Profiling yeast cell surface proteins of S. cerevisae by magnetic Profiling yeast cell surface proteins of S.

. cerevisae by magnetic Profiling yeast cell surface proteins of S. cerevisae by magnetic

nanoparticles based covalent labeling of amine groups nanoparticles based covalent labeling of amine groups
Ujwal S. Patil
1
; ParisaPirani; YangCai
1,3
Ujwal S. Patil
1
; ParisaPirani; YangCai
1,3
; Matthew A. Tarr
1,2
1
Department of Chemistry, University of New Orleans, New Orleans, Louisiana, 70148, USA
1
Department of Chemistry, University of New Orleans, New Orleans, Louisiana, 70148, USA
2
Advanced Materials Research Institute,University of New Orleans, New Orleans, Louisiana 70148, USA
2
Advanced Materials Research Institute,University of New Orleans, New Orleans, Louisiana 70148, USA
3
The Research Institute for Children, Childrens Hospital, New Orleans, LA, USA
3
The Research Institute for Children, Childrens Hospital, New Orleans, LA, USA
Overview of the project
LabelingCSPs of S. cerevisaeusingsulfo-NHSester modified
Fluorometriccharacterization of sulfo-NHSester coated
Overview of the project
LabelingCSPs of S. cerevisaeusingsulfo-NHSester modified
FeO@SiO NPs
Fluorometriccharacterization of sulfo-NHSester coated
Fe
3
O
4
@SiO
2
NPs
Overview of the project
Synthesisand characterization of sulfo-N-hydrosuccinimidyl (NHS)
b
Fe
3
O
4
@SiO
2
NPs
Fe
3
O
4
@SiO
2
NPs
Synthesisand characterization of sulfo-N-hydrosuccinimidyl (NHS)
ester conjugated magnetic nanoparticles a
b
ester conjugated magnetic nanoparticles
Quantification of free amine reactive groupspresent on the surface
a
Quantification of free amine reactive groupspresent on the surface
of magnetic nanoparticles of magnetic nanoparticles
Confirmingcell surface attachment of magnetic nanoparticlesusing
TEM TEM
Labelingamine groupsof yeast cell surface proteinsusingsulfo-NHS
Fluorescenceimagesof a) Dansylcadaverineconjugatedsulfo-NHSester
Labelingamine groupsof yeast cell surface proteinsusingsulfo-NHS
ester modified Fe
3
O
4
@SiO
2
NPs
Fluorescenceimagesof a) Dansylcadaverineconjugatedsulfo-NHSester
modifiedFe
3
O
4
@SiO
2
NPsb) NPsafter removal of dansylcadaverinevia
Introduction
modifiedFe
3
O
4
@SiO
2
NPsb) NPsafter removal of dansylcadaverinevia
disulfide cleavage. Dansylcadaverine was covalenlty conjugated to
sulfo-NHSester NPs andafter imaging, danylcadaverinewasremoved
Cell surfacelabelingwasperformedinaqueousenvironment at 3C. Upon
Introduction
Theexposedportionsofcell Surfaceproteins(CSPs)playmajorroles
sulfo-NHSester NPs andafter imaging, danylcadaverinewasremoved
by disulfide bond cleavage resulting into disappearance of green
Cell surfacelabelingwasperformedinaqueousenvironment at 3C. Upon
labeling, CSPs conjugated to NPs were magnetically separated and
Theexposedportionsofcell Surfaceproteins(CSPs)playmajorroles
incell-cell communicationandattachmenttohost.
by disulfide bond cleavage resulting into disappearance of green
fluorescenceofdanylcadaverine(
ex
of335nmand
em
of512nm).
labeling, CSPs conjugated to NPs were magnetically separated and
subjected to SDS wash and tryptic digestion. The last step involved
incell-cell communicationandattachmenttohost.
The popular biotinylation agent available to target free amine
fluorescenceofdanylcadaverine(
ex
of335nmand
em
of512nm).
