Single-Bead Quantification of Peptide Loading Distribution for One-Bead One-Compound Library Synthesis Using Confocal Raman Spectroscopy

Article abstract of DOI:10.1021/acs.analchem.7b00516

We report an analytical method to determine peptide loading of "one-bead one-compound" (OBOC) combinatorial peptide libraries at single-bead level. The quantification is based on a linear relationship between the amount of N-terminal amino groups on indiv

Full text of DOI:10.1021/acs.analchem.7b00516

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Article
Single-bead quantification of peptide loading distribution for One-Bead
One-Compound library synthesis using confocal Raman spectroscopy
Yuchen Tang, Yann Thillier, Ruiwu Liu, Xiaocen Li, Kit S. Lam, and Tingjuan Gao
Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 22 May 2017
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Analytical Chemistry
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Single-bead Quantification of Peptide Loading Distribution for One-
Bead One-Compound Library Synthesis Using Confocal Raman
Spectroscopy
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1,2,*
Yuchen Tang,
Yann Thillier, Ruiwu Liu, Xiaocen Li, Kit S. Lam, Tingjuan Gao,
1
College of Chemistry, Central China Normal University, Wuhan 430079, China
2
3
China Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, Wuhan 430079, China
Department of Biochemistry and Molecular Medicine, University of California at Davis, Sacramento 95817, United States
ABSTRACT: We report an analytical method to determine peptide loading of “one-bead one-compound” (OBOC)
combinatorial peptide libraries at single-bead level. The quantification is based on a linear relationship between the amount
of N-terminal amino groups on individual peptide beads and the intensity of Raman signal obtained from a specifically
designed reporter labeled on amino groups. Confocal Raman spectroscopy was employed to characterize peptide loading of
beads with defined peptide sequences and from OBOC combinatorial peptide libraries. Although amine loading of blank
TentaGel beads was found to be uniform, peptide loading among beads of OBOC peptide libraries varied substantially,
particularly for those libraries with long sequences. Construction of OBOC libraries can be monitored with this novel
analytical technique so that synthetic conditions can be optimized for the preparation of high-quality OBOC peptide
libraries. As the variability of peptide loading of individual library beads can significantly influence the screening results,
quantitative information obtained by this method will allow us to gain insight into the complexity and challenge of OBOC
library synthesis and screening.
The oneꢀbead oneꢀcompound (OBOC) combinatorial library
method has proven to be a powerful tool for identifying
synthetic compounds, such as peptides, peptidomimetics,
peptoids and small molecules, against a wide variety of
molecular targets. [1ꢀ5] The OBOC library is prepared via a
sequences appeared more than once when screening a
redundant library. [8] This latter approach, however, may
greatly decrease the diversity of the OBOC library.
One approach to minimize the number of false positive
beads is to perform orthogonal screening of the initial positive
beads at least once prior to microsequencing or decoding. [10]
However, analytical tools for singleꢀbead analysis are needed
in order to adequately address the enigma of false positive
“
splitꢀandꢀpool” strategy on resin beads where each bead
displays multiple copies of a single chemical entity. Millions
of compounds can be readily synthesized and screened
simultaneously within a few weeks. While powerful, the
OBOC technology has limitations. It is not uncommon that
some strong positive hits identified from the primary screen,
when resynthesized on beads or in solution, are no longer
positive. Some possible reasons for this enigma are: (i) errors
in decoding, (ii) minor impurity, rather than the library
compound on the bead, is the positive hit, (iii) overestimation
of binding activity of hits on positive beads with substantially
high compound loading. [6ꢀ8]
beads
in
OBOC
libraries.
