Rosin Nicole L, Winstone Tara M L, Kelley Margaret, Biernaskie Jeff, Dufour Antoine, Orton Dennis J
Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada.
Department of Pathology and Laboratory Medicine, University of Calgary, 3535 Research Rd NW, Room 1E-415, Calgary, AB, T2I 2K8, Canada.
Sci Rep. 2024 Aug 1;14(1):17769. doi: 10.1038/s41598-024-68377-9.
Collagen is the most abundant protein in mammals and a major structural component of the extracellular matrix (ECM). Changes to ECM composition occur as a result of numerous physiological and pathophysiological causes, and a common means to evaluate these changes is the collagen 3 (Col3) to collagen 1 (Col1) ratio. Current methods to measure the Col3/1 ratio suffer from a lack of specificity and often under- or over-estimate collagen composition and quantity. This manuscript presents a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantification of Col3 and Col1 in FFPE tissues. Using surrogate peptides to generate calibration curves, Col3 and Col1 are readily quantified in FFPE tissue sections with high accuracy and precision. The method is applied to several tissue types from both human and reindeer sources, demonstrating its generalizability. In addition, the targeted LC-MS/MS method permits quantitation of the hydroxyprolinated form of Col3, which has significant implications for understanding not only the quantity of Col3 in tissue, but also understanding of the pathophysiology underlying many causes of ECM changes. This manuscript presents a straightforward, accurate, precise, and generalizable method for quantifying the Col3/1 ratio in a variety of tissue types and organisms.
胶原蛋白是哺乳动物中含量最丰富的蛋白质，也是细胞外基质（ECM）的主要结构成分。由于多种生理和病理生理原因，ECM的组成会发生变化，评估这些变化的常用方法是胶原蛋白3（Col3）与胶原蛋白1（Col1）的比值。目前测量Col3/1比值的方法缺乏特异性，常常低估或高估胶原蛋白的组成和数量。本文介绍了一种靶向液相色谱串联质谱（LC-MS/MS）方法，用于定量福尔马林固定石蜡包埋（FFPE）组织中的Col3和Col1。使用替代肽生成校准曲线，可在FFPE组织切片中轻松、高精度地定量Col3和Col1。该方法应用于来自人类和驯鹿的多种组织类型，证明了其通用性。此外，靶向LC-MS/MS方法允许对Col3的羟脯氨酸化形式进行定量，这不仅对了解组织中Col3的数量具有重要意义，而且对理解ECM变化的许多原因背后的病理生理学也具有重要意义。本文介绍了一种直接、准确、精确且通用的方法，用于定量多种组织类型和生物体中的Col3/1比值。