TEM of S. cerevisaecells labeled by sulfoNHSester
subjected to SDS wash and tryptic digestion. The last step involved
magnetic separation of tryptic peptides conjugated to NPs followed by
cleavageof disulfidebond. Labelingbysulfo-NHSester NPsadded201.28
The popular biotinylation agent available to target free amine
residues of CSPs, sulfo-NHS-LCbiotin have shown some drawbacks
includingpoorrecoveryof biotinconjugatedproteinsandnonspecific
TEM of S. cerevisaecells labeled by sulfoNHSester
coated Fe
3
O
4
@SiO
2
NPs
cleavageof disulfidebond. Labelingbysulfo-NHSester NPsadded201.28
DaonlysineresiduesofCSPs.The labeledpeptidemixturewasanalyzedby
includingpoorrecoveryof biotinconjugatedproteinsandnonspecific
bindingofproteinstoavidin.
coated Fe
3
O
4
@SiO
2
NPs
DaonlysineresiduesofCSPs.The labeledpeptidemixturewasanalyzedby
LC-MS/MSand SEQUEST database was performed for peptide sequence
bindingofproteinstoavidin.
Magnetic nanoparticles functionalized with amine reactive groups LC-MS/MSand SEQUEST database was performed for peptide sequence
andlabeledsiteidentification
The S. Cerevisae cells were manually
Magnetic nanoparticles functionalized with amine reactive groups
couldserve as a potential tool to label the surface exposedamine
groupsofCSPs.
Results
The S. Cerevisae cells were manually
fixed for imaging. Cell surface
groupsofCSPs.
Inourlab, wehavedevelopeddisulfidelinkedN-hydrosuccininimdyl
Results
fixed for imaging. Cell surface
conjugation can be clearly seen in the
Inourlab, wehavedevelopeddisulfidelinkedN-hydrosuccininimdyl
(NHS) ester/sulfo-NHSester silicacoatedironoxide(Fe
3
O
4
@SiO
2
) to
Results conjugation can be clearly seen in the
image.
Characterization of FeO@SiO NPs and thiol coated
(NHS) ester/sulfo-NHSester silicacoatedironoxide(Fe
3
O
4
@SiO
2
) to
covalentlylabel aminegroupsofpeptides/proteins.
Modifiedcell surfacepeptideswereanalyzedbyLC-MS/MSfollowed
Characterization of Fe
3
O
4
@SiO
2
NPs and thiol coated
FeO@SiO NPs
Modifiedcell surfacepeptideswereanalyzedbyLC-MS/MSfollowed
bySEQUESTdatabasesearchtodeterminethelabeledsites.
Methods
3 4 2
Fe
3
O
4
@SiO
2
NPs
An exposed peptide segment of S. cerevisae protein
bySEQUESTdatabasesearchtodeterminethelabeledsites.
Methods
MVRVAINGFGRIGRLVMRIALSRPNVEVVANDPFITNDYAAYMFKYDSTHGRYAGEVSHDDKHIIVDGKKIATYQER
An exposed peptide segment of S. cerevisae protein
1200
a
b
MVRVAINGFGRIGRLVMRIALSRPNVEVVANDPFITNDYAAYMFKYDSTHGRYAGEVSHDDKHIIVDGKKIATYQER
DPANLPWGSSNVDIAIDSTGVFKELDTAQKHIDAGAKKVVITAPSSTAPMFVMGVNEEKYTSDLKIVSNASCTTNCL
APLAKVINDAFGIEEGLMTTVHSLTATQKTVDGPSHKDWRGGRTASGNIIPSSTGAAKAVGKVLPELQGKLTGMAFR Synthesis of sulfo-NHSester modified Fe
3
O
4
@SiO
2
NPs
1200
Si
a
b
APLAKVINDAFGIEEGLMTTVHSLTATQKTVDGPSHKDWRGGRTASGNIIPSSTGAAKAVGKVLPELQGKLTGMAFR
VPTVDVSVVDLTVKLNKETTYDEIKKVVKAAAEGKLKGVLGYTEDAVVSSDFLGDSHSSIFDASAGIQLSPKFVKLVS
WYDNEYGYSTVVDLVEHVAKA
Synthesis of sulfo-NHSester modified Fe
3
O
4
@SiO
2
NPs
1000
O
WYDNEYGYSTVVDLVEHVAKA
SEQUEST database search yielded few proteins with modified lysine
800
Counts
Fe
O
SEQUEST database search yielded few proteins with modified lysine
residues. One of the labeled protein is shown above which is
600
Counts
residues. One of the labeled protein is shown above which is
glyceraldehyde-3- phosphate dehydrogenase (Accession:
SGDID:S000003424), lysineresidue(K)islabeledbysulfo-NHSestertag.