Singleꢀbead
Edman
microsequencing [4, 11] allows one to quantify amino acids
released from each Edman cycle, and therefore estimate the
quality and quantity of the peptide on a single bead. However,
this method is slow, expensive and not feasible for analysis of
large population of beads. Mass spectrometry decoding of
peptides released from individual beads can be done
automatically with high speed, but the result is still not
quantitative. [12] There is a need for the development of
analytical method that allows rapid evaluation of the
distribution of peptide loading among beads within an OBOC
library. Such a singleꢀbead analytical method will have a
major impact on the quality control and application of OBOC
combinatorial libraries. In principle, the technology can also
be applied for quantifying peptide arrays on chip or
monitoring peptide derivatization of solid support. [13ꢀ15]
To face this challenge, Auer group developed a method to
label the library compound on isolated beads with a
fluorescent tag, and then use a fluorescence polarization (FP)
assay to determine their affinity for the target (protein of
interest) after they were cleaved and released from individual
beads. [9] This strategy is not feasible when the target is
difficult to produce in sufficient amount for FP, is a minor
component of a complex mixture, or is an integral membrane
protein. Kodadek and coꢀworkers designed redundant
combinatorial libraries to eliminate false hits, by proving that
the odds is extremely low for the false positives if identical
Raman spectroscopy is a wellꢀestablished, nonꢀdestructive
method for determining the chemical composition of a variety
of samples. It detects inelastic scattering of molecules undergo
vibrational, rotational, and other lowꢀfrequency modes. Due to
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this nature, Raman signals are very stable/repeatable when several investigators have applied such alkyneꢀcontaining
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experimental conditions (sample preparation, optical set up
etc.) remain the same. However Raman scattering signals are
typically very low (~10 lower), compared with fluorescent
reporters for Raman imaging of chemical compounds or
metabolites within subcellular structures. [31ꢀ35]
6
In this study, we utilized confocal Raman spectroscopy to
quantify the chemical content of individual TentaGel beads
from OBOC combinatorial libraries (Figure 1a and 1b). A
diyne molecule was prepared and used as the Raman reporter
coupled to the Nꢀterminal amino group of peptides through the
formation of an amide bond. The results present a linear
relationship between the amount of available amine on beads
and the Relative Raman intensity (RRI: intensity ratio of two
specific peaks from the Raman reporter and polystyrene,
respectively). We found that the RRI distribution shifted to the
lowerꢀloading ranges and became less uniform as the
sequences of the libraries elongated. Through this technique,
the RRI plot can be used as a unique and useful method to
assess the quality of OBOC peptide libraries, by determining
the distribution of peptide loading of individual beads among
various libraries. In the future, this method may be applied in
highꢀthroughput onꢀbead or arrayꢀbased screening of other
combinatorial libraries, by monitoring compound loading of
the solid support.
signals caused by transition between electronic states.
Confocal Raman spectroscopy provides a particular solution to
this challenge, due to the use of highꢀpower laser focusing on
a small precise volume and thus the capability of determining
chemical content from very small objects. [16] This allows
measurements on small amounts of material for analysis, such
as trace volumes of chemicals, small numbers of cells and
subcellular organelles etc. [17ꢀ22] When quantitative analysis
is desired, confocal microscopy typically utilizes a reporter
molecule possessing a specific signal peak whose intensity is
used to characterize the compound’s concentration. Unlike
confocal fluorescence microscopy, which normally exhibits
signal variations caused by photobleaching or other effects,
[23ꢀ29] Raman spectrum is much more reliable for
characterizing concentrations by measuring the intensity of a
specific Raman peak. In 2011, Sodeoka and coꢀworkers found
that alkyne molecules show an intense Raman peak in a
Ramanꢀsilent region for biological samples, and this peak is
free of interference from other Raman bands. [30] Since then,
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Figure 1. Experimental schematic of singleꢀbead quantification of peptide loading for OBOC libraries. (a) Peptide synthesis on TentaGel
beads and Raman reporter coupling to peptides synthesized on TentaGel beads. (b) Schematic of measuring Raman spectrum of an
individual bead within the laser focus using Confocal Raman Spectroscopy. (c) Raman spectra of 0.1 M Raman reporter candidates in
DMF.
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EXPERIMENTAL SECTION
Analytical Chemistry
minutes, followed by thorough wash with DMF, methanol and
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DMF before the next cycle of coupling. For OBOC library
synthesis, a typical synthetic cycle is described as follows:
beads were split into aliquots as desired, and each aliquot of
beads were coupled with a specific Fmocꢀprotected amino acid
in the presence of DIC and HOBt for 2 hours. All aliquots of
beads were then mixed together and washed with DMF 5
times. After Fmoc was removed as described above, the beads
were split again into separate containers for the next cycle of
coupling.
Materials. TentaGel S NH resin (130 ꢀm diameter, 0.29
2
mmol/g) was purchased from Rapp Polymere. Amino acid
derivatives, hydroxybenzotriazole (HOBt), and N, Nꢀ
diisopropylcarbodiimide (DIC) were obtained from multiple
companies including GL Biochem, Anaspec and ChemꢀImpex.
Methyl 4ꢀethynylbenzoate and phenylacetylene were
purchased from AK Scientific. 4ꢀCyanobenzoic acid, 2ꢀ
butynoic acid and all organic solvents were purchased from
Sigma Aldrich. The solvents were used without further
purification.
