HeaJI Lee (Korea Basic Science Institute (KBSI))

Jin Young Kim (Korea Basic Science Institute (KBSI), )

HeaJI Lee (Korea Basic Science Institute (KBSI))

Yeji Yang (Korea Basic Science Institute (KBSI), )

Jin Young Kim (Korea Basic Science Institute (KBSI), )

 Phosphorylation is a key post-translational modification (PTM) that regulates various biological processes such as signal transduction, cell cycle progression, stress response, and metabolism. Due to its dynamic nature, phosphorylation is closely associated with diseases including cancer and neurological disorders. As a result, phosphoproteomics has become a powerful tool for deciphering signaling pathways and disease mechanisms. However, the inherently low abundance and poor ionization efficiency of phosphopeptides make mass spectrometry (MS)-based detection challenging, necessitating effective enrichment strategies.

Conventional approaches to enhance analytical depth have relied on offline HPLC-based fractionation or online 2D-LC systems. While these methods provide high resolution and deep proteome coverage, they require specialized instrumentation such as multidimensional HPLC setups and often involve complex workflows.

In this study, we developed a simple phosphopeptide enrichment workflow tailored for low amount samples. This method integrates IMAC-based phosphopeptide enrichment with stepwise acetonitrile (ACN) gradient fractionation (10%, 25%, 50%) during the desalting step, enabling both enrichment and fractionation without the need for additional instrumentation.

We applied this workflow to both TMT-labeled and label-free samples (10–200 µg) and evaluated its performance across various input amounts. The results demonstrated high reproducibility and effective enrichment, supporting the utility of this approach for phosphoproteomic analysis of low-abundance samples.