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

Protein glycosylation and phosphorylation are two of
the most common
post-translational modifications (PTMs), which play an important role
in many biological processes. However, low abundance and poor ionization
efficiency of phosphopeptides and glycopeptides make direct MS analysis
challenging. In this study, we developed a hydrophilicity-enhanced
bifunctional Ti-IMAC (IMAC: immobilized metal affinity chromatography)
material with grafted adenosine triphosphate (denoted as epoxy-ATP-Ti4+) to enable simultaneous enrichment and separation of common
N-glycopeptides, phosphopeptides, and M6P glycopeptides from tissue/cells.
The enrichment was achieved through a dual-mode mechanism based on
the electrostatic and hydrophilic properties of the material. The
epoxy-ATP-Ti4+ IMAC material was prepared from epoxy-functionalized
silica particles via a convenient two-step process. The ATP molecule
provided strong and active phosphate sites for binding phosphopeptides
in the conventional IMAC mode and also contributed significantly to
the hydrophilicity, which permitted the enrichment of glycopeptides
via hydrophilic interaction chromatography. The two modes could be
implemented simultaneously, allowing glycopeptides and phosphopeptides
to be collected sequentially in a single experiment from the same
sample. In addition to standard protein samples, the material was
further applied to glycopeptide and phosphopeptide enrichment and
characterization from HeLa cell digests and mouse lung tissue samples.
In total, 2928 glycopeptides and 3051 phosphopeptides were identified
from the mouse lung tissue sample, supporting the utility of this
material for large-scale PTM analysis of complex biological samples.
Overall, the newly developed epoxy-ATP-Ti4+ IMAC material
and associated fractionation method enable simple and effective enrichment
and separation of glycopeptides and phosphopeptides, offering a useful
tool to study potential crosstalk between these two important PTMs
in biological systems. The MS data have been deposited to the ProteomeXchange
Consortium via the PRIDE partner repository with the data set identifier
PXD029775.