Multari Dylan H, Shatouri Mohadeseh Montazeri, de Oliveira Sabrina Grizzi, Ali Zeshan, Habibpourmehraban Fatemeh, Masoomi-Aladizgeh Farhad, Haynes Paul A
School of Natural Sciences, Macquarie University, North Ryde, NSW, Australia.
Methods Mol Biol. 2025;2884:25-41. doi: 10.1007/978-1-0716-4298-6_3.
Identifying proteins from living organisms helps us understand the biological functions of cells, discover new molecular mechanisms, and interrogate known mechanisms for improving our understanding. For a comprehensive understanding of cellular functions, identifying the whole protein content, or proteome, of a cell is desirable but challenging. Here, we describe in detail two methods of proteome fractionation at either the protein (SDS-PAGE) or peptide (high-pH reversed-phase fractionation) level, which can be used to maximize the identification of proteins from complex biological samples. We apply these methods to two different sample types commonly processed in our laboratory to demonstrate the versatility of these protocols. These methods produce many more peptide identifications when compared to conventional single-shot analysis and can also be used in combination to generate larger complementary datasets with greater depth of proteome coverage.
识别来自生物体的蛋白质有助于我们理解细胞的生物学功能、发现新的分子机制，并探究已知机制以增进我们的理解。为全面了解细胞功能，确定细胞的完整蛋白质含量即蛋白质组是理想的，但具有挑战性。在此，我们详细描述了两种在蛋白质（SDS-PAGE）或肽（高pH反相分级分离）水平上进行蛋白质组分级分离的方法，这些方法可用于最大限度地从复杂生物样品中鉴定蛋白质。我们将这些方法应用于我们实验室通常处理的两种不同样品类型，以证明这些方案的通用性。与传统的单次分析相比，这些方法能产生更多的肽鉴定结果，并且还可结合使用以生成具有更大蛋白质组覆盖深度的更大互补数据集。