The peptide sequences with different length and library
designs are shown in Table 1. Bead_5mer, Bead_8mer,
Bead_11mer, Bead_15mer and Bead_20mer are 5ꢀmer, 8ꢀmer,
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Synthesis of peptide-beads and OBOC libraries. Peptide
and
library
beads
were
prepared
with
9ꢀ
1
1ꢀmer, 15ꢀmer and 20ꢀmer defined peptide sequences
fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide
synthesis method. [36] For peptide synthesis, a typical
synthetic cycle is described as follows: beads were coupled
with a specific Fmocꢀprotected amino acid (molar ratio 3:1 to
amine loading of TentaGel) in the presence of 3 equiv. DIC
and 3.3 equiv. HOBt for 2 hours and then thoroughly washed
with N, Nꢀdimethylformamide (DMF), methanol and DMF 3
times each, respectively. The beads were then Fmocꢀ
deprotected twice with 20% piperidine in DMF for 5 and 15
Table 1. Peptide sequences and library designs.
synthesized on TentaGel beads, respectively. Lib_8 and
Lib_10 are 8ꢀmer and 10ꢀmer peptide libraries synthesized on
TentaGel beads, respectively. These 5 peptide beads and
Lib_10 were N ꢀcapped with the Raman reporter. For Lib_8,
α
the Raman reporter was introduced to the side chain of Nꢀ
terminal Lysine. X can be any of the 19 amino acids except
Cysteine.
Peptide bead or peptide library
Bead_5mer
Peptide sequence or library design
TagꢀQPFSPꢀTentaGel
Bead_8mer
TagꢀRPWQPFSPꢀTentaGel
Bead_11mer
Bead_15mer
Bead_20mer
Lib_8
TagꢀWPYRPWQPFSPꢀTentaGel
TagꢀPAPTWPYRPWQPFSPꢀTentaGel
TagꢀQPFSPPAPTWPYRPWQPFSPꢀTentaGel
H NꢀK(ꢀTag)ꢀX X X X X X X ꢀTentaGel
2
7
6
5
4
3
2
1
Lib_10
TagꢀX X X X X X X X X X ꢀTentaGel
10 9 8 7 6 5 4 3 2 1
G: Glycine; P: Proline; A: Alanine; V: Valine; L: Leucine; I: Isoleucine; M: Methionine; F: Phenylalanine; Y: Tyrosine: W: Tryptophan;
H: Histidine; K: Lysine; R: Arginine; Q: Glutamine; N: Asparagine; E: Glutamic Acid; D: Aspartic Acid; S: Serine; T: Threonine.
ꢀ
ꢀ
Raman reporter preparation and coupling to peptides.
ꢀ(Phenylbutadiynyl)benzoic acid was synthesized through
terminal alkyne coupling. [37] For the details, a mixture of
40 mg methyl 4ꢀethynylbenzoate (4 mmol), 2.2 mL
for [M ꢀ H] , C17
H
9
O
2
, calculated 245.0608, found 245.0603.
1
4
H NMR (800 MHz, DMSOꢀd ) δ 7.98 (d, J = 7.9 Hz, 2H),
6
7.73 (d, J = 7.8 Hz, 2H), 7.66 – 7.62 (m, 2H), 7.52 (t, J = 7.4
13
6
Hz, 1H), 7.46 (t, J = 7.6 Hz, 2H). C NMR (201 MHz,
phenylacetylene (20 mmol), 2.86 mL piperidine (24 mmol),
and 426 mg copper acetate monohydrate (2.4 mmol) in 30 mL
dichloromethane (DCM) was stirred in open air at 25°C for 3
hours (monitored by TLC). The mixture was concentrated in
vacuo and the residue was purified by column chromatography
using a linear gradient of hexaneꢀethyl acetate (95:5 v/v) to
afford 1.1 g methyl 4ꢀ(phenylbutadiynyl)benzoate (85% yield,
based on the amount of methyl 4ꢀethynylbenzoate). 850 mg
methyl 4ꢀ(phenylbutadiynyl)benzoate (3.4 mmol) was then
dissolved in the mixture of methanol/tetrahydrofuran(THF)
1:1, 10 mL). 1.0 mL NaOH (10 M) was added to the mixture
and the resulting solution was stirred at room temperature for
.5 hours (monitored by TLC). The solution was concentrated
under reduced pressure, and then 20 mL of 1 M HCl was
added to the solution. The white product was filtered under
reduced pressure and washed with water and then cold
methanol. The product was dried under reduced pressure and
DMSOꢀd ) δ 166.96, 133.07, 132.98, 132.03, 130.74, 130.04,
6
129.44, 125.17, 120.59, 83.56, 81.34, 76.28, 73.65. Please
refer to the Supporting Information for more details.
