7 research outputs found
Synthesis of a Highly Azide-Reactive and Thermosensitive Biofunctional Reagent for Efficient Enrichment and Large-Scale Identification of O‑GlcNAc Proteins by Mass Spectrometry
O-linked
β-N-acetylglucosamine (O-GlcNAc) is a ubiquitous
post-translational modification of proteins in eukaryotic cells. Despite
their low abundance, O-GlcNAc-modified proteins play many important
roles in regulating gene expression, signal transduction, and cell
cycle. Aberrant O-GlcNAc proteins are correlated with many major human
diseases, such as Alzheimer’s disease, diabetes, and cancer.
Because of the extremely low stoichiometry of O-GlcNAc proteins, enrichment
is required before mass spectrometry analysis for large-scale identification
and in-depth understanding of their cellular function. In this work,
we designed and synthesized a novel thermosensitive immobilized triarylphosphine
reagent as a convenient tool for efficient enrichment of azide-labeled
O-GlcNAc proteins from complex biological samples. Immobilization
of triarylphosphine on highly water-soluble thermosensitive polymer
largely increases its solubility and reactivity in aqueous solution.
As a result, facilitated coupling is achieved between triarylphosphine
and azide-labeled O-GlcNAc proteins via Staudinger ligation, due to
the increased triarylphosphine concentration, reduced interfacial
mass transfer resistance, and steric hindrance in homogeneous reaction.
Furthermore, solubility of the polymer from complete dissolution to
full precipitation can be easily controlled by simply adjusting the
environmental temperature. Therefore, facile sample recovery can be
achieved by increasing the temperature to precipitate the polymer-O-GlcNAc
protein conjugates from solution. This novel immobilized triarylphosphine
reagent enables efficient enrichment and sensitive detection of more
than 1700 potential O-GlcNAc proteins from HeLa cell using mass spectrometry,
demonstrating its potential as a general strategy for low-abundance
target enrichment
Dual Matrix-Based Immobilized Trypsin for Complementary Proteolytic Digestion and Fast Proteomics Analysis with Higher Protein Sequence Coverage
In
an age of whole-genome analysis, the mass spectrometry-based
bottom-up strategy is now considered to be the most powerful method
for in-depth proteomics analysis. As part of this strategy, highly
efficient and complete proteolytic digestion of proteins into peptides
is crucial for successful proteome profiling with deep coverage. To
achieve this goal, prolonged digestion time and the use of multiple
proteases have been adopted. The long digestion time required and
tedious sample treatment steps severely limit the sample processing
throughput. Though utilization of immobilized protease greatly reduces
the digestion time, highly efficient proteolysis of extremely complex
proteomic samples remains a challenging task. Here, we propose a dual
matrix-based complementary digestion method using two types of immobilized
trypsin with opposite matrix hydrophobicity prepared by attaching
trypsin on hydrophobic or hydrophilic polymer-brush-modified nanoparticles.
The polymer brushes on the nanoparticles serve as three-dimensional
supports for a large amount of trypsin immobilization and lead to
ultrafast and highly efficient protein digestion. More importantly,
the two types of immobilized trypsin show high complementarity in
protein digestion with only ∼60% overlap in peptide identification
for yeast and membrane protein of mouse liver. Complementary digestion
by applying these two types of immobilized trypsin together leads
to obviously enhanced protein and peptide identification. Furthermore,
the dual matrix-based complementary digestion shows particular advantage
in the digestion of membrane proteins, as twice the number of identified
peptides is obtained compared with solution digestion using free proteases,
demonstrating its potential as a promising alternative to promote
proteomics analysis with higher protein sequence coverage