Fundamental Sessions
Day 1, June 10（Wed.） 14:26-14:44 Room D (5F 511+512)
- 1D-O1-1426（2P-03）
Highly Sensitive Proteomics Enabled by NanoHILIC/MS/MS Using Direct Injection of the Entire Microvolume Eluate from a Reversed-Phase StageTip
(Kyoto Univ.)
oKoshin Akamatsu, Eisuke Kanao, Yasushi Ishihama
Nanoscale hydrophilic interaction chromatography coupled with tandem mass spectrometry (nanoHILIC/MS/MS) is a promising alternative to nanoscale reversed-phase LC/MS/MS (nanoRPLC/MS/MS) for high-sensitivity MS-based proteomics. However, HILIC sample preparation requires acetonitrile-rich solvents, which complicates handling of trace samples. Although our previously reported two-step solubilization improves peptide recovery, it requires a solvent volume of ≥100 µL and therefore limits applications to rare, ultra-low-input samples. Here, we leveraged the reverse polarity of mobile phases between reversed-phase StageTip desalting and nanoHILIC to develop DiReCT (Dissolution from Reversed-phase Chromatography Tip), which enables solubilization of low-input peptides in a minimal-volume HILIC sample solvent. Compared with conventional nanoRPLC/MS/MS, DiReCT-enabled nanoHILIC/MS/MS achieved 5–8-fold higher peptide identifications at 0.25–2.5 ng inputs. This improvement is attributed to the inherently efficient ESI desolvation in nanoHILIC and reduced nonspecific adsorption losses by avoiding speedvaccing in the DiReCT workflow.