Synthesis and characterization of a 'fluorous' (fluorinated alkyl) affinity reagent that labels primary amine groups in proteins/peptides

Article abstract of DOI:10.1002/jms.1854

Strong non-covalent interactions such as biotin-avidin affinity play critical roles in protein/peptide purification. A new type of 'fluorous' (fluorinated alkyl) affinity approach has gained popularity due especially to its low level of non-specific binding to proteins/peptides. We have developed a novel water-soluble fluorous labeling reagent that is reactive (via an active sulfo-N-hydroxylsuccinimidyl ester group) to primary amine groups in proteins/peptides. After fluorous affinity purification, the bulky fluorous tag moiety and the long oligoethylene glycol (OEG) spacer of this labeling reagent can be trimmed via the cleavage of an acid labile linker. Upon collision-induced dissociation, the labeled peptide ion yields a characteristic fragment that can be retrieved from the residual portion of the fluorous affinity tag, and this fragment ion can serve as a marker to indicate that the relevant peptide has been successfully labeled. As a proof of principle, the newly synthesized fluorous labeling reagent was evaluated for peptide/protein labeling ability in phosphate-buffered saline (PBS). Results show that both the aqueous environment protein/peptide labeling and the affinity enrichment/separation process were highly efficient.

Full text of DOI:10.1002/jms.1854

Research Article
Received: 28 April 2010
Accepted: 27 September 2010
Published online in Wiley Online Library: 21 October 2010
(wileyonlinelibrary.com) DOI 10.1002/jms.1854
Synthesis and characterization of a ‘fluorous’
(fluorinated alkyl) affinity reagent that labels
primary amine groups in proteins/peptides
Jiang Qian,a,b Richard B. Coleb and Yang Caia,b∗
Strong non-covalent interactions such as biotin-avidin affinity play critical roles in protein/peptide purification. A new type of
‘fluorous’ (fluorinated alkyl) affinity approach has gained popularity due especially to its low level of non-specific binding to
proteins/peptides. We have developed a novel water-soluble fluorous labeling reagent that is reactive (via an active sulfo-N-
hydroxylsuccinimidyl ester group) to primary amine groups in proteins/peptides. After fluorous affinity purification, the bulky
fluorous tag moiety and the long oligoethylene glycol (OEG) spacer of this labeling reagent can be trimmed via the cleavage
of an acid labile linker. Upon collision-induced dissociation, the labeled peptide ion yields a characteristic fragment that can
be retrieved from the residual portion of the fluorous affinity tag, and this fragment ion can serve as a marker to indicate that
the relevant peptide has been successfully labeled. As a proof of principle, the newly synthesized fluorous labeling reagent
was evaluated for peptide/protein labeling ability in phosphate-buffered saline (PBS). Results show that both the aqueous
c
environment protein/peptide labeling and the affinity enrichment/separation process were highly efficient. Copyright ꢀ 2010
John Wiley & Sons, Ltd.
Keywords: fluorinated alkyl tag; primary amine labeling; flourous affinity tag; protein/peptide labeling; acid cleavable tag
Introduction
ing and separation was first demonstrated in the fluorous
biphasic catalysis technique.[8] Since then, fluorous affinity meth-
ods were successfully utilized in a wide spectrum of organic
syntheses.[9] Recently, fluorous affinity techniques were intro-
ducedtoproteomics,[10] metabolomics,[11] microarraysforprobing
smallbiomolecules[12–14] andfabricationof‘clickable’fluorousthin
films.[15] No water-soluble fluorous affinity-based labeling reagent
has been developed for primary amine group, an important con-
jugation target that exists in almost all proteins/peptides in the
form of lysine side chains and unmodified N-termini.
Flourous affinity is characterized by strong non-covalent
interactions between the fluorous affinity tag (which can be
conjugated to molecules of interest) and a fluorous resin. The
interactions between fluorous resins and proteins/peptides are
generally weak, and the non-specific binding of protein/peptides
to the fluorous resin, if any, can be easily disrupted. Compared
to the biotin group, which is rather small (244 Da), fluorous
tags are usually larger (448 Da for a commonly used tag
C8F17CH2CH3). The strength of fluorous affinity increases with
the size (i.e. the number of fluorine atoms) of the fluorous
tag.[16] Moreover, fluorous compounds are usually insoluble in
water due to the hydrophobicity of the fluorous tag moiety.
This main caveat limits the application of fluorous compounds in
biological systems when the physiological environment needs to
be maintained [e.g. in phosphate-buffered saline (PBS)], and this
Proteins and peptides are present in living organisms at widely
different levels. Given the complexity of biological samples,
extraction and fractionation of proteins/peptides of interest
can be very challenging. It is usually necessary to enrich
and/or purify native proteins and peptides for their further
characterization. Conjugating an affinity tag to protein/peptide
is an attractive approach to protein/peptide enrichment and
purification. Biotin is almost exclusively chosen as the affinity
tag moiety in protein/peptide conjugating reagents due to the
biotin–avidin interaction, which is one of the strongest non-
covalent interactions in nature with a dissociation constant (Kd)
of 10−15. For affinity purification purposes, however, this well-
known biotin–avidin interaction also has several limitations: First,
some proteins/peptides may non-specifically bind to avidin;[1]
second, low recovery of the biotin-tagged molecules in affinity
purification is common because of the incomplete elution of
biotinylated molecules from avidin resins;[2,3] third, the biotin
moiety tends to reduce the ability to distinguish different peptides
based upon hydrophobicity of the tagged peptides and thus
compromisestheseparationofthesepeptidesonreversed-phased
liquid chromatography (LC) columns[4] and fourth, fragments
originating from the biotin moiety can complicate the mass
spectral interpretation of biotin-tagged peptides.[5] Incorporation
of a cleavable linker between the biotin moiety and the peptide
reactive group allows the removal of the biotin moiety, and this
approach has been employed in cleavable ICAT reagents[4,6] that
target the free thiol group in proteins/peptides.[7]
∗
Correspondence to: Yang Cai, The Research Institute for Children, Children’s
Hospital, 200 Henry Clay Ave., New Orleans, LA 70118, USA.
E-mail: ycai@chnola-research.org
The substantial non-covalent interaction between fluorinated
alkyl groups (makes the fluorine-fluorine interaction an alterna-
tive approach to biotin–avidin for affinity-based binding and
separation. Fluorine–fluorine (‘fluorous affinity’) based bind-
a
The Research Institute for Children, Children’s Hospital, New Orleans, LA, USA
Department of Chemistry, University of New Orleans, New Orleans, LA, USA
b
c
J. Mass. Spectrom. 2011, 46, 1–11
Copyright ꢀ 2010 John Wiley & Sons, Ltd.

