Trypsin Immobilization
on Hairy Polymer Chains Hybrid
Magnetic Nanoparticles for Ultra Fast, Highly Efficient Proteome Digestion,
Facile 18O Labeling and Absolute Protein Quantification
In recent years, quantitative proteomic research attracts
great
attention because of the urgent needs in biological and clinical research,
such as biomarker discovery and verification. Currently, mass spectrometry
(MS) based bottom up strategy has become the method of choice for
proteomic quantification. In this strategy, the amount of proteins
is determined by quantifying the corresponding proteolytic peptides
of the proteins, therefore highly efficient and complete protein digestion
is crucial for achieving accurate quantification results. However,
the digestion efficiency and completeness obtained using conventional
free protease digestion is not satisfactory for highly complex proteomic
samples. In this work, we developed a new type of immobilized trypsin
using hairy noncross-linked polymer chains hybrid magnetic nanoparticle
as the matrix aiming at ultra fast, highly efficient proteomic digestion
and facile 18O labeling for absolution protein quantification.
The hybrid nanoparticle is synthesized by in situ growth of hairy
polymer chains from the magnetic nanoparticle surface using surface
initiated atom transfer radical polymerization technique. The flexible
noncross-linked polymer chains not only provide large amount of binding
sites but also work as scaffolds to support three-dimensional trypsin
immobilization which leads to increased loading amount and improved
accessibility of the immobilized trypsin. For complex proteomic samples,
obviously increased digestion efficiency and completeness was demonstrated
by 27.2% and 40.8% increase in the number of identified proteins and
peptides as well as remarkably reduced undigested proteins residues
compared with that obtained using conventional free trypsin digestion.
The successful application in absolute protein quantification of enolase
from Thermoanaerobacter tengcongensis protein extracts
using 18O labeling and MRM strategy further demonstrated
the potential of this hybrid nanoparticle immobilized trypsin for
high throughput proteome quantification.