For Raman reporter coupling with peptide beads and
Lib_10, the Fmocꢀprotected peptidylꢀresin was treated twice
with 20% piperidine in DMF for 5 minutes and then 15
minutes. The beads were washed with DMF for 3 times,
methanol for 3 times, and then DMF for 3 times. 4ꢀ
(Phenylbutadiynyl)benzoic acid (3 equiv.), HOBt (3 equiv.),
DIC (3.3 equiv.) were dissolved in DMF to make 0.2 M
reactant. The mixture was shaken at room temperature for 5
minutes. The activated mixture was added to resin and shaken
for 2 hours. Completion of the coupling reaction was
monitored by Kaiser test. The reaction solution was removed
by filtration, and the resin was then washed again with DMF
for 3 times, methanol for 3 times, and then DMF for 3 times.
For Lib_8, 4ꢀ(phenylbutadiynyl)benzoic acid was introduced
to the side chain of a Nꢀterminal Lys. As will be seen below, a
(
1
4
ꢀ(phenylbutadiynyl)benzoic acid (701 mg, 84% yield) was
obtained as a white solid. Mp 295ꢀ297 C. HRMS (ESI) ꢀ m/z
°
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free Nꢀterminus has allowed us to perform automatic Edman relative Raman intensity (RRI) by normalizing raw intensity of
ꢀ
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microsequencing on beads isolated from this library.
the peak at 2232 cm against that at 1002 cm (a
characteristic peak of polystyrene). Using this method, we
were able to avoid influence from small fluctuations of
measurement volume, resolution of the system etc. The
calculated mean RRI and their standard deviation (SD) are
listed in Table 2. Figure 2c shows brightꢀfield images at
different x, y positions (z stayed at the center). Their mean
RRI and SD are listed in Table 3. From the data, consistent
RRI were found at different positions of the same bead. This
result confirms that 4ꢀ(phenylbutadiynyl) benzoic acid was
homogeneously labeled to different regions within a single
bead.
Confocal Raman spectroscopy and peptide sequencing.
All measurements were made using the homeꢀbuilt confocal
Raman spectroscopy system. Briefly, 25 mW of light from a
single mode, 785 nm laser (CrystalLaser, Reno, NV), was
coupled into an inverted microscope (IXꢀ71, Olympus, Center
Valley, PA) outfitted with a 60X, 1.2ꢀNA water immersion
objective. The light was focused by the objective to a
diffractionꢀlimited spot (∼1×1×3 ꢀm in size). The laser excited
Raman scattering from the confocal volume, and backscattered
light was collected back through the objective. A dichroic
beamꢀsplitter and edge filter (Chroma Technology, Bellows
Falls, VT) separated the Raman scattered light from the
backscattered excitation light. The filtered Raman signal was
focused into a multimode optical fiber where it was delivered
to a SpectraPro 2300i spectrograph (Princeton Instruments,
Trenton, NJ). The dispersed spectrum was measured by a TEꢀ
cooled Pixis 100 CCD (Princeton Instruments).
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Before measurement, compounds were dissolved in DMF,
or beads were suspended in DMF. Then a glassꢀbottom petriꢀ
dish containing 10 ꢀL of sample was put above the objective.
The laser focus was levitated away from the glass bottom to
reduce the signal strength of the substrate. Normally we
focused in the compound solution, typically ~10 ꢀm above the
glass surface, or focused at the center of TentaGel beads,
typically ~65 ꢀm above the glass surface. Five 2ꢀsecond
spectral frames were collected for each sample in order to
acquire data with acceptable signalꢀtoꢀnoise ratios. All spectra
were background corrected by subtracting off autoꢀ
fluorescence and other background deviations. [38] The 5
frames from each bead were averaged for an equivalent
integration time of
2 seconds. For microsequencing,
representative Beads with low and high Raman signals were
picked from libraries, and then sequenced using a previously
reported automated method. [11]
RESULTS AND DISCUSSION
Validation of Raman reporter labeling. We selected 4
Raman reporter candidates and measured their Raman spectra
in solution and on beads (Figure 1c). 4ꢀ(Pehenylbutadiynyl)
benzoic acid is not soluble and had to be made as 4ꢀ
(phenylbutadiynyl) benzoate for Raman characterization. As
shown in Figure 1c, peak intensity generated from diꢀaromatic
ꢀ1
substituted diyne (ArꢀC≡C−C≡CꢀAr, 2232 cm ) is much
higher than that from aromatic substituted alkyne (ArꢀC≡CꢀH,
ꢀ1
ꢀ1
2
115 cm ), aromatic substituted nitrile (ArꢀC≡N, 2244 cm )
and aliphatic substituted alkyne (RꢀC≡Cꢀ, peak very low).
Figure 2a shows the Raman spectra of TentaGel beads
derivatized with each of these 4 Raman tags. It is clear that 4ꢀ
(phenylbutadiynyl) benzoic amide is a more qualified reporter
than 4ꢀethynylbenzoic amide, 4ꢀcyanobenzoic amide, and 2ꢀ
Figure 2. Characterization of Raman reporter labeling on beads.