400
SGDID:S000003424), lysineresidue(K)islabeledbysulfo-NHSestertag.
Conclusion
200
Conclusion
0
200
Fe S Cu
Sulfo-NHSester groupswerecovalentlyconjugatedtothesurfaceof Fe
3
O
4
@SiO
2
NPsviathiol-disulfideexchangereaction.
500 1000
0
Energy(keV)
NPsviathiol-disulfideexchangereaction.
TEMimagingconfirmedthe cell surface attachment of sulfo-NHSester NPs to S
cerevisae.
Energy(keV)
(a) TEMimageof FeO@SiO NPs.(Averagesize: 150nm). TEManalysisof
cerevisae.
LC-MS/MSanalysisconfirmedlabelingof exposedcell surfacelysineresiduesby (a) TEMimageof Fe
3
O
4
@SiO
2
NPs.(Averagesize: 150nm). TEManalysisof
Fe
3
O
4
confirmedtheouter silicacoating. TheFe
3
O
4
@SiO
2
NPswerenearly
LC-MS/MSanalysisconfirmedlabelingof exposedcell surfacelysineresiduesby
Sulfo-NHSestermodifiedFe
3
O
4
@SiO
2
NPs. Protocol forsynthesisof sulfo-NHSestermodifiedFe
3
O
4
@SiO
2
NPs. 1)
Synthesisof ironcore(FeO)byhydrothermal method2)Preparation
Fe
3
O
4
confirmedtheouter silicacoating. TheFe
3
O
4
@SiO
2
NPswerenearly
uniform, monodisperseandstableinaqueoussolvents(c)TEM-EDXof thiol
coated FeO@SiO NPs showing the peak of sulfur to confirmMPTMS
Acknowledgements
3 4 2
Synthesisof ironcore(Fe
3
O
4
)byhydrothermal method2)Preparation
of FeO@SiO NPsbysol-gel approach3)Introductionof thiol groups
coated Fe
3
O
4
@SiO
2
NPs showing the peak of sulfur to confirmMPTMS
conjugationtoFeO@SiO NPs.
ThisworkwassupportedbytheLouisianaBoardof RegentsgrantLEQSF(2007-12)-ENH-PKSFI-PRS-
Acknowledgements
of Fe
3
O
4
@SiO
2
NPsbysol-gel approach3)Introductionof thiol groups
onthesurfaceof Fe
3
O
4
@SiO
2
NPs4) Conjugationof sulfo-NHSester
conjugationtoFe
3
O
4
@SiO
2
NPs.
ThisworkwassupportedbytheLouisianaBoardof RegentsgrantLEQSF(2007-12)-ENH-PKSFI-PRS-
04toMAT, theResearchInstituteforChildren, ChildrensHospital NewOrleans, NIHgrant
P01HL076100, Intramural fundfromRICandChildrensHospital toYC
onthesurfaceof Fe
3
O
4
@SiO
2
NPs4) Conjugationof sulfo-NHSester
group to thiol coated Fe
3
O
4
@SiO
2
NPs by thiol-disulfide exchange
reaction.
P01HL076100, Intramural fundfromRICandChildrensHospital toYC
3 4 2
reaction.