立队列(PAIM,n = 11;lmCRC,n = 19)中通过免疫化学(IHC)进行了进一步验证,以确认其诊断性能。在7871个蛋白质中,共有105个蛋白质在PAIM和lmCRC样本之间出现了明显的调控失调,并通过统一表层逼近和投影(UMAP)将两组样本很好地区分开来。PAIM中上调的蛋白质参与肌动蛋白细胞骨架组织、血小板脱颗粒和白细胞趋化调节,而下调的蛋白质参与线粒体跨膜转运、血管发育和干细胞增殖。通过机器学习算法,我们最终选出了一组 10 个候选蛋白(在 lmCRC 中高水平表达:CDH17、ATP1B3、GLB1、OXNAD1、LYST、FABP1;在 PAIM 中高水平表达:CK7(已确立的标记物)、NARR、MLPH、S100A14)来区分 PAIM 和 lmCRC。我们通过 IHC 进一步证实,CDH17、CK7、MLPH、FABP1 和 NARR 这五种蛋白质生物标记物是区分 PAIM 和 lmCRC 的有效生物标记物。我们的研究描述了PAIM特异性的蛋白质组特征,并证明了新蛋白质生物标志物在鉴别诊断PAIM和lmCRC方面的潜在作用。这些发现可能有助于提高诊断的准确性,并为这些患者的适当治疗提供指导。
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引用次数: 0
Systematic Investigation of the Trafficking of Glycoproteins on the Cell Surface. 细胞表面糖蛋白迁移的系统研究
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-01 Epub Date: 2024-04-07 DOI: 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.

位于细胞表面的糖蛋白在几乎所有细胞外活动中都起着举足轻重的作用。N-糖基化是真核细胞中最常见、最重要的蛋白质修饰之一,它经常调节蛋白质的折叠和运输。细胞表面蛋白质的糖基化要经过内质网(ER)和高尔基体中各种酶的精心调控,以确保其正确折叠并转运到细胞表面。然而,蛋白质 N-糖基化、N-糖成熟度和蛋白质折叠状态对细胞表面糖蛋白转运的影响仍有待探索。在这项研究中,我们对人体细胞中细胞表面糖蛋白的迁移进行了全面的、特定位点的研究。我们整合了代谢标记、生物正交化学和多重蛋白质组学,研究了单核细胞中 396 种细胞表面糖蛋白上的 706 个 N-糖基化位点,通过抑制蛋白质的 N-糖基化、干扰 N-糖的成熟或扰乱蛋白质在 ER 中的折叠。目前的研究结果揭示了它们对表面糖蛋白迁移的不同影响。抑制蛋白质N-糖基化会显著抑制许多细胞表面糖蛋白的运输。N-糖基不成熟对N-糖基化位点密度高的蛋白质影响更大,而对蛋白质在ER中折叠的干扰对体积较大的表面糖蛋白影响更明显。此外,N-糖基化蛋白质向细胞表面的迁移与糖基化位点的二级结构和相邻氨基酸残基有关。对表面糖蛋白迁移的系统分析有助于我们了解蛋白质分泌和表面呈现的机制。
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引用次数: 0
High-Throughput Antigen Microarray Identifies Longitudinal Prognostic Autoantibody for Chemoimmunotherapy in Advanced Non-Small Cell Lung Cancer. 高通量抗原芯片确定晚期非小细胞肺癌化疗免疫疗法的纵向预后自身抗体。
IF 7 2区 生物学 Q1 Chemistry Pub Date : 2024-05-01 Epub Date: 2024-03-20 DOI: 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.

化学免疫疗法已发展成为晚期非小细胞肺癌(aNSCLC)的标准疗法。然而,不可避免的耐药性限制了它的疗效,这凸显了对化学免疫疗法生物标志物的迫切需求。我们采用了一种三阶段策略,在化疗免疫疗法前后发现、验证和确认aNSCLC的纵向预测性自身抗体(AAbs)。在抗PD1免疫疗法前后,共收集了267名aNSCLC患者的528份血浆样本,以及30份独立的福尔马林固定石蜡包埋样本。在发现阶段,首先使用包含 21,000 个蛋白质的 HuProtTM 高密度芯片筛选出候选 AAbs,然后使用以 aNSCLC 为重点的芯片进行验证。根据应答者与非应答者的比较和无进展生存期(PFS)生存分析,选择了纵向预测性 AAbs 进行酶联免疫吸附试验(ELISA)。我们还利用免疫组化技术和公开的免疫疗法数据集对预后标志物进行了验证。在接受化疗免疫疗法的非小细胞肺癌患者中,我们发现并验证了由两种 AAbs(MAX 和 DHX29)组成的治疗前生物标记物,以及由两种 AAbs(MAX 和 TAPBP)组成的治疗中预测标记物。所有三种 AAb 都与早期反应和 PFS 呈正相关(p < 0.05)。MAX AAb的动力学在应答者中呈上升趋势(p < 0.05),而在非应答者中呈先升后降的趋势(p < 0.05)。重要的是,MAX蛋白和mRNA水平能有效区分接受免疫疗法治疗的非小细胞肺癌患者的PFS(p < 0.05)。我们的研究结果对接受化疗免疫治疗的 aNSCLC 患者预后 AAbs 的变化进行了纵向分析。
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引用次数: 0
Fast and Accurate Disulfide Bridge Detection. 快速准确的二硫桥检测。
IF 7 2区 生物学 Q1 Chemistry Pub Date : 2024-05-01 Epub Date: 2024-04-02 DOI: 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.