(a) Spectra of 4 Raman reporter candidates coupled to TentaGel
beads, compared with spectrum of blank TentaGel beads
(Bead_TentaGel). (b) Brightꢀfield images of a labeled bead at
different z foci (x and y focused at the center of the bead where
the white crosses are) and their corresponding RRI (Table 2). RRI
ꢀ
1
butynoic amide, with a significantly intense peak at 2232 cm .
We therefore selected 4ꢀ(phenylbutadiynyl) benzoic acid as
the Raman reporter for the work presented in this paper.
In order to demonstrate the homogeneity of coupling
efficiency on beads, we focused at different areas of the same
bead and measured their spectra. Figure 2b shows brightꢀfield
images of an example bead when the focus was at 5 different z
positions (x, y dimensions were centered). We calculated the
ꢀ1
is the peak intensity at 2232 cm (left arrowꢀpointed peak in
ꢀ1
Figure 2a) normalized against that at 1002 cm (right arrowꢀ
pointed peak in Figure 2a). 0 is the image of laser beam. 1~5 are
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Analytical Chemistry
images focused sequentially from up to down across the zꢀ
foci (z focused at the center of the bead) and their corresponding
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direction of the bead. 3 is focused exactly at the center of the
bead. The focus position of each image is different in a few
micrometers. (c) Brightꢀfield images of a bead at different x, y
RRI (Table 3). The white crosses highlight positions of x, y foci.
Part of the bead edges are highlighted with dotted red lines, in
order to clarify the location of the bead.
Table 2. Measured RRI at 5 different z-position foci.
zꢀfocus
1
2
3
4
5
4
.19
4.16
4.10
4.12
4.13
RRI
±
0.053 ±0.082 ±0.068 ±0.058 ±0.024
Table 3. Measured RRI at 9 different x, y-position foci.
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4.03
4.11
4.20
RRI
±0.077
±0.074
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±0.066
±0.087
±0.086
±0.11
Coupling efficiencies of 4-(phenylbutadiynyl) benzoic
acid to different amino acids. To determine the efficiency of
coupling 4ꢀ(phenylbutadiynyl) benzoic acid to terminal amino
group of amino acids on beads, we first coupled 11 different
Fmocꢀamino acids to TentaGel beads. Following Fmocꢀ
deprotection, the Nα amine was coupled with 4ꢀ
Pro, Lys (N ꢀ and N ꢀ); somewhat high (~94%) for Aib
α ε
(probably due to steric hindrance); and modest (~85%) for 3ꢀ
Abz (due to weak reactivity of aromatic amine). Interestingly,
protected amino acid, Cys(Mbzl) and Tyr(tꢀBu), yielded even
higher RRI (>100%) than the control. The extra signal might
result from small amount of coupling with unprotected ꢀSH
and –OH presented in the starting material or generated during
the reaction process. These results indicate that coupling of the
Raman reporter with aliphatic primary amine and cyclic
secondary amine (e.g. proline) gives accountable efficiency,
and thus the proposed singleꢀbead Raman spectroscopy
analysis will enable us to quantify peptide loading on
individual peptide beads within an OBOC library.
(phenylbutadiynyl) benzoic acid and then quantified with
confocal Raman spectroscopy. To normalize the data, we
derivatized blank TentaGel beads with 4ꢀ(phenylbutadiynyl)
benzoic acid, determined the RRI, and used it as the control
for 100% coupling. As shown in Table 4, coupling efficiency
of the Raman reporter to the aminoꢀacid derivatized bead was
highest (≥99%) for Gln, Leu, and Val; high (≥95%) for Ala,
Table 4. Measured RRI of coupling different amino acids.
Amine
groups
Bead
amine
Cys
(MBzl)
Lys
(Ac)
Acꢀ
Lys
Tyr
tꢀBu)
3ꢀAbz
Aib
Ala
Gln
Leu
Pro
Val
(
Relative
Raman
Intensity
4
.16
3.53
3.89
3.95
4.31
4.10
4.13
4.02
4.03
4.00
4.29
4.10
±
0.077 ±0.020 ±0.063 ±0.065 ±0.049 ±0.028 ±0.031 ±0.082 ±0.022 ±0.050 ±0.013 ±0.024
Coupling
efficiency
1
00%
85% 94% 95% 104% 99% 99% 97% 97% 96% 103% 99%
3ꢀAbz: 3ꢀaminobenzoic acid; Aib: αꢀaminoisobutyric acid; Ala: alanine; Cys (Mbzl): Sꢀ(4ꢀmethylbenzyl)ꢀcysteine; Gln: glutamine; Leu:
leucine; Lys (Ac): N ꢀacetylꢀlysine; AcꢀLys: N ꢀacetylꢀlysine; Pro: proline; Tyr (tꢀBu): Oꢀt-butylꢀtyrosine; Val: valine.
