Akamatsu Koshin, Kanao Eisuke, Tomioka Ayana, 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. 2025 May 20;97(19):10227-10235. doi: 10.1021/acs.analchem.5c00011. Epub 2025 May 6.
Nanoscale hydrophilic-interaction chromatography coupled with tandem mass spectrometry (nanoHILIC/MS/MS) is a promising alternative to reversed-phase liquid chromatography for proteomics, but its application is limited by the poor solubility of peptides in organic solvent-rich sample solutions. To overcome this issue, we have developed a two-step solubilization method, in which peptides are first solubilized in a solvent with an optimal acetonitrile (ACN) concentration of 25% and then diluted into a high ACN concentration solution of 95%. This procedure increases the peptide solubility without compromising compatibility with nanoHILIC/MS/MS. Compared to direct solubilization in 95% ACN, this approach increased the intensity of 82.8% of commonly quantified peptides in nanoHILIC/MS/MS, with an average intensity gain of 20.9%. Furthermore, nanoHILIC/MS/MS with this two-step solubilization outperformed nanoRPLC/MS/MS, identifying 8.47 times more peptides and 3.54 times more protein groups from 2.5 ng of tryptic peptides extracted from HeLa cells. The high sensitivity of nanoHILIC/MS/MS can be attributed to the enhanced loading of peptides as a result of the two-step solubilization, together with superior ESI efficiency arising from the use of the ACN-rich mobile phase. This high-sensitivity proteomics system is a promising platform for clinical and single-cell applications.
纳米级亲水相互作用色谱-串联质谱联用技术（nanoHILIC/MS/MS）是蛋白质组学中反相液相色谱的一种有前景的替代方法，但由于肽段在富含有机溶剂的样品溶液中溶解度较差，其应用受到限制。为克服这一问题，我们开发了一种两步溶解方法，即首先将肽段溶解在乙腈（ACN）最佳浓度为25%的溶剂中，然后稀释到ACN浓度为95%的高浓度溶液中。该方法在不影响与nanoHILIC/MS/MS兼容性的情况下提高了肽段溶解度。与直接溶解在95%的ACN中相比，这种方法使nanoHILIC/MS/MS中82.8%的常用定量肽段强度增加，平均强度增益为20.9%。此外，采用这种两步溶解法的nanoHILIC/MS/MS优于nanoRPLC/MS/MS，从2.5 ng从HeLa细胞中提取的胰蛋白酶肽段中鉴定出的肽段数量多出8.47倍，蛋白质组数量多出3.54倍。nanoHILIC/MS/MS的高灵敏度可归因于两步溶解导致的肽段加载量增加，以及使用富含ACN的流动相产生的卓越电喷雾电离（ESI）效率。这种高灵敏度蛋白质组学系统是临床和单细胞应用的一个有前景的平台。
