biomolecules

PEPscreen
An Enabling Technology for
Peptide-Based Drug Discovery

biomolecules

PEPscreen
An Enabling Technology for Peptide-Based Drug Discovery

Peptide-Based Drug Discovery

Successful drug discovery invariably involves protein studies,
because most drugs are designed either to interact with specific
target proteins, or to alter target protein-protein interactions.
Conventional one protein-one experiment strategy is time
consuming and expensive. Current approaches toward a successful
lead development and drug discovery requires high throughput
screening (HTS), that is, a fast and efficient screening of a large
number of compounds in a parallel manner. High throughput
screening is made possible as a result of the merging of three
distinct technologies.

Genomics and Proteomics High throughput genomic analysis

each other in the three-dimensional protein structure. The first step

in drug development is to map these active sequences of a protein
in a process called epitope mapping (Figure 1). The purpose is to
determine the minimum sequence of a peptide that constitutes the
active domain of the protein, thus avoiding the inherent problems of
delivering and stabilizing whole proteins as drug candidates. Once
the epitope is identified the peptide sequence is then optimized and
stabilized into a final drug product. Epitope mapping and sequence
optimization involve the use of large numbers of peptides that
constitute libraries, which are then synthesized and assayed in a
parallel, high throughput manner.

resulted in the identification of thousands of functionally important

genes, with several of the protein products not isolated or identified.
On the other hand, high throughput proteomic analysis resulted in
the isolation and identification of a large number of proteins, but
mostly with unknown functions. These dilemmas serve as driving
forces to search for high throughput systems for identifying proteins
and analyzing their structure and function relationships.

Target Protein
The sequence is known either from gene
sequencing or from protein sequencing.
Functional assay is also well-characterized.

Combinatorial Peptide Synthesis Technologies that allow

Step 1. Epitope Mapping

Synthesize overlapping peptide libraries
spanning the whole protein sequence and
perform functional assays to determine the
active sequence(s) or epitope(s).

combinatorial synthesis of large libraries of different organic

compounds are now available. Although combinatorial peptide
synthesis platforms have been in existence for some time, the
PEPscreen platform is the first truly flexible system with the ability
to incorporate non-standard amino acids, molecular dyes, isotopic
labels, varied peptide lengths, and various numbers of peptides
all in a single run. This flexibility enables synthesis of a variety
of peptide libraries and addresses drug discovery research in the
framework of high throughput, parallel screening.

Epitope Sequence
The newly-discovered peptide epitope(s)
constitute leads for peptide-based drug
development.

Step 2. Sequence Optimization

Synthesize and test peptide libraries representing
a systematic substitution of amino acids in the
lead peptide sequence(s) to optimize functionality.

Software Programming and Robotics Advances in software

programming and robotics enable automation in all aspects of

drug discovery: in genomic and proteomic analysis, in the synthesis
of peptide libraries and in high throughput functional assays.
More importantly, software programming enables fast processing
of extremely large amounts of data and customizing reports
according to the experimental design.

Optimized Sequence
The optimized sequence represents a drug
candidate, but may not yet be in its final form.

Step 3. Sequence Stabilization

Synthesize and test peptide libraries with various
conformational modifications to determine the
most stable peptide drug presentation. (In these
examples intramolecular bridges are incorporated
as one of the many different stabilization strategies).

Peptide-Based Drug Discovery

Proteins are large molecules and are usually ineffective as drugs due
to delivery and stability issues. As a large molecule, a single protein
may have multiple biological functions, where each function is
defined by localized interactions of a specific sequence of amino
acids in the protein with another protein or a non-protein ligand.
A specific sequence of the protein that represents the active site
is called an epitope. An epitope can be a continuous sequence of
amino acids or discontinuous sequences that are in close proximity to

Stabilized Sequence
Final form of peptide drug

Figure 1
General steps in peptide-based drug development. Each color represents a specific amino acid.

wherebiobegins.com/pepscreen

Design of Peptide Libraries for Epitope Mapping

The basic strategy in epitope mapping is to synthesize a peptide
library consisting of overlapping peptide sequences that together
cover the entire native protein. Designing peptide libraries is a critical
consideration in ensuring the success of the project. While the
bottom line is the eventual selection of a set of overlapping peptide
sequences of specific lengths and of specific offset number, in reality
it is a delicate balance between the cost of the entire experiment
versus the potential usefulness of the data obtained.
The offset number is analogous to the frame shift, that is, the number
of residues that the peptide sequence is shifted along the native
protein sequence.
Figure 2 is an illustration of a hypothetical sequence of a native
protein and four different strategies in selecting the sequences for a
peptide library. It is obvious from this illustration that:

The longer the peptide length, the fewer the number of peptides
to synthesize, and this is especially magnified over longer protein
sequences.

