Web Release Date: September 5,
Nitrilotriacetic Acid-Coated Magnetic Nanoparticles as Affinity Probes for Enrichment of Histidine-Tagged Proteins and Phosphorylated Peptides
and
Department of Applied Chemistry and Institute of Molecular Science, National Chiao Tung University, Hsinchu 300, Taiwan, and Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien 970, Taiwan
Received for review May 31, 2007. Accepted July 31, 2007.
Abstract:
We herein demonstrate superparamagnetic Fe3O4 nanoparticles coated with nitrilotriacetic acid derivative (NTA)
that can bind with different immobilized metal ions are
capable of probing diverse target species. Immobilized
Ni(II) on the surfaces of the NTA-magnetic nanoparticles
have the capability of selectively trapping histidine (His)-tagged proteins such as a mutated streptopain tagged with
6× His, i.e., C192S (MW ~42 kDa), from cell lysates.
Enrichment was achieved by vigorously mixing the sample
solution and the nanoparticles by pipetting in and out of
a sample vial for only 30 s. After enrichment, the probe-target species could be readily isolated by magnetic
separation. We also characterized the proteins enriched
on the affinity probes using on-probe tryptic digestion
under microwave irradiation for only 2 min, followed by
matrix-assisted laser desorption/ionization mass spectrometry analysis. Using this enrichment and tryptic
digestion, the target species can be rapidly enriched and
characterized, reducing the time required for carrying out
the complete analysis to less than 10 min. Furthermore,
when either Zr(IV) or Ga (III) ions are immobilized on
the surfaces of the NTA-magnetic nanoparticles, the
nanoparticles have the capability of selectively enriching
phosphorylated peptides from tryptic digests of 
-,
-caseins, and diluted milk. The detection limit for the
tryptic digests of - and -caseins is ~50 fmol.
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