SynthesisandcharacterizationofNHSesterconjugatedmagneticnanoparticlesfor

labelingprimaryaminegroupsinproteins/peptides
Ujwal S.Patil
1
;Haiou Qu
2
;DanielaCaruntu
2
;CharlesJ.O'Connor
2
;Arjun Sharma
1,3
;MatthewA.Tarr
1,2
;YangCai
1,3
1
DepartmentofChemistry,UniversityofNewOrleans,NewOrleans,Louisiana,70148,USA
2
Advanced Materials Research Institute University of New Orleans New Orleans Louisiana 70148 USA AdvancedMaterialsResearchInstitute, UniversityofNewOrleans,NewOrleans,Louisiana70148,USA
3
TheResearchInstituteforChildren,ChildrensHospital,NewOrleans,LA,USA
Overviewoftheproject Methods
+
Incubation(Room
temp,20minutes)
Magnetic separation
Position of amino
acid residues in the
sequence
a
Peptide sequence
b
Xcorr
c
Mature
91
SynthesisofNHSesterconjugatedmagneticnanoparticles
Qualitative/quantitativecharacterizationofthemagneticnanoparticle labeling
t
Identifiedtrypticpeptidesderivedfrombovineserumalbumin
(BSA)labeledbyNHSestermodifiedFe
3
O
4
@SiO
2
NPs
Mass
spectrometric
NHSestercoated
Fe
3
O
4
@SiO
2
nanoparticles
Magneticseparation
ofnanoparticle
peptideconjugates
TCEPreduction
(Roomtemp,30
minutes)
Peptide
sample
65-76 SLHTLFGDELC
75-91
K 3.64
82-93 ETYGDMADC
90-101
C
91-75
EK 2.93
82-98 ETYGDMADC
90-101
C
91-75
EKQEPER 3.36
160-173 YNGVFQEC
167-176
C
168-123
QAEDK 3.81
257-273 ADLAK*YIC
264-278
DNQDTISSK 4.09
262-273 YIC
264-278
DNQDTISSK 4.08
348-362 LAK*EYEATLEEC
359-368
C
360-315
AK 4.36
351-362 EYEATLEEC
359-368
C
360-315
AK 2.65
363-375 DDPHAC
368-359
YSTVFDK 2.93
397 409 LGEYGFQNALIVR 3 6
reagent
Peptide/proteinlabelingbythenovelmagneticnanoparticle labelingreagent
spectrometric
analysis Removalof
magnetic
nanoparticles
Peptideswith
labeledamine
groups
397-409 LGEYGFQNALIVR 3.6
413-427 K*VPQVSTPTLVEVSR 3.8
428-444 SLGK*VGTRC
436-447
C
437-391
TKPESER 3.61
445-458 MPC
447-436
TEDYLSLILNR 4.54
466-483 TPVSEK*VTKC
475-486
C
476-460
TESLVNR 4.92
466-483 TPVSEKVTK*C
475-486
C
476-460
TESLVNR 3.95
472-483 VTKC
475-486
C
476-460
TESLVNR 3.85
484-499 RPC
486-475
FSALTPDETYVPK 4.16
505-520 LFTFHADIC
513-558
TLPDTEK 4.2
509-520 HADIC
513-558
TLPDTEK 2 72
ProtocolforlabelingprimaryaminegroupsofpeptideusingNHSestermodifiedFe
3
O
4
@SiO
2
nanoparticles
Preparation of NHS ester coated Fe3O4@SiO2 NPs using DSP
Results
509 520 HADIC TLPDTEK 2.72
Functionalized by various peptide reactive groups, magnetic nanoparticles have potential
for proteome enrichment.
High magnetization values, relatively easy control over particle size, and narrow particle
size distributions make iron oxide nanoparticles effective for conjugating peptides and
proteins for isolation/separation purposes
Introduction
0.0
0.1
0.2


e
n
t

(
e
m
u
)
*lysineresiduesmodifiedbyNHSestertag.Thepeptideshighlightedingreencontainsat
leastonelabeledlysineresidue
aPositionofaminoacidresiduesinthesequenceofmaturedBSA
bPeptidesequenceinmaturedBSAwithpositionsofcysteineresidues
cCrosscorrelationscoreprovidedbySEQUESTalgorithm
PreparationofNHSestercoatedFe3O4@SiO2NPsusingDSP
c
(a) TEM image of bare Fe
3
O
4
NPs synthesized by coprecipitation method and (b) Fe
3
O
4
@SiO
2
NPs
coated by solgel method . TEM analysis of Fe
3
O
4
and showed the agglomeration of nanoparticles,
thereby increasing the diameter from 15 5 nm to ~ 150 nm. (c) Magnetization studies of APTES
coated Fe3O4@SiO2 NPs showing superparamgnetic nature of the nanoparticles.
100
y
5
HomodimerofmatureBSA
showinglysineresidues
(highlightedinred)labeledby
NHSestermodifiedFe
3
O
4
@SiO
2
NPs
proteins for isolation/separation purposes.
Abundance of lysine residues in living organisms makes them the better site for protein
labeling.
In this research, the surface of superparamagnetic silica coated iron oxide (Fe
3
O
4
@SiO
2
)
nanoparticles was functionalized with a disulfide linked NHS ester group in order to develop
a method for labeling primary amines in peptides/proteins.
The labeled peptides were analyzed using LCMS/MS for sequence and labeled site
identification.
-10000 -5000 0 5000 10000
-0.2
-0.1
M
o
m
e
Field(Oe)
Roomtemp.and
LabelinginPBS
TCEPcleavage
NHSestercoated
Fe
3
O
4
@SiO
2
NPs
Peptide
100
e
l
a
t
i
v
e

A
b
u
n
d
a
n
c
e
y
2
292.1
y
6
2+
459.6
y
5
781.2
y
4
NH
3
617.1
y
1
234 8
b
5
789.1
b
7
1032.2
y 1047 3
*M E F H R W G K*
b2 b1 b5 b4 b6 b3 b7
y3 y7 y4 y2 y6 y5 Conclusion
NHS ester groups were conjugated on the surface of Fe
3
O
4
@SiO
2
NPs through covalent
bond formation.
The novel NHS ester coated Fe
3
O
4
@SiO
2
NPs were successfully applied to label
peptide/protein samples at room temperature and in aqueous environment, conditions
under which the native structures of protein/peptide were preserved.
CID
SchematicillustrationofprimaryaminelabelingusingNHSestercoated
Fe
3
O
4
@SiO
2
NPs
200 400 600 800 1000 m/z
0
R
e
234.8 y
7
1047.3
ESIMS/MS of doubly charged peptide ACTH (411) ion at m/z 633.9 Da. The precursor ion at m/z
633.9 yielded a series of fragment ions that corresponded to cleavages at peptide bonds in the
peptide, i.e. b and y ions. NHS ester label added 88.01 Da to the lysine residue and the Nterminus
of ACTH (411).
ThisworkwassupportedbytheLouisianaBoardofRegentsgrant LEQSF(200712)ENH
PKSFIPRS04toMAT,alsoinpartbyanintramuralfundfromtheResearchInstitutefor
Children,ChildrensHospitalNewOrleansandNIHP01HL076100toYC,andbyUNOCollege
ofSciencesgrant,UNODissertationImprovementGranttoUSP.
WewouldliketothankDr.LeonardSpinuforhishelpwiththisresearch.
Acknowledgements
CID
61st ASMS