J. Qian, R. B. Cole and Y. Cai
D
E
O
O
O
O
C8F17
O
O
O
N
H
O
O
N
H
N
N
F
C
O
SO3Na
S
O
N
N
O
O
B
O
O
A
Figure 1. The structure of the synthesized fluorous labeling reagent sulfo-NHS-(OEG)3-perfluorooctane. Building block A is the leaving group when the
labeling reagent conjugates protein/peptide via a primary amine group. The tertiary carbamate group in blocks C and D is acid labile.
hydrophobicity may cause the fluorous tag conjugated proteins to
aggregate. Incorporation of a polar, hydrophilic spacer between
the fluorinated alkyl moiety and the protein/peptide reactive
group will increase the solubility of the reagent to various extents
and thereby alleviate aggregation of labeled proteins. However,
theenlargedfluoroustagmoietysignificantlyincreasesthesizesof
labeled peptides and may further complicate their fragmentation
patterns in tandem mass spectrometry experiments. To avoid this
problem, it can be helpful to trim fluorinated alkyl and spacer
moieties from labeled peptides before LC–MS analysis. Trimming
part of the detergent/matrix molecule that is useful in sample
preparation, but may otherwise interfere with MS analysis, was
shown as an effective strategy to increase signal-to-noise ratios in
a MALDI-MS study of membrane proteins.[17]
and [N-(ε-maleimidocaproyloxy) sulfosuccinimide ester] (sulfo-
EMCS) were purchased from Molecular Bioscience (Boulder, CO).
N-(3-dimethylaminopropyl)-Nꢁ-ethylcarbodiimide hydrochloride
(EDCI) was purchased from Advanced ChemTech (Louisville, KY).
1-Hydroxybenzotriazole, anhydrous (HOBt) was purchased from
Chem-Impex International, Inc. (Wood Dale, IL). N,Nꢁ-disuccini-
midyl carbonate and 1-Boc-piperazine were purchased from Oak-
wood Products, Inc. (West Columbia, SC), tris(2-carboxyethyl)
phosphine hydrochloride (TCEP·HCl) was purchased from EMD
Chemicals, Inc. (Gibbstown, NJ). All chemicals were used as re-
ceived without further purification.
Synthesis of sulfo-NHS-(OEG)3-perfluorooctane (Scheme 1)
4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-Heptadecafluoro-N-(2-(2-(2-(2-
hydroxyethoxy)ethoxy)ethoxy)ethyl)undecanamide (1)
In this study, we have developed a novel water-soluble
fluorous labeling reagent (sulfo-NHS-(OEG)3-perfluorooctane in
Fig. 1) that includes a protein/peptide reactive group (sulfo-
N-hydroxylsuccinimidyl ester in blocks A and B, Fig. 1),[18] an
acid labile linker (a tertiary carbamate group), an oligoethylene
glycol (OEG)3 spacer and a perfluorinated alkane moiety (C8F17).
Efficiency in labeling peptide/protein was demonstrated in PBS.
Fluorous affinity enrichment of labeled peptide/protein and
cleavage of the acid labile linker were also tested as part of
characterization of this novel labeling reagent.
EDCI (0.64 g, 3.3 mM, 1.1 eq.) and HOBt (0.68 g, 4.5 mM, 1.5
eq.) were added to a stirred mixture of 2-(2-(2-(2-aminoethoxy)
ethoxy)ethoxy)ethanol (0.62 g, 3.2 mM, 1.05 eq.) and 4,4,5,5,6,6,
7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecanoic acid (1.50
g, 3.0 mM, 1.0 eq.) in 20 ml anhydrous dichloromethane (CH2Cl2,
Sigma) at room temperature. After 16 h stirring, the solution was
diluted with CH2Cl2, washed by saturated sodium bicarbonate
and extracted by CH2Cl2. The combined organic layer was
washed by brine, dried over MgSO4 and concentrated under
reduced pressure. The crude product was purified by flash column
chromatography (SiO2, eluent: 9 : 1 CH2Cl2/MeOH) as pale brown
oil 1 (1.56 g, yield: 78%). 1H NMR (400 MHz, CDCl3) δ 2.49 (m, 2H),
2.58 (m, 2H), 3.49 (t, J = 5.0 Hz, 2H), 3.56 (t, J = 5.1 Hz, 2H), 3.67
(m, 4H), 3.71 (m, 4H), 3.81 (t, J = 6.0 Hz, 2H), 4.45 (t, J = 6.0 Hz,
2H), 6.26 (s, 1H). ESI-MS: (M + H)+ = m/z 668.1 (calculated MW of
C19H22F15NO5: 667.4 Da).
Experimental
Materials
3-Hydroxy-3-methylbutyronitrile, (fluorenylmethoxycarbonyloxy)
succinimide (Fmoc-OSu), LiAlH4, pyridine, N,N-tetrabutylam-
monium fluoride (TBAF), trifluoroacetic acid (TFA), ammonium
formate, bovine serum albumin (BSA), DL-dithiothreitol (DTT),
iodoacetamide (IA) and 3,3ꢁ-disulfanediyldipropanoic acid were
purchased from Sigma (St. Louis, MO). Diisopropylethylamine
(DiEA) and p-nitrophenylchloroformate were purchased from
Acros (Raritan, NJ). ACTH (4–11) was purchased from Ameri-
can Peptide Company, Inc. (Sunnyvale, CA). Sequencing grade
modified trypsin was purchased from Promega (Madison, WI). Flu-
orous silica beads (5 µm) and 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-
heptadecafluoroundecanoic acid were purchased from Fluorous
Technologies, Inc. (Pittsburg, PA). A cartridge (fitting the car-
tridge holder in c-ICAT kit, Applied Biosystems, Foster City, CA)
packed with fluorous silica beads was manufactured under 400 PSI
with a bed volume of 100 µl by Optimize Technologies (Ore-
gon City, OR). 2-{2-[2-(2-Amino-ethoxy)-ethoxy]-ethoxy}-ethanol
16,16,17,17,18,18,19,19,20,20,21,21,22,22,23,23,23-heptade-
cafluoro-13-oxo-3,6,9-trioxa-12-azatricosyl4-nitrobenzoate (2)
4-Nitrophenyl chloroformate (0.72 g, 3.6 mM, 1.5 eq.) in 5 ml
anhydrous CH2Cl2 was slowly added to a solution of 1 (1.4 g,
2.4 mM, 1.0 eq.) and DiEA (0.93 g, 7.2 mM, 3.0 eq.) in 15 ml
anhydrous CH2Cl2 at 4 ◦C. The mixture was stirred for 16 h
while gradually warmed to room temperature. The reaction was
quenched by adding ice-cold 0.5 N HCl, followed by CH2Cl2
extraction. The organic layer was washed by H2O, brine, dried
over MgSO4 and concentrated in vacuo. The crude product
was purified by flash column chromatography (SiO2, eluent: 1 : 1
hexane/ethylacetate) as pale yellow waxy solid 2 (1.80 g, yield:
c
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Copyright ꢀ 2010 John Wiley & Sons, Ltd.
