A novel quantitative proteomics reagent based on soluble nanopolymers

Article abstract of DOI:10.1039/b614926j

Bi-functionalized dendrimers leads to highly efficient quantitative proteomics and the determination of protease activities in snake venoms. The Royal Society of Chemistry.

Full text of DOI:10.1039/b614926j

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A novel quantitative proteomics reagent based on soluble
nanopolymers{
Minjie Guo, Jacob Galan and W. Andy Tao*
Received (in Cambridge, MA, USA) 13th October 2006, Accepted 14th December 2006
First published as an Advance Article on the web 10th January 2007
DOI: 10.1039/b614926j
order to isotopically tag and then recover tagged samples for mass
Bi-functionalized dendrimers leads to highly efficient quantita-
tive proteomics and the determination of protease activities in
snake venoms.
spectrometry (MS) analysis, we introduced an acid-cleavable
linker, 4-(4-formyl-3,5-dimethoxyphenoxy)butyric acid, and an
isotope tag (aniline-12C6 or -13C6) between dendrimer and the
reactive group (Fig. 1B). The dendrimer surface was also
functionalized with the terminal alkyne group as the ‘handle’ to
achieve efficient isolation through the click chemistry,9 a highly
potent bioconjugation between terminal alkyne group and
azide group. Selective isolation, identification and quantification
of Cys-containing peptides is illustrated in Fig. 1C.
Quantitative proteomics holds significant promise for the discovery
of diagnostic or prognostic protein markers and for the detection
of new therapeutic targets.1 The isotope-coded affinity tagging
(ICAT) method is perhaps the best characterized approach for
quantitative proteomics that combines stable isotope labeling with
affinity purification.2 Among other popular methods3,4 developed
since then, the adaptation of solid-phase capture and release
process is an important improvement for isotope tagging and
selective peptide isolation.5,6 However, the most notable liability of
solid phase capture is the heterogeneous reaction conditions, which
can exhibit several of the following problems. Due to extreme
complexity and proteins in low-abundance, the heterogeneous
nature of solid-phase reaction presents a significant issue for
proteomic sample recovery.
Here we devise a new strategy, termed Soluble Polymer-based
Isotopic Labeling (SoPIL). The design was based on the concept
that the derivatization with proteomic samples in limited amount
was carried out in the solution phase for maximum yield and in the
second step, samples tagged with the nanopolymer were isolated
on a solid phase by choosing a highly efficient reaction between a
pair of bioorthogonal groups on the soluble polymer and on the
solid phase (Fig. 1A). The cornerstone of SoPIL is a soluble,
globular nanopolymer (e.g. dendrimers) which was functionalized
with reactive groups for site-specific, stable isotopic labeling of
proteins/peptides of interest. Unmodified dendrimer was recently
used for phosphoproteomics.7 Here, a ‘handle’ group on the
polymer facilitates the isolation of tagged samples through highly
efficient bio-conjugations. The method takes advantage of the
homogeneity of solution-phase reaction, convenience of solid-
phase capture and release process, and characteristics of cell-
permeable nanoparticles.8 High concentration ratio of the reactive
group to the ‘handle’ group facilitates the completion of solution
phase reaction while eliminating extra steps to remove excess
reagents which is the case for small chemical reagents such as
ICAT reagents.2 In this initial report, bromoaceto group was
employed to selectively capture cysteine-containing peptides/
proteins for a direct comparison with solid-phase reagents. In
Fig. 1 Schematic representation of the SoPIL method. A: Modular
composition of the SoPIL strategy which consists of two step reactions: a
homogeneous reaction between proteins/peptides and the SoPIL reagent
and a heterogeneous reaction between the solid-phase and the SoPIL
reagent. B: Chemical composition of the SH-reactive solid-phase. C:
Strategy for quantitative proteome analysis. Two protein samples to be
comparatively analyzed were proteolyzed. The Cys-containing peptides
were reduced and captured on SoPIL reagents carrying either the light or
heavy isotope tag. The samples were then combined and immobilized by
the azide beads through the efficient click chemistry. After stringent
washing of the beads, the peptides were released by acid-cleavage and
analyzed by mLC-MS/MS.
Department of Biochemistry and Purdue Cancer Center, Purdue
University, West Lafayette, IN 47907, USA.
E-mail: watao@purdue.edu; Fax: +1 (765)494-7898;
Tel: +1 (765)494-9605
{ Electronic supplementary information (ESI) available: Detailed synthesis
and characterization data. Quantitative analysis of standard protein
mixtures and two snake venoms. See DOI: 10.1039/b614926j
This journal is ß The Royal Society of Chemistry 2007
Chem. Commun., 2007, 1251–1253 | 1251

