Kanao Eisuke, Tanaka Shunsuke, Tomioka Ayana, Ogata Kosuke, Tanigawa Tetsuya, Kubo Takuya, Ishihama Yasushi
Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
Laboratory of Proteomics for Drug Discovery, National Institute of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka 567-0085, Japan.
Anal Chem. 2024 Dec 31;96(52):20390-20397. doi: 10.1021/acs.analchem.4c03753. Epub 2024 Dec 16.
In mass spectrometry-based proteomics, loss-minimized peptide purification techniques play a key role in improving sensitivity and coverage. We have developed a desalting tip column packed with thermoplastic polymer-coated chromatographic particles, named ChocoTip, to achieve high recoveries in peptide purification by pipet-tip-based LC with centrifugation (tipLC). ChocoTip identified more than twice as many peptides from 20 ng of tryptic peptides from Hela cell lysate compared to a typical StageTip packed with chromatographic particles entangled in a Teflon mesh in tipLC. The high recovery of ChocoTip in tipLC was maintained for peptides with a wide variety of physical properties over the entire retention time range of the LC-MS/MS analysis, and was especially noteworthy for peptides with long retention times. These excellent properties are attributable to the unique morphology of ChocoTip, in which the thermoplastic polymer covers the pores, thereby inhibiting irreversible adsorption of peptides into mesopores of the chromatographic particles. ChocoTip is expected to find applications, especially in clinical proteomics and single-cell proteomics, where sample amounts are limited.
在基于质谱的蛋白质组学中，损失最小化的肽段纯化技术在提高灵敏度和覆盖率方面发挥着关键作用。我们开发了一种填充有热塑性聚合物涂层色谱颗粒的脱盐尖柱，名为ChocoTip，通过基于移液器吸头的离心液相色谱（tipLC）在肽段纯化中实现高回收率。与在tipLC中填充有缠绕在聚四氟乙烯网中的色谱颗粒的典型StageTip相比，ChocoTip从20 ng的Hela细胞裂解物胰蛋白酶肽段中鉴定出的肽段数量多出两倍以上。在LC-MS/MS分析的整个保留时间范围内，ChocoTip在tipLC中对具有各种物理性质的肽段都保持了高回收率，对于保留时间长的肽段尤为显著。这些优异的性能归因于ChocoTip独特的形态，其中热塑性聚合物覆盖了孔隙，从而抑制了肽段不可逆地吸附到色谱颗粒的中孔中。ChocoTip有望得到应用，特别是在临床蛋白质组学和单细胞蛋白质组学中，因为这些领域的样品量有限。