J. Mass. Spectrom. 2011, 46, 1–11

Synthesis and characterization of a fluorous affinity tag
90%). 1H NMR (400 MHz, CDCl3) δ 2.46 (m, 2H), 2.49 (m, 2H), 3.47 (t,
J = 5.0 Hz, 2H), 3.57 (t, J = 5.0 Hz, 2H), 3.65 (m, 4H), 3.70 (m, 4H),
3.81 (t, J = 6.0 Hz, 2H), 4.44 (t, J = 6.0 Hz, 2H), 6.25 (s, 1H), 7.38 (d,
J = 9.2 Hz, 2H), 8.28 (d, J = 9.2 Hz, 2H). ESI-MS: (M + H)+ = m/z
817.1 (calculated MW of C26H25F17N2O8: 816.5 Da).
were filtered and washed thoroughly with 70 ml THF three times.
After the removal of THF in vacuo, 80 ml of 15% NaOH was added
to the residue followed by extraction with 90 ml of CH2Cl2 three
times. The organic phases were combined, washed by brine and
dried over MgSO4 overnight. Solvents were evaporated to afford
product as pale yellow oil 3 (1.40 g, yield: 92%), which was used
for the next step without further purification.
4-Amino-2-methyl-butane-2-ol(3)
(3-Hydroxy-3-methyl-butyl)-carbamic acid 9H-fluoren-9-ylmethyl
ester (4)
To a vigorously stirred solution of LiAlH4 (2.8 g, 74 mM, 5.0 eq.) in
140 mlice-colddryether, 2-methylpent-4-yn-2-ol(1.45 g, 14.8 mM,
1.0 eq.) in 50 ml dry ether was slowly added. After stirring for 1 h
at room temperature, the mixture was refluxed for 10 h. After
cooling, 15 ml ice-cold 10% NaOH was slowly added under stirring
to quench the reaction. After an additional 2 h stirring, precipitates
Compound 3 (0.60 g, 5.8 mM, 1.0 eq.) was taken up in 20 ml of
5% Na2CO3 and cooled in an ice-water slurry bath. Fmoc-Osu
(2.16 g, 6.4 mM, 1.1 eq.) in 25 ml of THF was slowly added to the
mixture. After stirring for 16 h, THF was removed in vacuo and
F
F
F
F
F
F
F
F
F
F
O
F
F
F
F
F
F
F
OH
HOBt/EDCI
(C8F17
)
+
DCM
78%
H2N
O
OH
O
O
O
DiEA,DCM
O
O
N
O
OH
C8F17
p-nitrophenyl
chloroformate
H
1
90%
H
N
C8F17
O
O
O
O
O
O
2
O
NO2
NH2
N
1. LiAlH4
Fmoc-OSu
HO
HO
ether, reflux
92%
5% Na2CO3 , THF
93%
3
O
O
C
NH
p-nitrophenyl chloroformate
pyridine
96%
HO
Fmoc
4
NHFmoc
O2N
O
O
O
5
Scheme 1. Synthesis of sulfo-NHS-(OEG)3-perfluorooctane.
c
J. Mass. Spectrom. 2011, 46, 1–11
Copyright ꢀ 2010 John Wiley & Sons, Ltd.
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J. Qian, R. B. Cole and Y. Cai
NHFmoc
O
O
O
C
N
N
O
OH
O
FmocHN
O
O
O
N
N
O
S
S
DSC, pyridine
DiEA, DCM
50% TFA
S
S
O
O
O
then 5
DCM
93%
tert-butyl
piperazine-1-carboxylate
DiEA/HOBt
8
94%
N
73%
N
S
S
O
O
N
O
S
O
N
OH
O
O
O
S
N
O
6
7
then 2 and DiEA
in DCM
0.3 M TBAF
in DMF
N
C
84%
O
O
O
O
O
C8F17
O
N
O
O
O
S
S
N
N
N
O
O
O
O
N
H
O
O
H
9
H
N
H
N
O
O
N
O
C8F17
O
O
O
TCEP in Sat. NaHCO3/ THF
93%
C8F17
O
O
N
O
O
10
O
SH
N
N
O
O
N
O
H
H
O
O
O
N
SO3Na
O
O
N
sulfo-EMCS
Quant.
O
O
C8F17
O
O
N
O
O
S
O
N
N
O
O
N
H
O
H
O
O
O
N
11
SO3Na
O
O
N
O
Scheme 1. (Continued).
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Synthesis and characterization of a fluorous affinity tag
the aqueous phase was extracted by 70 ml of CH2Cl2 three times.
The extracts were combined, washed by brine, dried over MgSO4
and evaporated to dryness. Crude product was purified by flash
column chromatography (SiO2, eluent: 6 : 1 CH2Cl2/ethylacetate)
of cleavage reaction, solvents were evaporated under reduced
pressure and the resulting residue was mixed with 6 ml of CH2Cl2,
1.7 ml of DiEA and HOBt (27.6 mg, 0.20 eq.). To this mixture was
added compound 5 (0.98 g, 2.0 mM, 2.2 eq.) in 5 ml of CH2Cl2.
After stirring for 16 h, 30 ml of cold 0.5 N HCl was added to
quench the reaction and 200 ml of CH2Cl2 was then used for three
extractions. The extracts were washed by brine, dried by MgSO4
and evaporated to dryness. Crude product was purified by flash
column chromatography (SiO2, eluent: ethylacetate) as white solid
8 (0.7 g, yield: 73%). 1H NMR (400 MHz, CDCl3) δ 1.49 (s, 12H), 2.30
(t, J = 6.8 Hz, 4H), 2.74 (t, J = 7.0 Hz, 4H), 2.95 (t, J = 7.0 Hz, 4H),
3.28 (t, J = 6.8 Hz, 4H), 3.41 (m, 4H), 3.44 (m, 8H), 3.57 (m, 4H),
4.20 (t, J = 6.4 Hz, 2H), 4.40 (d, J = 6.4 Hz, 4H), 4.80 (s, 2H), 7.31 (t,
J = 7.6 Hz, 4H), 7.40 (t, J = 7.6 Hz, 4H), 7.58 (d, J = 7.6 Hz, 4H), 7.62
(d, J = 7.6 Hz, 4H). ESI-MS: (M + H)+ = m/z 1049.9 (calculated
MW of C56H68N6O10S2: 1049.3 Da).
1
as white solid 4 (1.75 g, yield: 93%). H NMR (400 MHz, CDCl3) δ
1.27 (s, 6H), 1.71 (t, J = 6.8 Hz, 2H), 3.64 (t, J = 6.8 Hz, 2H), 4.22
(t, J = 6.8 Hz, 1H), 4.38 (d, J = 6.8 Hz, 2H), 5.37 (s, 1H), 7.31 (t,
J = 7.2 Hz, 2H), 7.40 (t, J = 7.2 Hz, 2H), 7.60 (d, J = 7.2 Hz, 2H),
7.76 (d, J = 7.2 Hz, 2H). ESI-MS: (M + H)+ = m/z 326.2 (calculated
MW of C20H23NO3: 325.4 Da).
