Mapes James H, Stover Julia, Stout Heather D, Folsom Tucker M, Babcock Emily, Loudwig Sandra, Martin Christopher, Austin Mariah J, Tu Fan, Howdieshell Casey J, Simpson Zachary B, Blom Thomas, Weaver Daniel, Winkler Daniel, Vander Velden Kent, Ossareh Parham M, Beierle John M, Somekh Talli, Bardo Angela M, Anslyn Eric V, Marcotte Edward M, Swaminathan Jagannath
Erisyon, Inc. Austin, TX, 78752.
Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712.
bioRxiv. 2023 Sep 16:2023.09.15.558007. doi: 10.1101/2023.09.15.558007.
The need to accurately survey proteins and their modifications with ever higher sensitivities, particularly in clinical settings with limited samples, is spurring development of new single molecule proteomics technologies. Fluorosequencing is one such highly parallelized single molecule peptide sequencing platform, based on determining the sequence positions of select amino acid types within peptides to enable their identification and quantification from a reference database. Here, we describe substantial improvements to fluorosequencing, including identifying fluorophores compatible with the sequencing chemistry, mitigating dye-dye interactions through the use of extended polyproline linkers, and developing an end-to-end workflow for sample preparation and sequencing. We demonstrate by fluorosequencing peptides in mixtures and identifying a target neoantigen from a database of decoy MHC peptides, highlighting the potential of the technology for high sensitivity clinical applications.
对蛋白质及其修饰进行更高灵敏度的精确检测的需求，尤其是在样本有限的临床环境中，正在推动新型单分子蛋白质组学技术的发展。荧光测序就是这样一种高度并行的单分子肽测序平台，它基于确定肽段内特定氨基酸类型的序列位置，以便从参考数据库中对其进行识别和定量。在此，我们描述了对荧光测序的重大改进，包括鉴定与测序化学兼容的荧光团、通过使用延长的多聚脯氨酸接头减轻染料-染料相互作用，以及开发用于样本制备和测序的端到端工作流程。我们通过对混合物中的肽段进行荧光测序，并从诱饵MHC肽数据库中鉴定出目标新抗原，突出了该技术在高灵敏度临床应用中的潜力。
