High-Throughput, Quantitative Analysis of Peptide-Exchanged MHCI Complexes by Native Mass Spectrometry.
High-Throughput, Quantitative Analysis of Peptide-Exchanged MHCI Complexes by Native Mass Spectrometry.
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DOI：
10.1021/acs.analchem.2c02423
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发表时间：
2022-10-25
影响因子：
7.4
通讯作者：
Sandoval, Wendy
中科院分区：
文献类型：
--
作者：
Schachner, Luis F.;Phung, Wilson;Han, Guanghui;Darwish, Martine;Bell, Ashley;Mellors, J. Scott;Srzentic, Kristina;Huguet, Romain;Blanchette, Craig;Sandoval, Wendy
癌症免疫治疗中的免疫监测涉及筛选 CD8+ T 细胞针对新抗原的反应，新抗原是细胞表面主要组织相容性复合物 I 类 (MHCI) 呈递的肿瘤特异性肽。高通量免疫监测需要生产和表征少量数千个 MHCI-肽复合物的方法，这些复合物可以用于测试患者的 T 细胞反应。 MHCI 的合成是通过新抗原交换的光裂解肽实现的；然而，测量肽交换的测定目前在分析之前分解复合物，排除了直接的分子表征。在这里，我们使用天然质谱 (MS) 来分析完整的重组 MHCI 复合物并直接测量肽交换。与尺寸排阻色谱法或毛细管区带电泳相结合，该测定法以最短的运行时间鉴定了纳摩尔至皮摩尔范围内的所有测试的人类白细胞抗原 (HLA)/肽组合，使 MHCI 肽筛选的合成和分析要求与下游 T 细胞测定相一致。我们进一步表明，该测定可以通过同时测量多个肽的交换来“多重化”，并且还可以计算 Vc50（气相稳定性的量度）。此外，通过自上而下的测序对 MHCI 复合物进行片段化，证明可以在单次分析中确定完整的复合物、肽序列及其结合亲和力。这种 MHCI-新抗原复合物的筛选工具代表了在转化环境中应用最先进的 MS 技术的一步。该检测不仅已经为免疫疗法在实践中的可行性提供了信息，该平台还有望激发新的 MS 读数，用于疾病诊断和治疗中日益复杂的生物分子。
Immune monitoring in cancer immunotherapy involves screening CD8+ T-cell responses against neoantigens, the tumor-specific peptides presented by Major histocompatibility complex Class I (MHCI) on the cell surface. High-throughput immune monitoring requires methods to produce and characterize small quantities of thousands of MHCI–peptide complexes that may be tested for a patient’s T-cell response. MHCI synthesis has been achieved using a photocleavable peptide that is exchanged by the neoantigen; however, assays that measure peptide exchange currently disassemble the complex prior to analysis—precluding direct molecular characterization. Here, we use native mass spectrometry (MS) to profile intact recombinant MHCI complexes and directly measure peptide exchange. Coupled with size-exclusion chromatography or capillary-zone electrophoresis, the assay identified all tested human leukocyte antigen (HLA)/peptide combinations in the nanomole to picomole range with minimal run time, reconciling the synthetic and analytical requirements of MHCI–peptide screening with the downstream T-cell assays. We further show that the assay can be “multiplexed” by measuring exchange of multiple peptides simultaneously and also enables calculation of Vc50, a measure of gas-phase stability. Additionally, MHCI complexes were fragmented by top-down sequencing, demonstrating that the intact complex, peptide sequence, and their binding affinity can be determined in a single analysis. This screening tool for MHCI–neoantigen complexes represents a step toward the application of state-of-the-art MS technology in translational settings. Not only is this assay already informing on the viability of immunotherapy in practice, the platform also holds promise to inspire novel MS readouts for increasingly complex biomolecules used in the diagnosis and treatment of disease.
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影响因子：
4.4
作者：
Hoek M;Demmers LC;Wu W;Heck AJR
通讯作者：
Heck AJR
一种集成的天然质谱和自上而下的蛋白质组学方法，该方法将序列连接到大分子复合物的结构和功能。
影响因子：
21.8
作者：
Li H;Nguyen HH;Ogorzalek Loo RR;Campuzano IDG;Loo JA
通讯作者：
Loo JA
DOI：
10.4049/jimmunol.1700893
发表时间：
2017-11-01
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
--
作者：
Jurtz V;Paul S;Andreatta M;Marcatili P;Peters B;Nielsen M
通讯作者：
Nielsen M
影响因子：
14.9
作者：
Robinson, James;Barker, Dominic J.;Marsh, Steven G. E.
通讯作者：
Marsh, Steven G. E.
影响因子：
3.4
作者：
Schumacher, Frances-Rose;Delamarre, Lelia;Lill, Jennie R.
通讯作者：
Lill, Jennie R.