Carbonic acid 3-(9H-fluoren-9-ylmethoxycarbonylamino)-1,
1-dimethyl-propyl ester 4-nitro-phenyl ester (5)
To a solution of 4 (0.76 g, 2.3 mM, 1.0 eq.) and pyridine (5 ml) in
25 ml of CH2Cl2 was slowly added 4-nitrophenyl chloroformate
(0.94 g, 4.6 mM, 2.0 eq.) in 9 ml of CH2Cl2 at 0 ◦C. The mixture was
stirred for 8 h at room temperature and poured into 90 ml of cold
0.5 N HCl. CH2Cl2 (300 ml) was used three times for extraction and
the combined extracts were washed by brine, dried over MgSO4
beforeevaporationtodryness. Crudeproductwaspurifiedbyflash
column chromatography (SiO2, eluent: 5 : 1 hexane/ethylacetate)
as colorless to light yellow oil 5 (1.10 g, yield: 96%). 1H NMR
(400 MHz, CDCl3) δ 1.60 (s, 6H), 2.07 (t, J = 7.6 Hz, 2H), 3.40 (t,
J = 7.6 Hz, 2H), 4.22 (t, J = 6.8 Hz, 1H), 4.42 (d, J = 6.8 Hz, 2H),
4.93 (s, 1H), 7.28 (t, J = 7.2 Hz, 2H), 7.23 (t, J = 7.2 Hz, 2H), 7.35
(d, J = 8.4 Hz, 2H), 7.40 (t, J = 7.2 Hz, 2H), 7.58 (d, J = 7.2 Hz,
2H), 7.76 (d, J = 7.2 Hz, 2H), 8.24 (d, J = 8.4 Hz, 2H). ESI-MS: (M +
H)+ = m/z 491.2 (calculated MW of C27H26N2O7: 490.5 Da).
23,23,24,24,25,25,26,26,27,27,28,28,29,29,30,30,30-
Heptadecafluoro-2-methyl-6,20-dioxo-7,10,13,16-
tetraoxa-5,19-diazatriacontan-2-yl4-(3-((3-(4-
(24,24,25,25,26,26,27,27,28,28,29,29,30,30,31,31,31-
heptadecafluoro-3,3-dimethyl-7,21-dioxo-2,8,11,14,17-
pentaoxa-6,20-diazahentriacontan-1-oyl)piperazin-1-yl)-3-
oxopropyl)disulfanyl)propanoyl)piperazine-1-carboxylate (9)
To a solution of 8 (84 mg, 0.8 mM, 1.0 eq.) in 0.7 ml anhydrous
DMF was added 66 mg TBAF. After 2 h, TLC showed complete
de-protection and compound 2 (135 mg, 0.16 mM, 2.0 eq.) in
4 ml anhydrous CH2Cl2 was slowly added at room temperature,
followed by the addition of DiEA (0.03 ml). The mixture was stirred
overnight, and then the reaction was quenched by adding 10 ml
of ice-cold 0.2 N HCl. The separated aqueous phase was further
extracted by using 60 ml of CH2Cl2 three times. The extracts were
washed by brine, dried by MgSO4 and evaporated to dryness.
Crude product was purified by flash column chromatography
(SiO2, eluent: 13 : 1 CH2Cl2/MeOH) as pale oil 9 (135 mg, yield:
84%). 1H NMR (400 MHz, CDCl3) δ 1.47 (s, 12H), 2.00 (t, J = 7.2 Hz,
4H), 2.47 (m, 4H), 2.51 (m, 4H), 2.76 (t, J = 7.1 Hz, 4H), 2.95 (t,
J = 7.0 Hz, 4H), 3.25 (q, J = 7.2 Hz, 4H), 3.40 (m, 16H), 3.56 (t,
J = 5.0 Hz, 4H), 3.60 (m, 24H), 4.19 (t, J = 5.0 Hz, 4H), 4.99 (s, 2H),
6.63 (s, 2H). ESI-MS: (M + H)+ = m/z 1992.5 (calculated MW of
Bis(2,5-dioxopyrrolidin-1-yl) 3,3ꢁ-disulfanediyldipropanoate (6)
To a solution of 3,3ꢁ-disulfanediyldipropanoic acid (50 mg,
0.24 mM, 1.0 eq.) and pyridine (0.11 ml, 6.0 eq.) in 3 ml of CH2Cl2
was added N,Nꢁ-disuccinimidyl carbonate (182 mg, 0.72 mM, 3.0
eq.). The mixture was stirred at room temperature overnight and
then diluted by 10 ml of CH2Cl2 before pouring into 10 ml of cold
0.2 N HCl. An additional 50 ml of CH2Cl2 was used for extractions
and the extracts were washed by brine, dried over MgSO4 before
evaporation to dryness. Crude product was purified by flash col-
umn chromatography (SiO2, eluent: 2 : 3 hexane/ethylacetate) as
white solid 6 (89 mg, yield 93%). ESI-MS: (M + H)+ = m/z 405.5
(calculated MW of C14H16N2O8S2: 404.4 Da).
C
66H88F34N8O18S2: 1991.5 Da).
Di-tert-butyl 4,4ꢁ-(3,3ꢁ-disulfanediylbis(propanoyl))bis(piperazine-1-
carboxylate) (7)
23,23,24,24,25,25,26,26,27,27,28,28,29,29,30,30,30-Heptadeca-
fluoro-2-methyl-6,20-dioxo-7,10,13,16-tetraoxa-5,19-diazatria-
contan-2-yl 4-(3-mercaptopropanoyl)piperazine-1-carboxylate(10)
To a solution of 6 (0.5 g, 1.23 mM, 1.0 eq.) and DiEA (0.47 ml) in
11 mlCH2Cl2 wasslowlyaddedtert-butylpiperazine-1-carboxylate
(0.51 g, 2.72 mM, 2.2 eq.) in 5 ml of CH2Cl2 at room temperature.
After 3 h stirring, solvents were evaporated and crude product was
recrystallizedbyhexane/ethylacetateaswhitesolid7(0.62 g, yield:
94%). 1H NMR (400 MHz, CDCl3) δ 1.47 (s, 18H), 2.76 (t, J = 6.8 Hz,
4H), 2.97 (t, J = 6.8 Hz, 4H), 3.46 (m, 8H), 3.48 (t, J = 4.9 Hz, 4H),
3.60 (t, J = 4.9 Hz, 4H). ESI-MS: (M + H)+ = m/z 547.4 (calculated
MW of C24H42N4O6S2: 546.7 Da).