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quantified and sequenced by mLC-MS/MS experiments. Using a
limited amount of proteins (1–400 fmols), all four proteins
were unambiguously identified and accurately quantified (ESI
Table S1{). Multiple tagged peptides were encountered for each
protein. The mean differences between the observed and expected
quantities for the four proteins ranged 2–6%.
The SoPIL strategy was applied to study differences in protein
abundance in two snake venoms. Snake venom contains complex
mixtures of pharmacologically active molecules including small
peptides and proteins. The biological effects of venom are complex
because different components have distinct but sometimes
synergistic actions.10 The fact that members of the same protein
family show remarkable structure similarity but diverge in their
biological targeting makes them valuable biotechnological tools
for studying physiological processes and for drug discovery. In
addition, a number of snake venom proteins are extremely
cysteine-rich, which makes a perfect paradigm for us to study using
cysteine-specific SoPIL reagents.
The same amount of two characteristically different snake
venoms A and B from Crotalus scutulatus scutulatus (Mohave
rattlesnake) were labeled on light and heavy SoPIL reagents,
respectively, combined and processed as described in Fig. 1C.
Although the snake genome has not been sequenced, the analysis
identified and quantified over 250 unique peptides representing
51 snake toxins in Swiss Protein Database, by far the largest
presentation of snake venoms (Selected cysteine-containing pep-
tides from snake venoms in Table 1 and a complete list is provided
in ESI Table S2{). Consistent with previous reports, quantitative
measurements indicated that several classes of cysteine-rich
proteins dominantly exist in venom A but not in venom B. A
few proteins, such as disintegrin, were only observed in venom B.
Fig. 3 illustrates the identification of one peptide from L-amino
acid oxidase and its relatively abundance in two snake venoms. We
also observed for the first time extensive cleavage products by
proteases in snake venoms (two examples were shown in ESI S3{).
Quantitative analysis by SoPIL reagents allowed us to measure
difference of protease activities in two snake venoms (Table 1 and
S2{).
Dendrimers have been shown to cross cell membranes at
sufficient rates to act as potential carrier/delivery systems.8,11
Therefore, SoPIL reagents have the potential to directly tag and
label proteins in living cells and in vivo. We examined the efficiency
of SoPIL reagents to cross cell membranes. To facilitate the
observation of the delivery efficiency, SoPIL reagents were
functionalized with fluorescence groups. The delivery of SoPIL
reagents into HeLa cells was monitored directly under a
fluorescence microscope as a function of SoPIL concentration
and incubation time with the living cells (ESI Fig. S2{). Using a
concentration of 5 mM for four hour treatment, a maximal of 80%
of cells displayed bright fluorescence signals. After 4 h incubation
of specific SoPIL reagents with cells, cells were lysed and protein
samples labeled by SoPIL reagents were recovered and analyzed
by mass spectrometry. Only a few cysteine-containing proteins
were identified (data not shown). We reason that, although we
used Cys- and Met-free media to culture the cells, an intracellular
reducing environment was still present and the SoPIL reagents
predominantly reacted with glutathione in living cells. We are
currently synthesizing SoPIL reagents with more specificity to
target a specific class of proteins.
Fig. 2 Comparison between the one-step solid phase method and the
SoPIL method. MALDI-TOF MS was used to analyze a peptide mixture
consisting of a Cys-containing peptide laminin B (m/z 967.4) and a non
Cys-containing peptide angiotensin II (m/z 1046.6). (A) Prior to the
reaction; (B) right after the addition of solid phase beads (1 min); (C) right
after the addition of the SoPIL reagent (1 min); (D) acid cleaved product
from the one-step solid phase method; (E) acid cleaved product from the
SoPIL method. The ion of m/z 1100.6 is the product after the modification
on the cysteine residue. The m/z 1046.6 ion was added for comparison.
To demonstrate the whole strategy and also compare it to the
direct solid-phase isotope tagging method, a standard peptide
mixture consisting of cysteine-containing peptide laminin B (m/z
967) and non-cysteine-containing angiotensin II (m/z 1046) was
used (Fig. 2). The solid phase reagent was synthesized in a similar
fashion to directly incorporate the acid-cleavable linker, the
isotope tag aniline, and bromoacetyl group as the thiol-specific
group on the aminopropyl controlled pore glass beads (See ESI for
detailed synthesis{). Laminin B was attached to the polymer in less
than 1 min after the SoPIL reagent was added into the peptide
