ATP-Coated Dual-functionalized Titanium (IV) IMAC Material for Simultaneous Enrichment and Separation of Glycopeptides and Phosphopeptides

ABSTRACT: Protein glycosylation and phosphorylation are two of the most common post-translational modifications (PTMs), which plays an important role in many biological processes. However, low abundance and poor ionization efficiency of phosphopeptides and glycopeptides make direct MS analysis challenging. Previously, we explored the electrostatic and hydrophilic properties of commercial centrifuge-assisted-extraction Titanium (IV) IMAC (CAE-Ti-IMAC) material and its application in simultaneously enriching and separating common glycopeptides, phosphopeptides, and M6P glycopeptides in dual-mode. In this study, we developed a hydrophilicity enhanced dual-functional Ti-IMAC material with adenosine triphosphate as grafted group (denoted as: epoxy-ATP-Ti4+) to achieve better enrichment performance in dual-mode separation. The epoxy-ATP-Ti4+ IMAC material was prepared from commercially available epoxy functionalized silica particles in a facile way, which only required two steps of reaction. The ATP molecule not only provided superiorly strong and active metal phosphate sites to bind phosphopeptides, but also contributed significantly to the hydrophilicity to enrich glycopeptides. The epoxy-ATP-Ti4+ IMAC material showed great selectivity and sensitivity for phosphopeptide enrichment in conventional IMAC mode. With optimized buffer and fractionation, the material successfully separated glycopeptides and phosphopeptides with high specificity. Besides standard protein samples, the material was further applied to HeLa cells and mouse lung tissue samples. Our method allows simple and effective enrichment and separation of glycopeptides and phosphopeptides, which paves the way for studying the potential crosstalk between these two PTMs.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Lung, Hela Cell

SUBMITTER: Danqing Wang  

LAB HEAD: Lingjun Li

PROVIDER: PXD029775 | Pride | 2023-07-20

REPOSITORIES: Pride