A volume of 0.6 ml of TCEP·HCl (65 mg, 0.23 mM, 4.0 eq.) in
saturatedNaHCO3 wasaddedto3 mlofTHFsolutionofcompound
9 (113 mg, 0.057 mM, 1.0 eq.). After stirring for 9 h, solvents
were evaporated under reduced pressure. The crude product
was purified by flash column chromatography (SiO2, eluent: 20 : 1
CH2Cl2/MeOH) as pale brown oil 10 (52 mg, yield: 93%). 1H NMR
(400 MHz, CDCl3) δ 1.47 (s, 6H), 1.73 (t, J = 8.0 Hz, 1H), 2.00 (t,
J = 7.2 Hz, 2H), 2.49 (m, 2H), 2.52 (m, 2H), 2.65 (t, J = 6.8 Hz, 2H),
2.81 (q, J = 7.2 Hz, 2H), 3.24 (q, J = 7.2 Hz, 2H), 3.40 (m, 8H), 3.56
(t, J = 5.2 Hz, 2H), 3.63 (m, 12H), 4.19 (t, J = 5.2 Hz, 2H), 4.99 (s,
1H), 6.63 (s, 1H). ESI-MS: (M + H)+ = m/z 997.3 (calculated MW of
Bis(4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-methylbutan-
2-yl)4,4ꢁ-(3,3ꢁ-disulfanediylbis(propanoyl))bis(piperazine-1-
carboxylate) (8)
Compound 7 (0.5 g, 0.91 mM, 1.0 eq.) was taken up by 2.5 ml of
50% TFA in CH2Cl2 under stirring at room temperature. After 1 h
C33H45 F17N4O9S: 996.8 Da).
c
J. Mass. Spectrom. 2011, 46, 1–11
Copyright ꢀ 2010 John Wiley & Sons, Ltd.
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J. Qian, R. B. Cole and Y. Cai
Sodium 1-((6-(3-((3-(4-(24,24,25,25,26,26,27,27,28,28,29,29,
30,30,31,31,31-heptadecafluoro-3,3-dimethyl-7,21-dioxo-2,8,11,
14,17-pentaoxa-6,20-diazahentriacontan-1-oyl)piperazin-1-yl)-
3-oxopropyl)thio)-2,5-dioxopyrrolidin-1-yl)hexanoyl)oxy)-2,5-
dioxopyrrolidine-3-sulfonate(sulfo-NHS-(OEG)3-perfluorooctane)
(11)
formate, 0.1 M acetic acid in 60% MeOH) for fluorous affinity
purification. Tryptic BSA mixture was loaded onto a cartridge
packedwithfluoroussilicagelbeadsat10 µl/minbysyringepump.
After washing with buffer A (3 ml), the retained modified peptides
were eluted by 2 ml of buffer B (20 mM ammonium formate, 0.1 M
acetic acid in 100% MeOH) at a flow rate of 10 µl/min. The eluate
was dried under vacuum and resuspended in 95% TFA with 5%
anisole for incubation at 37 ◦C for 2 h. After removal of solvents,
cleaved peptides were resuspended in 50 µl of 5% FA, 2% ACN
and 6 µl was injected for LC–MS analysis.
To a solution of 10 (10 mg, 24 mM, 1.0 eq.) in anhydrous DMF was
added sulfo-EMCS (sodium salt, 25.5 mg, 25.6 mM, 1.05 eq.) and
the mixture was stirred at room temperature for 1 h and then for
an additional 3 h at 40 ◦C. Solvents were removed under reduced
pressure and final product 11 was obtained as pale yellow waxy
solid. 1H NMR (400 MHz, DMSO-d6) δ 1.30 (q, J = 7.2 Hz, 2H),
1.38 (s, 6H), 1.49 (q, J = 7.2 Hz, 2H), 1.60 (q, J = 7.2 Hz, 2H), 1.84
(t, J = 7.6 Hz, 2H), 2.41 (m, 4H), 2.44 (t, J = 7.2 Hz, 2H), 2.62 (t,
J = 6.8 Hz, 2H), 2.70 (m, 2H), 2.84 (m, 2H), 3.02 (m, 2H), 3.20 (q,
J = 6.0 Hz, 2H), 3.30–3.51 (m, 24H), 4.02 (m, 4H), 7.18 (t, J = 5.6 Hz,
1H), 8.12 (t, J = 5.6 Hz, 1H). Negative ion ESI-MS: (M − H)− = m/z
1383.2 (calculated MW of C47H60F17N6NaO18S2: 1407.1 Da).
LC–MS/MS analysis and database searching
LC–ESI MS/MS was performed on a Finnigan LTQ-ion trap mass
spectrometer (Thermo Electron, San Jose, CA). Peptide sample
(8 µl) was first loaded onto a C18 trapping column and washed with
3% acetonitrile and 0.1% formic acid for 60 min for desalting, then
the peptides were eluted onto a reversed-phase C18 analytical
column (PicoFrit Column:75 µm ID, 15 µm tip ID, packed with
5 µm BioBasic C18, 10 cm length, New Objective, Woburn, MA)
by a 60-min gradient made of A buffer (0.1% formic acid/97%
water/3% acetonitrile, v/v/v) and B buffer (0.1% formic acid/3%
acetonitrile/97% water, v/v/v) at a flow rate of 200–500 nl/min.
Separated peptides were analyzed under the data-dependent
acquisition mode controlled by Xcalibur, 2.2 version (Thermo
Electron). After a survey scan in the mass range m/z 300–2000, the
seven most intense precursor ions were selected and subjected
to fragmentation by collision-induced dissociation (CID). The
normalized collision energy was set at 35% with activation Q
value being 0.25 and dynamic exclusion of 100 s. The acquired raw
data were processed by BioWorks software, version 3.3 (Thermo
Electron). The parameters for SEQUEST database searching were
set as follows: differential mass increase of 57.02 Da on cysteinyl
residue, 15.99 Da on methionine and 367.02 Da on lysine. The
number of missed cleavage sites was set to three. The search
results were filtered by cross-correlation score (XCorr), i.e. 2.0 for
singly charged peptide ions, 2.5 for doubly charged peptide ions
and 3.0 for the triply charged ions. MS/MS spectra for detected
modified peptides were manually examined to ensure the quality
of identifications.
Labeling of ACTH (4–11) using sulfo-NHS-(OEG)3-
perfluorooctane
A volume of 10 µl of 1 mg/ml ACTH (4–11) was diluted in
120 µl of PBS 8.0 and the solution was added to a 1.5-ml
vial containing 0.038 mg labeling reagent (sulfo-NHS-(OEG)3-
perfluorooctane, see structure in Fig. 1). The labeling proceeded
at room temperature for 1 h, and then excess labeling reagent was
consumed by reacting with approximate 0.5 mg polymer-bound
tris(2-aminoethyl)-amine for 1 h. After centrifugation, the polymer
beadswerewashedby100 µlof80%MeOHand100%MeOHtwice,
andsupernatantswerecombined. Avolumeof1 µlofthecollected
supernatant was resuspended in 100 µl of 2.5% FA in MeOH for
direct ESI-MS analysis. The rest of the supernatant was vacuum
dried and resuspended in 50 µl of TFA for cleavage. The cleavage
reaction was completed after 1 h at 37 ◦C. After the removal of the
TFA under reduced pressure, residues were dissolved in 100 µl of
50% MeOH containing 2.5% FA for ESI-MS analysis.