ε
α
Dynamic range of amine amount and peptide loading
quantification. In order to validate our idea of quantifying
peptide loading of individual beads, TentaGel amine was
reacted with a series of solutions containing mixtures of
FmocꢀAla and BocꢀAla (molar ratio: 0:100, 2:98, 5:95, 10:90,
20:80, 50:50, 75:25, 100:0), so that the percentage of free Nαꢀ
Ala on the beads was capped with 4ꢀ(phenylbutadiynyl)
benzoic acid. The Raman spectra of the 8 bead samples were
obtained. For each sample we randomly picked 5 beads, and
for each bead, we measured 5 frames (2ꢀsec laser exposure for
each frame). The averaged spectra (each averaged from the 5
frames) are shown in Figure 3a.
amine can be tuned. After Fmoc deprotection, the N amine of
α
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Figure 3. Quantitative measurements of 4ꢀ(phenylbutadiynyl) benzoic acid coupled to TentaGel Ala with different amounts. (a)
Representative spectra of beads after 4ꢀ(phenylbutadiynyl) benzoic acid was coupled with 100%, 75%, 50%, 20%, 10%, 5%, 2% and 0%
Ala on bead. For comparison, the reference is the curve of blank TentaGel beads (Bead_TentaGel) without 4ꢀ(phenylbutadiynyl) benzoic
acid coupling. (b) The calibration curve of RRI versus the expected molar percentage of available amine (based on ratio of FmocꢀAla and
BocꢀAla). Error bars represent the standard deviation of data from 5 randomly selected beads. (c) The curve of raw intensity at 2232 cm
versus the expected molar percentage of available amine.
ꢀ1
Figure 3b shows a plot of RRI versus expected molar
percentage (EMP) of available amine for coupling. Each data
point represents the average RRI of 5 randomly selected
beads, and the error bar is plotted as the SD of these 5 parallel
measurements. There was a small RRI (0.078, nonzero) at zero
EMP, possibly caused by small amount of nonꢀspecific
adsorption/reaction of the Raman reporter to impurities on
beads. However, this small effect does not affect the quality of
quantitative analysis of the calibration curve. Compared with
the analysis based only on the raw intensity of peak at 2232
Raman reporter (Bead_O; 100% coupling) as the control. The
same batch of blank TentaGel beads were used to synthesize 5
peptide beads for comparison, representing defined 5ꢀmer, 8ꢀ
mer, 11ꢀmer, 15ꢀmer and 20ꢀmer sequences, respectively
(Table 1). For each of these bead samples, 100 beads were
randomly picked and analyzed under the confocal Raman
microscope. For each measurement, 5 frames of 2ꢀsec spectra
were taken and averaged to produce one spectrum. The
distribution of backꢀcalculated peptide loading in percentage is
shown as the histogram graph (number of beads vs. peptide
yield, Figure 4).
ꢀ
1
cm in Figure 3c, the adjusted coefficient of determination
2
(adj. R , indicator of the accountability of data fitting) is
significantly increased from 0.988 to 0.996 using the
normalization method. The SD of data points are kept at a low
level of a few percent, e.g. less than 1% at some data points
(
Figure 3b).
The fitted curve in Figure 3b presents a linear relationship
between RRI and EMP (RRI = 0.039 × EMP + 0.182) Using
.
this linear relationship, we can easily deduce the quantitative
yield of peptides synthesized on TentaGel beads. Assuming
that the TentaGel beads have uniform amine density of 0.29
mmol/g (reported from the commercial provider), and the
coupling efficiency for reaction between 4ꢀ(phenylbutadiynyl)
benzoic acid and Nꢀterminal amine is ∼100%, final peptide
loading on TentaGel beads can then be backꢀcalculated and is
equal to (RRI – 0.182) / 0.039 × 0.029 mmol/g.
Figure 4. Peptide loading distributions of 100 randomly selected
beads from 5 different samples of peptide beads with defined
sequences (Bead_5mer, green; Bead_8mer, red; Bead_11mer,
blue; Bead_15mer, orange; Bead_20mer, grey), compared with
TentaGel amine distribution (Bead_O, purple). X axis is the
peptide loading represented in percentage (peptide yield). Y axis
Single-bead
quantification
of
peptide
loading
distribution for peptide beads. The results shown above
indicate that it is feasible to determine the yields of peptides
synthesized on TentaGel beads, using 4ꢀ(phenylbutadiynyl)
benzoic acid as the Raman reporter attached to the N terminus
of peptides. We used blank TentaGel beads coupled with the
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hypothesis, we synthesized Lib_8 and Lib_10, which
is the number of beads corresponding to their specific peptide
loading. The bin size is 2%.
