High-Efficiency Phosphopeptide and Glycopeptide Simultaneous Enrichment by Hydrogen Bond–based Bifunctional Smart Polymer

Aberrant
protein phosphorylation and glycosylation are closely
associated with a number of diseases. In particular, an interplay
between phosphorylation and glycosylation regulates the hyperphosphorylation
of protein tau, which is regarded as one of the pathologic features
of Alzheimer’s disease (AD). However, simultaneous characterization
of these two types of post-translational modifications (PTMs) in the
complex biological samples is challenging. TiO2 and the
immobilized ion affinity chromatography (IMAC)-based enrichment method
suffers from low selectivity and/or low recovery of phosphopeptides
and glycopeptides because of the inherent limitations in intermolecular
interactions. Here, we introduce a hydrogen bond-based poly­[(N-isopropylacrylamide-co-4-(3-acryloylthioureido)­benzoic
acid0.2] (referred to as PNI-co-ATBA0.2) as a bifunctional enrichment platform to solve this bottleneck
problem. Benefited from multiple hydrogen bonding interactions of
ATBA with N-acetylneuraminic acid (Neu5Ac) located
at the terminals of sialylated glycans and from favorable conformational
transition of the copolymer chains, the smart copolymer has high adsorption
capacity (370 mg·g–1) and high recovery (ranging
from 74.1% ± 7.0% to 106% ± 5.0% (n = 3))
of sialylated glycopeptides. The smart copolymer also has high selectivity
(79%) for simultaneous enrichment of glycopeptides and phosphopeptides
from 50 μg HeLa cell lysates, yielding 721 unique phosphorylation
sites from 631 phosphopeptides and 125 unique glycosylation sites
from 120 glycopeptides. This study will open a new avenue and provide
a novel insight for the design of enrichment materials used in PTM-proteomics.