Labeling of BSA using sulfo-NHS-(OEG)3-perfluorooctane
and affinity purification of the labeled tryptic BSA peptides
A volume of 10 µl of 10 mg/ml BSA stock solution was diluted
in 35 µl PBS buffer (pH 8.0) and the solution was transferred to
a vial containing 0.26 mg fluorous reagent, followed by a brief
vortex. The mixture was incubated for 2 h at room temperature
while shaking. The excess labeling reagent was consumed by
mixing with 15 µl of 50 mm tris–HCl for about 1 h. The labeled
BSA was then subjected to in-solution trypsin digestion. Briefly,
80 µl of 8.0 M urea was added to the labeling mixture, followed
by the addition of 1.7 µl of 200 mM DTT in 50 mM ammonium
bicarbonate for 1 h reduction at 45 ◦C. A volume of 6 µl of 200 mM
IA in 50 mM ammonium bicarbonate was added for 1 h alkylation
at room temperature in the dark, and the excess IA was consumed
by incubation with 6 µl of DTT for 1 h at room temperature.
The mixture was diluted by adding 1 ml of 50 mM ammonium
bicarbonate before trypsin (6 µg) digestion at 37 ◦C for 15 h. The
digestion was stopped by adding 5% FA until colorless precipitate
was observed (pH 3.0, hydrolysis by-product of the labeling
reagent);themixturewasthenseparatedbycentrifugationandthe
precipitates were further washed by 200 µl of 5% FA. Combined
supernatant was evaporated to dryness and one-sixth of the
sample was resuspended in 450 µl of buffer A (20 mM ammonium
Results and Discussion
The fluorous labeling reagent, sulfo-NHS-(OEG)3-perfluorooctane,
can be divided into six building blocks (Fig. 1). The synthesis of
this labeling reagent was based on a retrosynthetic analysis shown
in Fig. 2. The target product, sulfo-NHS-(OEG)3-perfluorooctane,
can be obtained through a highly efficient coupling reaction
between 1 (sulfo-EMCS, blocks A and B in Fig. 1) and a thiol
compound 2 (blocks C–F in Fig. 1), which can be derived from
the cleavage of the disulfide bond in its dimer, compound
3. Compound 3 can be synthesized through formation of a
carbamate using 4 and carbonate compound 5 after the removal
of the Fmoc group with TBAF in DMF. The acid labile linker in
compound 4 can be constructed through carbonate compound
7[19] and piperazine derivative 6, which can be easily prepared
from dithiobis(succinimidyl propionate) 9 and 1-Boc-piperazine
10. Compound 5 can be prepared from hydroxyl compound 8
by reacting with p-nitrophenylchloroformate. The (OEG)3 spacer
in compound 8 can be introduced by the incorporation of
(OEG)3-mono-amine 12 to fluorous compound 11 via amide bond
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Copyright ꢀ 2010 John Wiley & Sons, Ltd.
J. Mass. Spectrom. 2011, 46, 1–11

Synthesis and characterization of a fluorous affinity tag
C8F17
O
O
O
O
2
N
H
O
N
H
3
O
N
Target Compound
sulfo-EMCS
+
3
SH
N
sulfo-NHS-(OEG)3-perfluoroctane
O
1
C8F17
O
O
O
O
O
O
C8F17
O
N
H
O
N
H
3
N
O
O
O
N
N
H
3
S
)
+
3
HN
N
2
O
N
Fmoc
5
O
NO2
O
O
O
O
S
)
4
)
2
S
N
O
2
O
O
C8F17
HN
O
9
)
O
S
HO
N
N
H
3
2
+
8
6
Boc
+
N
Fmoc
NH
O
C8F17
O
NH
O
HO
NH2
3
HO
+
NO2
O
O
10
12
11
7
Figure 2. Retrosynthetic analysis of sulfo-NHS-(OEG)3-perfluorooctane.
formation. Experimental procedures for compiling those building
blocks are detailed in Scheme 1.
reagent was trimmed from the labeled ACTH peptide upon
treatment with TFA as expected, leaving a small tag of 367.2
Da (net mass increase to the amine group in the peptide by
adding building blocks B and C in Fig. 1). The cleavage of the
tertiary carbamate group (in block C and D, Fig. 1) by strong
acid is quantitative,[20] as further evidenced by the fact that no
ACTH peptide with uncleaved sulfo-NHS-(OEG)3-perfluorooctane
tagcouldbefoundontheESI-MSspectrumofthelabeling reaction
mixture after acid cleavage.
The positive ion ESI-MS/MS spectrum of the labeled ACTH
peptide (after TFA cleavage) is shown in Fig. 4. Most of the b
and y ions can be observed. Interestingly, a fragment ion at m/z
368.1 (which corresponds to the residual of sulfo-NHS-(OEG)3-
perfluorooctane cleaved from the amide bonds formed during
the labeling reaction, its structure is shown in Scheme 2) also
appeared on the MS/MS spectrum; and this ion could serve as a
marker of the labeling and fluorous tag moiety removal processes.
The newly synthesized fluorous labeling reagent indeed efficiently
modifiedthefreeaminegroupsinthepeptide;andthefluoroustag
moiety could be easily trimmed. The residual of sulfo-NHS-(OEG)3-
perfluorooctane (building blocks B and C in Fig. 1) after acid
cleavage did not interfere with the fragmentation of the labeled
peptide, which provided an informative decomposition pattern
for unambiguous identification of the peptide.
Thefluorouslabelingreagentwassynthesizedviaa10-stepreac-
tion(Scheme 1)withanoverallyieldof21%.Thisnewlysynthesized
sulfo-NHS-(OEG)3-perfluorooctane was characterized by negative
ion electrospray mass spectrometry and by NMR. Dissolved in 50%
MeOH, the labeling reagent was detected by negative ion mass
spectrometry as shown in Fig. 3 (bottom panel). Upon CID, the
molecular anion [sulfo-NHS-(OEG)3-perfluorooctane – H]− at m/z
1383.3 yielded two major product ions at m/z 605.0 and 560.9
(Fig. 3, middle panel) corresponding to the product ion from the
cleavage at the tertiary C–O bond in block D (Fig. 1) and subse-
quentlossofaCO2 Furtherfragmentationoftheproductionatm/z
605.0 yielded the fragment ion at m/z 560.9 (Fig. 3, top panel) after
the neutral loss of CO2. The product ion at m/z 560.9 in the MS/MS
spectrum of m/z 1383.3 and the ion at m/z 560.9 in the MS/MS/MS
spectrum of m/z 605.0 were the same, both ions yielded m/z 387.0
(spectrum not shown) upon CID, corresponding to the formation
of sulfo-EMCS as the result of thiol ether bond (between blocks B
and C in Fig. 1) cleavage. This mass spectral evidence supports the
structure of newly synthesized sulfo-NHS-(OEG)3-perfluorooctane
as shown in Fig. 1. It appears that the bonds that link the differ-
ent building blocks are also the favored sites of cleavage upon
fragmentation of the molecule.
