Cell surface proteomics has seen momentous developments in the past two decades but still faces major challenges in location verification of identified cell surface proteins (CSPs). Recent approaches focus on modification/labeling of CSPs by chemical reagents followed by mass spectrometric analysis of labeled CSPs. Popular biotinylation regents have shown some intrinsic disadvantages such as internalization in the cell cytoplasm, poor recovery of biotinylated proteins, presence of endogenous biotin and non-specific interactions between avidin and proteins. In our study, silica coated iron oxide (Fe3O4@SiO2) superparamagnetic nanoparticles (MNPs) of 100-150 nm were utilized to prepare an impermeable and magnetically separable cell surface labeling reagent. Sulfo-N-hydroxysuccinimidyl (NHS) ester group was conjugated to the surface of Fe3O4@SiO2 MNPs via a disulfide bond to facilitate removal of the magnetic nanoparticle moiety after separation. The surface exposed amine groups of Saccharomyces cerevisae were modified at physiological pH on ice to preserve the native structure of CSPs. Electron microscopic analysis of MNPs conjugated to the S. cerevisae cell surface confirmed the impermeable nature of sulfo-NHS ester Fe3O4@SiO2 MNPs. The labeled CSPs were easily separated by using a magnet and eluted from MNPs by cleaving a disulfide bond. The LC-MS/MS analysis of labeled peptides revealed 30 surface proteins located on solvent exposed surface of the S cerevisae. The sulfo-NHS ester modified Fe3O4@SiO2 MNPs offers benefits such as impermeability, quick magnetic separation of labeled peptides and labeling under physiological conditions.

1. Living cell surface labeling of exposed amine groups by membrane
impermeable and magnetically separable labeling reagent
Ujwal S. Patil1; Parisa Pirani; Yang Cai1,3; Matthew A. Tarr1,2
1Department of Chemistry, University of New Orleans, New Orleans, Louisiana, 70148, USA
2Advanced Materials Research Institute, University of New Orleans, New Orleans, Louisiana 70148, USA
3The Research Institute for Children, Children’s Hospital, New Orleans, Louisiana, 70118 USA
Abstract
Cell surface labeling was performed in aqueous environment at 3°C . Upon
labeling, CSPs conjugated to NPs were magnetically separated and
subjected to SDS wash and tryptic digestion. The last step involved
magnetic separation of tryptic peptides conjugated to NPs followed by
cleavage of disulfide bond. Labeling by sulfo-NHS ester NPs added 201.28
Da on lysine residues of CSPs. The labeled peptide mixture was analyzed by
LC-MS/MS and SEQUEST database was performed for peptide sequence
and labeled site identification
Introduction
Conclusion
• Sulfo-NHS ester groups were covalently conjugated to the surface of Fe3O4@SiO2
NPs via thiol-disulfide exchange reaction.
• TEM imaging confirmed the cell surface attachment of sulfo-NHS ester NPs to S
cerevisae.
• LC-MS/MS analysis confirmed labeling of exposed cell surface lysine residues by
Sulfo-NHS ester modified Fe3O4@SiO2 NPs.
This work was supported by the Louisiana Board of Regents grant LEQSF(2007-12)-ENH-PKSFI-PRS-
04 to MAT, the Research Institute for Children, Children’s Hospital New Orleans, NIH grant
P01HL076100, Intramural fund from RIC and Children’s Hospital to YC
Acknowledgements
Cell surface proteomics has seen momentous developments in
the past two decades but still faces major challenges in location
verification of identified cell surface proteins (CSPs). Recent
approaches focus on modification/labeling of CSPs by chemical
reagents followed by mass spectrometric analysis of labeled
CSPs. Popular biotinylation regents have shown some intrinsic
disadvantages such as internalization in the cell cytoplasm, poor
recovery of biotinylated proteins, presence of endogenous biotin
and non-specific interactions between avidin and proteins. In our
study, silica coated iron oxide (Fe3O4@SiO2) superparamagnetic
nanoparticles (MNPs) of 100-150 nm were utilized to prepare an
impermeable and magnetically separable cell surface labeling
reagent. Sulfo-N-hydroxysuccinimidyl (NHS) ester group was
conjugated to the surface of Fe3O4@SiO2 MNPs via a disulfide
bond to facilitate removal of the magnetic nanoparticle moiety
after separation. The surface exposed amine groups of
Saccharomyces cerevisae were modified at physiological pH on ice
to preserve the native structure of CSPs. Electron microscopic
analysis of MNPs conjugated to the S. cerevisae cell surface
confirmed the impermeable nature of sulfo-NHS ester
Fe3O4@SiO2 MNPs. The labeled CSPs were easily separated by
using a magnet and eluted from MNPs by cleaving a disulfide
bond. The LC-MS/MS analysis of labeled peptides revealed 30
surface proteins located on solvent exposed surface of the S
cerevisae. The sulfo-NHS ester modified Fe3O4@SiO2 MNPs offers
benefits such as impermeability, quick magnetic separation of
labeled peptides and labeling under physiological conditions.
Protocol for synthesis of sulfo-NHS ester modified Fe3O4@SiO2 MNPs. 1)
Synthesis of iron core (Fe3O4) by hydrothermal method 2) Preparation of
Fe3O4@SiO2 NPs by sol-gel approach 3) Introduction of thiol groups on the
surface of Fe3O4@SiO2 NPs 4) Conjugation of sulfo-NHS ester group to
thiol coated Fe3O4@SiO2 NPs by thiol-disulfide exchange reaction.
Fig: TEM image of a) Fe3O4@SiO2 MNPs and b) Sulfo NHS ester
modified Fe3O4@SiO2 MNPs treated with S. Cerevisae cells. The
S. Cerevisae cells were manually fixed for imaging. Cell surface
conjugation can be clearly seen in the image.
• The exposed portions of cell Surface proteins (CSPs) play major
roles in cell-cell communication and attachment to host.
• The popular biotinylation agent available to target free amine
residues of CSPs, sulfo-NHS-LC biotin have shown some drawbacks
including intracellular uptake, poor recovery of biotin conjugated
proteins and nonspecific binding of proteins to avidin.
• Magnetic nanoparticles functionalized with amine reactive groups
could serve as a potential tool to label the surface exposed amine
groups of CSPs.
• In our lab, we have developed disulfide linked N-
hydrosuccininimdyl (NHS) ester/sulfo-NHS ester silica coated iron
oxide (Fe3O4@SiO2) to covalently label amine groups of
peptides/proteins.
•Modified cell surface peptides were analyzed by LC-MS/MS
followed by SEQUEST database search to determine the labeled
sites.
TEM analysis of Fe3O4@SiO2 NPs and S. cerevisae cells
labeled by sulfo NHS ester coated Fe3O4@SiO2 NPs
Methods Results
a b
cytoplasm
mitochondrion
membrane
plasma membrane
mitochondrial envelope
nucleus
ribosome
cell wall
vacuole
chromosome
Labeled yeast cell
surface proteins by
location