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represents 2 libraries of 8ꢀmer sequences and 10ꢀmer
sequences, respectively. For Lib_10, the N amine of the Nꢀ
α
Two major observations have emerged from this study.
First, as the target peptide sequence gets longer, its peptide
loading decreases. The peptide loading maxima of
Bead_5mer, Bead_8mer, Bead_11mer, Bead_15mer and
Bead_20mer were determined to be 85%, 82%, 27%, 19% and
terminus of the peptide library was capped with the Raman
reporter. For Lib_8, the Raman reporter was coupled to the Nε
amine of Lys placed at the Nꢀterminus of the peptide library.
The result on distribution of peptide loading among library
beads is shown in Figure 5a. The tight distribution of TentaGel
beads (Bead_O) indicates that the initial amine loading of the
starting solid support is quite uniform. In contrast, both
libraries (Lib_8 and Lib_10) exhibited a wide spread of
peptide loading, ranging 3~10 times differences from the
lowest to the highest loading. This indicates that coupling rates
vary greatly along the reaction cycles during peptide synthesis,
as a result of the diversity of peptide sequences within an
OBOC library. For example, it is well known that some
peptide sequences are difficult to make, and special strategies
have to be developed to make those peptides. [39ꢀ41]
1
7%, respectively. Second, the decrease of peptide loading is
not linear to the decrease of the sequence length. As the 8ꢀmer
sequence was synthesized by adding RPW to the 5ꢀmer
sequence, the 11ꢀmer, 15ꢀmer and 20ꢀmer sequences were
synthesized using the same strategy by adding more amino
acids to the 8ꢀmer, 11ꢀmer and 20ꢀmer sequences, respectively
0
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4
5
6
7
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9
0
(
Table 1). Compared to that Bead_5mer and Bead_8mer
demonstrate high yields (narrow distribution), Bead_11mer
starts to have dramatically lower yield with broader
distribution after adding WPY to Bead_8mer. Bead_15mer
and Bead_20mer show relatively reasonable yield decrease
compared with Bead_11mer. Assuming the coupling
efficiency is the same during the process of adding amino
acids for each sequence synthesis, based on the Raman
quantification result, after the 1ꢀ5th, 6ꢀ8th, 9ꢀ11th, 12ꢀ15th and
To further explore the phenomena of wide distribution of
peptide loading in OBOC libraries, we compared the peptide
sequences of the highꢀyield beads and lowꢀyield beads. An Nꢀ
terminal Lys was added to Lib_8 and then conjugated with the
1
8
6ꢀ20th cycles of amino acid coupling, the final yields were
5%, 82%, 27%, 19% and 17%, respectively as reported
Raman reporter through the N amino group of the Lys,
ε
leaving the N amino group free for the subsequent Edman
α
above. Then the coupling efficiency of each cycle for adding
the 1ꢀ5th, 6ꢀ8th, 9ꢀ11th, 12ꢀ15th, and 16ꢀ20th amino acids can
microsequencing. Singleꢀbead analysis with Edman chemistry,
not only allows us to read peptide sequence, but also provide
information on peptide quality via “preview analysis”. Twelve
representative highꢀloading and lowꢀloading beads (6 beads
from each group) were picked for automatic Edman
microsequencing. The result is shown in Figure 5b. All of
them have unambiguous signals in sequencing HPLC
chromatogram except the one with the lowest yield (see the
Supporting Information). None of these 12 beads have
identical sequences. Their difference in sequences may be one
of the factors causing variance in each cycle of amide
formation and eventually resulting in the yield variance. Not
unexpected, the sequence/bead with the lowest peptide loading
identified from Raman characterization (K’YRARRRR:
5
3
3
be backꢀcalculated as 0. 85
,
0. 82 / 0. 85
. 19 / 0. 27 , and 5 0. 17 / 0. 19 , respectively. The results
,
0. 27 / 0. 82
,
4
0
are 97%, 99%, 69%, 92% and 98%, respectively. This
ratiocination indicates that while adding the 9ꢀ11th amino
acids, a significant fraction of the peptides had their Nꢀtermini
either physically or chemically blocked, so that WPY coupling
and the subsequent Raman tag coupling can no longer occur
efficiently.
Single-bead
quantification
of
peptide
loading
distribution for peptide libraries. Considering an OBOC
library including a large number of beads/peptides, there is
high possibility of yield variance among beads, resulting in
variance of peptide loading distribution. To confirm this
8
.51%), had the lowest signal during Edman microsequencing
as well.