Sulfo-NHS-(OEG)3-perfluorooctane was employed to label a
small peptide ACTH (4–11) (NH2-M-E-H-F-R-W-G-K) in PBS at room
temperature (Scheme 2). After 1 h reaction time, the free amine
groups (N-terminal and lysine side chain) in ACTH (4–11) were
almost completely tagged by the labeling reagent (approximately
29% of the peptides were singly tagged at either N-terminal or
lysine side chain, 68% were doubly tagged at both N-terminal
and lysine side chain and 3% were untagged). The bulky part
(corresponding to building blocks D–F in Fig. 1) of the labeling
To further demonstrate the potential of the sulfo-NHS-(OEG)3-
perfluorooctane in protein/peptide modification, it was employed
to label BSA in PBS. No precipitation was observed throughout
the labeling reaction, which indicated that the labeling can be
performed in aqueous solution without introducing any organic
solvent to the reaction mixture. After trypsin digestion, the
tagged peptides were easily enriched through fluorous affinity
purification. After TFA cleavage as detailed above, the tagged
peptides were analyzed by LC–MS/MS. About 95% of the
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J. Mass. Spectrom. 2011, 46, 1–11
Copyright ꢀ 2010 John Wiley & Sons, Ltd.
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J. Qian, R. B. Cole and Y. Cai
560.9
100
-
MS3
SO3
O
O
HN
S
O
N
N
N
CH2
O
5
O
O
O
m/z 560.9
1200
0
200
400
605.0
600
800
1000
1400
1600
1800
2000
OH
-
100
SO3
560.9
O
O
MS2
O
N
S
O
N
N
N
CH2 5
O
O
O
O
m/z 605.1
0
400
600
800
1000
1200
C8F17
1400
1600
1383.2
1800
2000
O
O
100
MS
O
O
N
H
O
N
H
3
-
SO3
O
N
O
N
O
S
O
N
N
CH2
5
O
O
O
O
m/z 1383.3
400
0
200
600
800
1000
m/z
1200
1400
1600
1800
2000
Figure 3. Negative ion electrospray mass spectrometry analysis of sulfo-NHS-(OEG)3-perfluorooctane and its fragmentation pattern. (Bottom panel) MS
spectrum of the labeling reagent. Singly charged anion at m/z 1383.3 corresponds to deprotonated sulfo-NHS-(OEG)3-perfluorooctane. (Middle panel)
MS2 spectrum of precursor ion at m/z 1383.3. Product ion at m/z 605.0 corresponds to cleavage of the precursor ion at the tertiary C–O bond in building
block D in Fig. 1. (Top panel) MS3 spectrum of ion at m/z 605.0. Upon CID, m/z 605.0 undergoes neutral loss of CO2 to yield product ion at m/z 560.9.
identified peptides were tagged (Table 1). This indicates that
affinity purification based on fluorine–fluorine interaction was
indeed highly effective in enriching labeled peptides; non-specific
binding was thus minor. The sulfo-NHS-(OEG)3-perfluorooctane
taggedpeptideshadlongerretentiontimesonthereversed-phase
UnlikeICATreagents[4,6,7] thattargetfreethiols(e.g.sidechainof
unmodified cysteine residue), sulfo-NHS-(OEG)3-perfluorooctane
targets primary amine groups (e.g. unmodified side chain of
the lysine residue) which is about threefold more abundant
than the cysteine residue in living organisms.[21] Moreover,
conjugating sulfo-NHS ester (sulfo-N-hydroxylsuccinimidyl ester)
to a primary amine is a robust reaction that has been widely
employed in many biological fields.[22–27] The overall size of
sulfo-NHS-(OEG)3-perfluorooctane is large, the reactive group
(sulfo-NHS ester) and the bulky fluorous tag moiety are linked
by a long spacer of OEG and alkyl chain, and this long spacer
minimizes the steric effect imposed by the fluorous tag moiety on
the reactive sulfo-NHS ester. Moreover, incorporation of the OEG
linker increases sulfo-NHS-(OEG)3-perfluorooctane’s solubility in
aqueous environments, which is critical in labeling proteins and
peptides, especially those located on living cell surfaces.
It is well documented that negatively charged sulfonate groups
(SO3−) in a molecule impede diffusion of such a molecule
into the cytosol because of the barrier of the non-polar lipid
bilayer.[18] Moreover, the OEG spacer and fluorous tag moiety in
sulfo-NHS-(OEG)3-perfluorooctane may strengthen the molecule’s
membrane impermeability due to a combination of OEG spacer’s
hydrophilicityandperfluorocarbons’lipophobicity.[28] Theseprop-
erties of sulfo-NHS-(OEG)3-perfluorooctane make this reagent
desirable for labeling live mammalian and bacterial cell surfaces.
Notably, it lacks certain limitations noted above caused by the
C
18 columnduetotheincreasedhydrophobicityintroducedbythe
fluorous alkyl chain. Once the fluorous tag moiety was trimmed,
the elution profile of tagged peptide was similar to that of the
unmodifiedpeptidesinreversed-phasedLCseparation.Fifteenout
of 16 tryptic peptides (Table 1) were identified as tagged peptides
in a single LC–MS/MS experiment followed by database searching
against BSA sequence using SEQUEST algorithm. All these tagged
tryptic peptides contain at least one labeled lysine residue in the
middle of their sequences, as trypsin does not cleave the protein
at labeled lysine residues. Most of the tryptic peptides in Table 1
have a C-terminal arginine. If the BSA was fully unfolded to allow
all lysine residues to be labeled at their free amino group, then
only peptides with C-terminal arginine (Arg-C) would be produced
by trypsin digestion, and in this case trypsin would serve as an
Arg-C protease. As shown in Fig. 5, the residual fluorous tag had
little influence on the fragmentation of the labeled BSA peptides.
In addition to the b and y ions that verify the peptide sequence,
a characteristic ion at m/z 368.1 (structure in Scheme 2) appeared
on the spectrum to confirm that this peptide segment of BSA was
indeed labeled by the fluorous reagent.
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Copyright ꢀ 2010 John Wiley & Sons, Ltd.
J. Mass. Spectrom. 2011, 46, 1–11

Synthesis and characterization of a fluorous affinity tag
O
O
O
fluorinated alkyl
N
N
OEG
O
N
N
fluorinated alkyl
OEG
O
O
SO3Na
S
O
N
O
Labeling
S
N
N
NH
O
O
O
O
O
+
O
O
in PBS
NH2
TFA cleavage
after enrichment
NH2
H
N
+ H+
CID
O
N
Peptide fragments
+
H
N
N
S
NH
O
O
O
N
O
S
N
O
O+
O
Residual of the labeling reagent at m/z 368.1
Scheme 2. Labeling of protein/peptide using sulfo-NHS-(OEG)3-perfluorooctane.
