pH-switchable polymer that pulls down N-glycopeptides
A soluble poly(acrylic acid-co-hydrazide) captures glycopeptides in homogeneous solution and precipitates them by dropping pH for MS.
Source
A pH-responsive soluble polymer-based homogeneous system for fast and highly efficient <i>N</i>-glycoprotein/glycopeptide enrichment and identification by mass spectrometry
Study at a glance
- Design
- Other — Homogeneous hydrazide polymer enrichment of periodate-oxidized N-glycopeptides for LC-MS
- N
- Method development; mouse-brain application identified 1,317 N-glycopeptides / 458 glycoproteins
- Population
- Model glycoproteins and mouse-brain digests
- Outcome
- Enrichment efficiency and N-glycoproteome identification performance
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Inspect source passages
pH-switchable polymer that pulls down N-glycopeptides
These passages occur verbatim in the stored paper text. They support the points listed below; they do not verify every statement in the explanation.
Fast enrichment with >95% efficiency can be achieved within 1 h.
First, fast enrichment with >95% efficiency can be achieved within 1 h due to the unrestricted mass transfer in the homogeneous enrichment system.
Signal intensity and S/N of the 1741.8 m/z glycopeptide increase 29 and 325 times after enrichment.
For example, the signal intensity and S / N of the glycopeptide at 1741.8 m / z increases 29 and 325 times after enrichment, respectively.
What they did
Authors copolymerized methyl acrylate and acrylic acid (1:8), converted esters to hydrazides, periodate-oxidized glycans, captured aldehydes in solution, cycled pH to wash, released peptides with PNGase F, and identified sites by LC-MS/MALDI.
What they found
Homogeneous capture exceeds 95% within 1 h. Asialofetuin recovery is ~90%; a 1741.8 m/z glycopeptide gains 29× intensity and 325× S/N. Mouse brain gave 1317 non-redundant N-glycopeptides and 458 glycoproteins (80% in ≥2 replicates) with 1.46% FDR.
The limits
What it doesn't show
The method uses mouse brain, not a clinical plasma cohort; periodate chemistry destroys some glycan structural information, and false deamidation still occurs at 1.46%.
Key terms
- pH-responsive polymer
- A polyacid that dissolves at high pH and precipitates at low pH for capture/release.
- Hydrazide enrichment
- Covalent capture of periodate-oxidized glycan aldehydes as hydrazones.
- PNGase F
- Enzyme that releases N-glycans and converts Asn to Asp (+0.984 Da).
- N-X-S/T sequon
- Canonical N-glycosylation motif used to assign sites after deamidation.
- Homogeneous enrichment
- Capture in solution to avoid solid-phase mass-transfer limits.
Flashcards
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Quiz yourself
Homogeneous hydrazide enrichment is claimed to reach:
Common questions
Why homogeneous instead of beads?
Unrestricted mass transfer; >95% enrichment within 1 h.
How is the polymer recovered?
Low-pH precipitation and high-pH dissolution cycles.
What tissue was profiled?
Mouse brain.
How are sites assigned?
0.984 Da Asn→Asp shift on an N-X-S/T sequon after PNGase F.
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