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Figure 5. Peptide loading distributions and peptide sequences of OBOC libraries. (a) Peptide loading distributions of 100 randomly
selected beads from OBOC peptide libraries (Lib_8, orange; Lib_10, red), compared with TentaGel amine distribution (Bead_O, purple).
X axis is the peptide yield (loading represented in percentage). Y axis is the number of beads corresponding to their specific peptide
loading. The bin size is 2%. (b) Peptide sequences of representative beads with low yield and high yield from Lib_8.
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No matter what happens on beads during library synthesis,
our results imply that a library may inevitably include beads
with very high and very low peptide loading, especially for
large libraries with long peptide sequences. Unlike the
synthesis of a specific peptide with unique sequence in which
each coupling cycle can be optimized for maximal yield,
OBOC library synthesis is much more challenging. Using
standard ninhydrin test [42] to monitor completion of coupling
at each reaction cycle may not be enough. The singleꢀbead
analytical method described here is expected to be useful for
optimizing conditions for the preparation of highꢀquality
OBOC combinatorial peptide libraries, with assessing
quantitative information of (i) high peptide loading and (ii)
narrow distribution of peptide loading among library beads.
distributions, while the loading distributions of peptide
libraries become even broader. These findings suggest that the
inhomogeneity of peptide loading from bead to bead has to be
taken into account when the synthetic quality has to be
monitored while or after the library synthesis is performed.
For example, if the subsequent onꢀbead library screening is
performed without the verification of the synthetic quality, it
will potentially happen that false positive beads are identified
or true positive beads are missed due to substantially high or
low peptide loading, respectively. Our method enables singleꢀ
bead determination of peptide loading within an OBOC
library, and therefore can be used for quality control of library
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construction using two standard parameters
ꢀ loading
maximum and distribution width. As long as the loading
maximum is reasonably large and the distribution width is
reasonably small, the synthesized library can pass the quality
assessment and be used for the subsequent screening studies.
CONCLUSIONS
We have developed a quantitative method capable of
determining peptide loading at singleꢀbead level. Bead
samples from OBOC libraries labeled with a sensitive Raman
reporter at the Nꢀterminus can be measured with confocal
Raman spectroscopy. A calibration curve of RRI (the Raman
intensity ratio between two specific peaks) vs. peptide loading
is generated so that we can use the linear relationship to backꢀ
calculate peptide yield at singleꢀbead level. This will allow us
to obtain more direct and relevant information during the
progress of OBOC library construction without the need to
cleave peptides off beads for analysis. Compared with offꢀ
bead quantification methods, our onꢀbead analysis can
efficiently monitor the quality of OBOC library construction,
especially when the subsequent library screen is performed on
beads.
Moreover, the data acquisition of this method is based on
measurement of confocal Raman microscopy, the entire
analytical process can potentially be automated using a
mechanical stage. As a result, hundreds to thousands of library
beads can be interrogated within a relatively short time. To
extend the application, the method may also be used for
quantifying microarrayꢀbased peptide screening using the
same principle.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Characterization data of Raman reporter molecules; HPLC elution
results for sequencing beads from Lib_8.
We use 4ꢀ(phenylbutadiynyl) benzoic acid as the Raman
reporter molecule. This type of molecule includes a core
structure of bisphenylꢀsubstituted diynes that has a strong
AUTHOR INFORMATION
ꢀ
1
Raman peak at ~2232 cm . This peak lies in a Raman silent
region where most of Raman signatures of TentaGel and
peptides are absent (they present at wavenumbers either lower
Corresponding Author
*Email: tingao@mail.ccnu.edu.cn
ꢀ1
ꢀ1
than ~1800 cm or higher than ~2700 cm ). We introduced
the Raman reporter to the Nꢀterminus of various amino acids
using the highly efficient amide coupling method. In order to
avoid derivatizing the Lys side chains with the Raman
reporter, tagging reaction has to be performed prior to peptide
deprotection, i.e. prior to screening. In some cases, it may be
desirable to introduce the Raman reporter after screening, i.e.
after sideꢀchain deprotection. This can be easily achieved by
introducing cysteine to the Nꢀterminus of the positive peptides
identified from screening, followed by the subsequent ligation
of the Raman reporter via glyoxylylꢀbased conjugation. [43,
ACKNOWLEDGEMENT
This work is supported by Central China Normal University startꢀ
up fund 210ꢀ31102 to TG and NIH R33CA196445 to KSL. The
authors thank Zachary James Smith from the University of
Science and Technology of China for helpful discussion in
understanding Raman spectra data. The authors also
acknowledge the UC Davis Campus Mass Spectrometry facilities
and the NMR spectrometer purchased by US NSF grant
DBIO722538.
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