Technology Advancements for Proteome Analysis Incorporating Surfactants and Mass Spectrometry

人类基因组计划带来了研究人类健康的革命性方法。该项目的一个惊人发现是，人类基因组中基因的数量很低（约20,500个），只是预期数量的一小部分。这证实了我们的生物多样性是通过基因产物（即蛋白质）的变异来捕获的。蛋白质组是一个动态系统；离散的蛋白质改变浓度或功能活性与特定的生理状态相关。这些变化可能标志着疾病的进展（生物标志物）；绘制蛋白质组可以定制新的治疗方法（个性化医疗）。我们推测可能存在超过100万种独特的人类蛋白质。这种多样性部分是通过化学修饰（如甲基化、磷酸化）产生的，这些修饰表现出独特的蛋白质形式。蛋白质形态表征（我们现在称这些化学上不同的蛋白质）是自上而下的蛋白质组学这一新兴领域背后的驱动因素。该方法结合了分离、高分辨率串联质谱（MS/MS）和计算工具来分析完整的蛋白质。蛋白质形态表征仍然充满挑战：蛋白质容易降解、聚集或意外的化学修饰；完整蛋白质的分离策略缺乏所需的分辨率，可能会增加样品损失；质谱检测容易受到干扰。迫切需要改进的分析技术来建立蛋白质分析作为诊断、监测和潜在治疗疾病的可行策略。我们的团队开创了多种使用ms进行蛋白质组分析的方法，其中值得注意的是完整蛋白质分离的平台，纯化蛋白质的方法，以及如何保持蛋白质组稳定性的改进见解。这样的发展推动了基于ms的蛋白质组学，特别是自上而下的模式。我们还有很长的路要走。当前建议的一个关键方面是使用十二烷基硫酸钠（SDS），一种有利于溶解、稳定和分离蛋白质组的表面活性剂。不幸的是，SDS也对MS有害，因此许多研究人员避免使用这种洗涤剂。我们提出了一个基于电泳的自动化平台，可以在ms之前从SDS中分离蛋白质。我们还评估了丙酮沉淀作为蛋白质组纯化和保存的一种有前途的方法，旨在提高我们对这一过程的基本理解（动力学研究，聚集机制）。提出了电泳和固相蛋白质捕获相结合的分离蛋白质形态的方法。这些研究旨在提供强大的工具来描述生物系统。最终，这些创新在基因检测的同时建立了蛋白质组学，从而改善了加拿大人的健康状况。

The Human Genome Project ushered transformative approaches to study human health. A startling reveal of this project was the low number of genes (~20,500) in our genome - being a fraction of what was anticipated. This confirmed that our biological diversity is captured through variations in gene products, i.e. proteins. A proteome is a dynamic system; discrete proteins change concentration or functional activity to correlate with a particular physiological state. Such changes may mark the progression of a disease (biomarkers); mapping the proteome may tailor new therapeutic approaches (personalized medicine). We speculate that over a million unique human proteins may exist. This diversity is created in part through chemical modifications (e.g. methylation, phosphorylation) which manifests unique protein forms. Proteoform' characterization (as we now call these chemically distinct proteins) is a driving feature behind the emerging field of top-down proteomics. This approach employs a combination of separation, high resolution tandem mass spectrometry (MS/MS) and computational tools to profile intact proteins. Proteoform characterization is still fraught with challenges: proteins are prone to degradation, aggregation or unintended chemical modification; separation strategies for intact proteins lack the needed resolution and can exaggerate sample loss; MS detection is subject to interference. Improved analytical technologies are urgently needed to establish proteoform analysis as a viable strategy to diagnose, monitor, and potentially cure diseases. Our group has pioneered multiple approaches for proteome analysis using MS. Of note are platforms for intact protein separation, methods to purify proteins, and improved insights on how to maintain proteome stability. Such developments have advance MS-based proteomics, particularly in top-down format. We still have a long way to go. A pivotal aspect of the current proposal is the use of sodium dodecyl sulfate (SDS), a surfactant beneficial to solubilize, stabilize and separate the proteome. Unfortunately, SDS is also detrimental to MS and for this reason many researchers avoid this detergent. We propose an automated platform based on electrophoresis to isolate proteins from SDS ahead of MS. We also evaluate acetone precipitation as a promising approach for proteome purification and preservation, aiming to improve our fundamental understanding of the process (kinetic studies, mechanism of aggregation). Methods to fractionate proteoforms are proposed, combining electrophoresis with solid phase protein capture. These studies aim to provide powerful tools to profile biological systems. Ultimately, these innovations establish proteomics alongside genetic testing, thus improving the health outcomes of Canadians.