Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft 2629 HZ, The Netherlands.
Laboratory of Organic Chemistry, Wageningen University & Research, Wageningen 6807 WE, The Netherlands.
ACS Nano. 2024 Oct 22;18(42):28999-29007. doi: 10.1021/acsnano.4c09872. Epub 2024 Oct 10.
Peptide hormones are decorated with post-translational modifications (PTMs) that are crucial for receptor recognition. Tyrosine sulfation on plant peptide hormones is, for example, essential for plant growth and development. Measuring the occurrence and position of sulfotyrosine is, however, compromised by major technical challenges during isolation and detection. Nanopores can sensitively detect protein PTMs at the single-molecule level. By translocating PTM variants of the plant pentapeptide hormone phytosulfokine (PSK) through a nanopore, we here demonstrate the accurate identification of sulfation and phosphorylation on the two tyrosine residues of PSK. Sulfation can be clearly detected and distinguished (>90%) from phosphorylation on the same residue. Moreover, the presence or absence of PTMs on the two close-by tyrosine residues can be accurately determined (>96% accuracy). Our findings demonstrate the extraordinary sensitivity of nanopore protein measurements, providing a powerful tool for identifying position-specific sulfation on peptide hormones and promising wider applications to identify protein PTMs.
肽激素经过翻译后修饰（PTMs），这些修饰对于受体识别至关重要。例如，植物肽激素的酪氨酸硫酸化对于植物的生长和发育是必不可少的。然而，在分离和检测过程中存在重大技术挑战，这使得测量硫酸酪氨酸的出现和位置变得复杂。纳米孔可以在单分子水平上灵敏地检测蛋白质 PTM。通过将植物五肽激素 phytosulfokine (PSK) 的 PTM 变体穿过纳米孔，我们在此证明了对 PSK 两个酪氨酸残基上的硫酸化和磷酸化的准确识别。可以清楚地区分（>90%）同一残基上的硫酸化和磷酸化。此外，可以准确确定两个附近酪氨酸残基上是否存在 PTM（>96%的准确率）。我们的发现证明了纳米孔蛋白质测量的非凡灵敏度，为识别肽激素上的位置特异性硫酸化提供了有力工具，并有望更广泛地应用于鉴定蛋白质 PTM。
