NanoHILIC/MS/MS provides high sensitivity for low-input peptide analysis, yet its use in bottom-up proteomics has been constrained by a persistent solvent mismatch: tryptic peptides exhibit poor solubility in the 95% acetonitrile (ACN) required for nanoHILIC injection. Our previously reported two-step solubilization method [Anal Chem 2025, 97 (19), 10227-10235] alleviated this issue but required large dilution volumes, limiting the amount of sample that could be injected. Here, we introduce DiReCT (Dissolution from Reverse-Phase Chromatography Tips), a StageTip-based workflow that integrates peptide solubilization, desalting, and nanoHILIC-compatible elution into a single operation. During elution from RP-StageTips, residual water on the stationary phase is rapidly displaced by a small volume of high-ACN solvent, generating a transient mid-ACN environment that maximizes peptide solubility without drying. This mechanism enables high-recovery peptide concentration and allows direct injection of the entire eluate onto nanoHILIC/MS/MS. Using ~0.25 ng of HeLa digest, DiReCT/nanoHILIC/MS/MS identified 1177 peptides and 410 proteins, representing 8.9- and 6.7-fold increases over nanoRPLC/MS/MS, respectively.
Akamatsu, K., Kanao, E., Ishihama, Y.
Advertisement
Stats
- Recommendations n/a n/a positive of 0 vote(s)
- Views 9
- Comments 0