Figure 4. Positive ion MS/MS of peptide ACTH (4–11) labeled by sulfo-NHS-(OEG)3-perfluorooctane and then treated with trifluoroacetic acid to trim the
fluorinated alkyl moiety. Approximately 10 µg of the labeled ACTH was analyzed by ESI-MS using direct injection. A triply charged precursor ion at m/z
609.5 was isolated and fragmented upon CID to generate this MS2 spectrum. N-terminus and the lysine side chain of the peptide were labeled. (ꢀ) The
unique fragment ion at m/z 368.1 corresponds to the residual of the labeling reagent. The series of b and y ions confirm the sequence of the peptide.
Fragment ion mass error tolerance: 0.4 Da.
biotin moiety in sulfo-NHS–biotin (Pierce, Rockford, IL). The rela-
tively large size, its hydrophilicity and its lipophobicity also make
sulfo-NHS-(OEG)3-perfluorooctane suitable to label fungal cell sur-
faces, and less likely to penetrate cell walls when it is compared
to other smaller sulfo-NHS esters, such as sulfo-NHS–LC–biotin
andsulfo-NHS–SS–biotin(Pierce).Thus,thereshouldbemuchless
ambiguitythatsulfo-NHS-(OEG)3-perfluorooctanelabeledpeptide
segments are indeed exposed portions of fungal cell wall proteins.
In conclusion, a novel fluorous labeling reagent sulfo-
NHS-(OEG)3-perfluorooctane has been synthesized through 11
steps of simple chemical reactions with an overall yield of
21%. Sulfo-NHS-(OEG)3-perfluorooctane reacts with free amine
groups from peptides/proteins through an active sulfo-N-
hydroxylsuccinimidyl ester group. The labeling reaction pro-
ceeded efficiently in PBS buffer without the presence of any
organic solvent, which must be added into the reaction mixture
in the case where water-insoluble fluorous labeling reagents are
employed. More importantly, a homogeneous solution of reaction
mixture was observed throughout the labeling process, which
indicated that the fluorous labeling reagent was not only soluble
c
J. Mass. Spectrom. 2011, 46, 1–11
Copyright ꢀ 2010 John Wiley & Sons, Ltd.
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J. Qian, R. B. Cole and Y. Cai
Figure 5. Positive ion collision-induced dissociation MS/MS of a BSA tryptic peptide tagged by labeling reagent sulfo-NHS-(OEG)3-perfluorooctane at
lysine residue (K∗). After fluorous affinity enrichment and treatment with trifluoroacetic acid, labeled peptides derived from 2 µg of BSA were loaded on
the HPLC column for LC–MS/MS analysis. The doubly charged precursor ion at m/z 890.7 yielded series of b and y ions that confirm the sequence of the
peptide. (ꢀ) The unique fragment ion at m/z 368.1 corresponds to the residual of the labeling reagent.
the fluorous affinity column were identified by LC–MS analysis
Table 1. Identified tryptic peptides derived from sulfo-NHS-(OEG)3-
as labeled peptides. Once the bulky fluorous tag moiety and the
OEG linker were trimmed by acid cleavage, a small residue (from
the fluorous labeling reagent) still attached to the peptides had
minimal influence on the elution profile on C18 chromatography
and on MS/MS fragmentation pattern of the labeled peptides. All
of these results indicate that labeling proteins/peptides by the
newly synthesized sulfo-NHS-(OEG)3-perfluorooctane followed by
affinity separation/enrichment can be readily incorporated into
most LC–MS-based proteomic approaches. The combination of
its size, solubility in water, hydrophilicity of its spacer moiety and
lipophobicity of its fluorinated alkyl moiety makes this novel sulfo-
N-hydroxylsuccinimidyl ester very suitable for labeling living cell
surface proteins/peptides.
perfluorooctane labeled bovine serum albumin (BSA)a
Sequence of detected peptide
XCorrb
K.NYQEAK∗DAFLGSFLYEYSR.R
R.ADLAK∗YIC@DNQDTISSK.L
K.GLVLIAFSQYLQQC@PFDEHVK.L
R.ADLAK∗YIC@DNQDTISSK∗LK.E
R.LAK∗EYEATLEEC@C@AK.D
K.EAC@FAVEGPK∗LVVSTQTALA
K.FWGK∗YLYEIAR.R
6.22
6.14
5.80
5.55
5.16
4.86
4.43
4.32
4.28
4.17
3.79
3.67
3.56
3.39
3.02
2.64
R.K∗VPQVSTPTLVEVSR.S
K.SLHTLFGDELC@K∗VASLR.E
R.ETYGDMADC@C@EK∗QEPER.N
K.VTK∗C@C@TESLVNR.R
R.ETYGDMADC@CEK∗QEPER.N
R.ETYGDM#ADC@CEK∗QEPER.N
R.ALK∗AWSVAR.L
References
R.C@ASIQK∗FGER.A
R.C@C@TK∗PESER.M
[1] J. Masuoka, L. N. Guthrie, K. C. Hazen. Complications in cell-surface
labelingbybiotinylationofCandidaalbicansduetoavidinconjugate
binding to cell-wall proteins. Microbiology 2002, 148, 1073.
[2] J. E. Cronan. Biotination of proteins in vivo – a posttranslational
modification to label, purify, and study proteins. J. Biol. Chem.
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[3] H. Steen, M. Mann. A new derivatization strategy for the analysis of
phosphopeptides by precursor ion scanning in positive ion mode.
J. Am. Soc. Mass Spectrom. 2002, 13, 996.
∗Lysine residue was modified by fluorous affinity tag, # methionine was
oxidized and @cysteine was modified by iodoacetamide.
a Bulky perfluorooctane moiety and OEG linker of the labeling reagent
were trimmed by acid cleavage, which resulted in a net mass increase
of 367.1 Da on labeled lysine residue.
b Cross-correlation score determined by searching peptide tandem
mass spectra against BSA sequence by the use of SEQUEST algorithm.
[4] J. X. Li, H. Steen, S. P. Gygi. Protein profiling with cleavable isotope-
coded affinity tag (cICAT) reagents – the yeast salinity stress
response. Mol. Cell Proteomics 2003, 2, 1198.
[5] Y. Oda, T. Nagasu, B. T. Chait. Enrichment analysis of phosphory-
lated proteins as a tool for probing the phosphoproteome. Nat.
Biotechnol. 2001, 19, 379.
[6] W. W. Wu, G. H. Wang, S. J. Baek, R. F. Shen. Comparative study of
three proteomic quantitative methods, DIGE, cICAT, and iTRAQ,
using 2D gel- or LC-MALDI TOF/TOF. J. Proteome Res. 2006, 5, 651.
in water, but also, due to the incorporation of a hydrophilic (OEG)3
linker, did not cause any aggregation of the labeled proteins.
Fluorous affinity purification was performed to isolate the tagged
tryptic peptides of BSA; the majority of peptides eluting from
c
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Synthesis and characterization of a fluorous affinity tag
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