Online Proteolysis and Glycopeptide Enrichment with
Thermoresponsive Porous Polymer Membrane Reactors for Nanoflow Liquid
Chromatography-Tandem Mass Spectrometry
Version 2 2018-02-20, 20:21
Version 1 2018-02-20, 20:17
Posted on 2018-02-20 - 20:21
In this Article,
we have reported a fully automated online method
to carry out proteolysis and glycopeptide enrichment in sequence for
nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS)
analysis. By implementing two serial thermoresponsive porous polymer
membrane reactors (TPPMRs), in which the TPPM could be immobilized
either with trypsin for proteolysis or with lectins for glycopeptide
enrichment, the entire pretreatment procedure can be performed online
in about an hour. The TPPM was fabricated by coating polystyrene-maleic
anhydride-N-isopropylacrylamide (PS-MAn-PNIPAm),
which was synthesized by reversible addition–fragmentation
chain transfer polymerization, on a Nylon sheet. Because of the thermoresponsive
nature of PNIPAm, it formed micelle cavities and changed its morphology
at elevated temperatures, resulting in enhanced interactions between
the enzyme or lectins and the proteins/peptides flowing through the
membrane. The performances of the TPPMs were evaluated by varying
the temperature conditions and the amount of standard proteins, showing
that both proteolysis and glycopeptide enrichment with online deglycosylation
were highly efficient at 37 °C. The developed online serial TPPMRs-nLC-ESI-MS/MS
method was applied to the human plasma sample (1.5 μL) and a
total of 262 N-glycopeptides could be identified
from 155 glycoproteins. Thus, the present work demonstrates a fully
automated high speed analytical protocol for online proteolysis and
glycopeptide enrichment, which is extremely useful for analyzing small
amounts of the proteome samples.
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Yang, Joon Seon; Qiao, Juan; Kim, Jin Yong; Zhao, Liping; Qi, Li; Moon, Myeong Hee (2018). Online Proteolysis and Glycopeptide Enrichment with
Thermoresponsive Porous Polymer Membrane Reactors for Nanoflow Liquid
Chromatography-Tandem Mass Spectrometry. ACS Publications. Collection. https://doi.org/10.1021/acs.analchem.7b04273
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AUTHORS (6)
JY
Joon Seon Yang
JQ
Juan Qiao
JK
Jin Yong Kim
LZ
Liping Zhao
LQ
Li Qi
MM
Myeong Hee Moon