Technology Advancements for Proteome Analysis Incorporating Surfactants and Mass Spectrometry

人类基因组计划开创了研究人类健康的变革性方法。 这个项目的一个令人吃惊的发现是我们基因组中的基因数量很低（约20，500个）-只是预期的一小部分。 这证实了我们的生物多样性是通过基因产物，即蛋白质的变化来捕获的。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以与特定的生理状态相关联。 这些变化可能标志着疾病的进展（生物标志物）;绘制蛋白质组可能会定制新的治疗方法（个性化医疗）。 我们推测可能存在超过一百万种独特的人类蛋白质。这种多样性部分是通过化学修饰（例如甲基化，磷酸化）产生的，这表明了独特的蛋白质形式。 Proteoform的表征（我们现在称这些化学上不同的蛋白质）是自上而下蛋白质组学新兴领域背后的驱动特征。 该方法采用分离、高分辨率串联质谱（MS/MS）和计算工具的组合来分析完整蛋白质。 Proteoform表征仍然充满挑战：蛋白质易于降解，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，可能会夸大样品损失; MS检测受到干扰。 迫切需要改进的分析技术来建立蛋白质型分析作为诊断、监测和潜在治愈疾病的可行策略。 我们的团队开创了使用MS进行蛋白质组分析的多种方法。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。 这些发展推动了基于MS的蛋白质组学，特别是自上而下的格式。 我们还有很长的路要走。 目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。 我们提出了一个自动化的平台，基于电泳分离蛋白质SDS之前MS。我们还评估丙酮沉淀作为一种有前途的方法，蛋白质组纯化和保存，旨在提高我们的基本理解的过程（动力学研究，聚集机制）。提出了将电泳与固相蛋白捕获相结合来分离蛋白质组的方法。 这些研究旨在提供强大的工具来分析生物系统。 最终，这些创新将与基因检测一起建立蛋白质组学，从而改善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.