在治疗性抗体的推动下,蛋白质的重组表达已发展成为一个价值数十亿美元的产业。其中至关重要的是对关键属性的质量控制评估,如序列保真度、适当折叠和翻译后修饰。错误会导致生物活性降低,对于治疗蛋白而言,还会增加免疫原性风险。多年来,人们开发并应用了许多技术,以标准化和高通量的方式验证蛋白质。然而,有一项参数的评估至今仍具有挑战性。二硫桥是连接两个半胱氨酸残基的共价键,有助于蛋白质的正确折叠和稳定性,因此对蛋白质的功效有重大影响。质谱技术有望成为快速、准确地揭示硫桥的最佳技术。在这项工作中,我们提出了一种独特的样品制备、数据采集和分析组合方法,有助于快速准确地评估纯化蛋白质中的二硫桥。通过微波辅助酸水解,蛋白质被快速、无人工痕迹地消化成肽段,并在序列上有很大程度的重叠。不过,这一过程的非特异性会带来化学背景,而在质谱测量之前整合离子迁移率可有效去除化学背景。消化步骤的非特异性还要求在数据分析方面有新的发展,为此我们扩展了蛋白质组发现者中的 XlinkX 节点,以高效处理数据,并通过有效的错误发现率校正确保数据的正确性。整个工作流程可在 1 小时内完成,从而实现高通量、高精度的二硫化物图谱分析。
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引用次数: 0
The Landscape and Perspectives of the Human Gut Metaproteomics. 人类肠道元蛋白组学的前景与展望。
IF 7 2区 生物学 Q1 Chemistry Pub Date : 2024-05-01 Epub Date: 2024-04-10 DOI: 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.

人类肠道微生物群与人类健康和疾病密切相关。通过分析微生物群落中存在的全部蛋白质,元蛋白质组学已成为研究肠道微生物组功能的重要工具。液相色谱和串联质谱(LC-MS/MS)技术的最新进展扩大了元蛋白质组学的检测范围。然而,元蛋白质组学对蛋白质组的总体覆盖范围仍然有限。虽然元基因组学研究揭示了人类肠道微生物组的大量微生物多样性和功能潜力,但很少有研究对元蛋白组学揭示的人类肠道微生物组景观进行总结和研究。在本文中,我们通过重新分析 15 项已发表研究的鉴定结果,介绍了目前人类肠道元蛋白组学研究的现状。我们量化了元蛋白质组学有限的蛋白质组覆盖率,并揭示了元蛋白质组学鉴定蛋白质的高比例注释覆盖率。我们对人类肠道微生物组的元蛋白组学观点和元基因组学观点进行了初步比较,确定了一致和分歧的关键领域。基于人类肠道元蛋白组学的现状,我们讨论了使用元蛋白组学研究功能未知蛋白质的可行性,并提出了以肽为中心的整个工作流程分析。此外,我们还建议通过完善分类学分类和计算置信度分数来加强元蛋白质组学分析,并开发用于分析分类学与功能之间相互作用的工具。
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引用次数: 0
Systematic Optimization of Automated Phosphopeptide Enrichment for High-Sensitivity Phosphoproteomics. 系统优化高灵敏度磷酸蛋白组学的自动磷酸肽富集。
IF 7 2区 生物学 Q1 Chemistry Pub Date : 2024-05-01 Epub Date: 2024-03-27 DOI: 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).

通过单次液相色谱串联质谱(LC-MS/MS)从最少的肽输入进行常规磷酸化蛋白质组学分析,提高覆盖率、稳健性和灵敏度至关重要。在此,我们系统地优化了珠上磷蛋白组学自动样品制备的关键实验参数,重点关注低输入样品。通过评估鉴定出的磷酸肽数量、富集效率、位点定位得分以及多重磷酸化肽的相对富集程度,我们找出了影响磷酸化蛋白质组的关键变量。通过优化上样缓冲液中乙醇酸的浓度、洗脱缓冲液中氢氧化铵的比例、肽与珠子的比例、结合时间、样品和上样缓冲液的体积,我们在 Orbitrap Exploris 480 上以 30 μg 肽为起始材料,在半小时的 LC-MS/MS 中鉴定出了 16,000 多条磷酸肽。此外,我们还评估了连续富集如何提高磷酸蛋白组的覆盖率,结果表明将馏分汇集到单个 LC-MS/MS 分析中可提高分析深度。我们还提出了另一种基于逐步添加珠子的磷酸肽富集策略,从而将磷酸蛋白组的覆盖率提高了 20%。最后,我们将优化后的策略应用于 Orbitrap Astral MS,使用细胞稀释系列来评估磷酸化蛋白质组的深度,并使用窄窗数据无关采集(nDIA)在半小时的 LC-MS/MS 中从 0.5 百万 HeLa 细胞中鉴定出了 >32,000 个磷酸肽。
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Molecular & Cellular Proteomics
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