mixture, and in contrast it took over 30 min for the solid phase
reagent to completely capture the peptide in the solution. Both
reactions were allowed to go to completion. The SoPIL reagent
was then captured on the azide-functionalized beads through click
chemistry. After 1 h of acid treatment, the tagged peptide was
cleaved off the SoPIL reagent and recovered into the solution
efficiently, while the recovery yield was much lower with the same
treatment of the solid phase reagent (compare Fig. 2D, 2E). Stable
isotope labeling was also used to accurately compare the yield
between two methods on the same spectrum (ESI Fig. S1{). The
yield using the SoPIL method was over 80% while the solid phase
method was less than 40%. Therefore, the data demonstrated that
the capture and release of laminin B using the SoPIL reagent was
specific and more efficient than the one-step solid phase isotopic
labeling reagent.
The SoPIL reagents for quantitative analysis were first
demonstrated with a standard protein mixture. Two mixtures
containing the same four proteins (bovine serum albumin,
a-lactalbumin, lysozyme C, and b-lactoglobulin) at different
concentration ratios were prepared and analyzed as illustrated in
Fig. 1C. The isolated labeled cysteine-containing peptides were
1252 | Chem. Commun., 2007, 1251–1253
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Table 1 A partial list of snake venom peptides analyzed
Prob
Peptide sequencea
Protein description
ASAP ratiob
1.00
0.99
0.92
0.99
D.VVVGGDECNINEH.R
Fibrinogen-clotting enzyme TL-BJ
Ancrod
Pallabin-2
2.56 (0.26)
9.68 (0.48)
1.08 (0.18)
2.12 0.22)
A.TLCAGILEGGKDTCK.E
A.DTCVGDSGGPLICNGQF.V
C.DCADIVINDLSLIHELPK.E
L-amino-acid oxidase
Phospholipase A2, acidic
Disintegrin ussuristatin-1
Crotoxin acid chain
Hemorrhagic metalloproteinase HT-E
Venom serine proteinase 3
Catrin-1\2
0.99
1.00
1.00
0.93
0.94
1.00
0.99
0.98
a
C.CFVHDCCYGK.V
D.C*TGQSADC*PR.N
D.PCGTQICECDK.A
E.FIMNQKPQCILK.K
E.HIAPLSLPSSPPSVGSACR.V
K.CGENIYMSPVPIK.W
K.FFCLSSK.N
3.54 (0.24)
0.11 (0.06)
0.54 (0.06)
0.00
1.75 (0.545)
2.34 (0.28)
4.42 (0.20)
5.30 (0.51)
Calobin
Venom serine proteinase A
b
K.FFCLSSR.N
C and C* refer to cysteine residues labeled with light or heavy SoPIL reagents, respectively. Ratio of intensities of peptides from snake
venom A and B, respectively. In parentheses are measurement errors.
the SoPIL reagents efficiently labeled multiple close-spaced
cysteine residues, a feature the solid phase method cannot achieve
due to steric hindrance. Using SoPIL reagents as the carrier of
functional groups, we demonstrated the possibility to deliver the
reagents into living cells to react with potential targets. Targeted
proteins were isolated and identified by MS in vitro. Compared to
current strategies using small chemical reagents,12 conjugating
functional groups to nanopolymers may improve their bioavail-
ability in living systems, particularly for hydrophobic molecules,
and also increases transmembrane permeation by bypassing the
cells’ efflux transporters. We expect the SoPIL strategy can be used
to discover various enzyme activities and screen specific inhibitors
for these enzymes using different reactive groups. It is also
reasonable to assume that the reagent can be used to study non-
covalent interactions such as drug/ligand-protein interactions.
This project has been funded in part by Purdue University. We
thank professor M. G. Finn in the Scripps Research Institute for
the supply of ligand for the click chemistry and for helpful
suggestions. The National Toxin Research Center at Texas A&M-
Kingsvile is also acknowledged for the supply of snake venom
samples. We acknowledge the use of software in the Institute for
Systems Biology developed using Federal funds from NHLBI,
NIH under contract No. N01-HV-28179.
Notes and references
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Fig. 3 (A) MS/MS of the peptide DC*ADIVINDLSLIHELPK derived
from L-amino acid oxidase (LAO; * refers the cysteine residues labeled by
the heavy isotope tag). Its characteristic peptide bond fragment ions, type
b and type y ions, are labeled. (B) Reconstructed ion chromatogram of the
precursor and its heavy version using ASAPRatio software.13 The
program draws a smoothed chromatogram based on the ion signal and
then calculates the ratio of the peak areas. Raw chromatograms are
plotted in red, smoothed chromatograms in blue and areas used for
calculating abundance ratio of the charge state in green.
7 W. A. Tao, B. Wollscheid, R. O’Brien, J. Eng, X. Li, B. Bodenmiller,
J. Watts, L. Hood and R. Aebersold, Nat. Methods, 2005, 2, 591–598.
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Overall, the promising data presented here clearly demonstrate
that the novel SoPIL method is simple and efficient for
quantitative proteomics. In the experiments using snake venoms
This journal is ß The Royal Society of Chemistry 2007
Chem. Commun., 2007, 1251–1253 | 1253