A C D E F G H

I K L M N P Q R S T V W Y

Length = 6
Offset = 2

A C D E F G H

The greater the offset number, the fewer the number of peptides to
synthesize.

The longer the peptide sequence, the more potential for multiple

hits. Hits are defined as peptide sequences that contain all of the
essential residues in the active site or epitope. The attributes for the
different combinations of peptide length and offset numbers are
summarized in Table 1.

The importance of properly choosing appropriate peptide length

and offset number can be illustrated in two extreme situations in
Figure 2. If one chooses a 10-mer peptide length at an offset of
2, at least three peptide sequences would constitute hits, or span
the epitope. Even if the epitope sequence is shifted in the protein
sequence, at least two peptides would still constitute as hits.
Obviously, six 10-mers need to be synthesized. On the other
extreme, if one chooses a 6-mer length at an offset of 4, only three
6-mers and one 8-mer need to be synthesized. However, the epitope
can potentially be missed outright, and such is the case if the
hypothetical epitope spans the sequence HIKLMN shown below.

I K L M N P Q R S T V W Y

Length = 10
Offset = 2

A C D E F G

A C D E F G H

D E F G H
F G H
H

D E F G H

I K L

F G H

I K L M N

K L M N P Q

I K L
I K L M N
I K L M N P Q
I K L M N P Q R S
K L M N P Q R S T V

M N P Q R S

M N P Q R S T V W Y

P Q R S T V
R S T V W Y

Length = 6
Offset = 4
A C D E F G
F G H

Length = 10
Offset = 4
A C D E F G H

I K L

F G H

K L M N P Q

Short Offset Number

Long Offset Number

I K L M N P Q
K L M N P Q R S T V

P Q R S T V W Y

Offset Number

I K L

P Q R S T V W Y

Figure 2
Examples of strategies in selecting sequences for peptide libraries. Residues inside the
dotted box are in the hypothetical active site or epitope. The last peptide sequence
must fulfill all three of the following requirements: (1) it must include the last residue in
the native sequence, (2) it must have more amino acids than the offset number, and (3)
it must have at least 6 residues, the minimum number of residues that can potentially
form an epitope.

Short Peptide Sequence

Long Peptide Sequence

1. Requires the most number of peptides to synthesize.

2. Shorter peptides are easier to synthesize, and often
result in higher purity.
3. More chances for multiple epitope hits.
1. Requires fewer number of peptides to synthesize.
2. Shorter peptides are easier to synthesize, and often
result in higher purity.
3. Least chance for multiple epitope hits.

1. Requires a larger number of peptides to be synthesized.

2. Longer peptides are more difficult to synthesize, and
may result in lower purity.
3. Most chances for multiple epitope hits.
1. Requires the fewest number of peptides to synthesize.
2. Longer peptides are more difficult to synthesize, and
may result in lower purity.
3. Less chance for multiple epitope hits.

Table 1
Summary of the potential effects of the different combinations between peptide length and offset number.

biomolecules

PEPscreen
An Enabling Technology for Peptide-Based Drug Discovery

It should also be noted that these strategies of epitope mapping

are more suited to mapping continuous (or contiguous) epitope
sequences. However, as previously mentioned, some epitopes are
discontinuous (or noncontiguous). In the latter case, one might be
lucky to obtain partial activities at different regions of the protein
sequence. It will take other structural and functional information
to allow the piecing together of the segments that constitute the
epitope. It will also require a different strategy of designing peptide
drugs based on non-contiguous peptide epitopes.
While ideally one would choose longer sequences and a shorter
offset number, the cost of synthesizing long peptides can potentially
Length
of Protein

100

200

400

600

800

1000

Length of Peptides
in the Library
9
12
15
18
9
12
15
18
9
12
15
18
9
12
15
18
9
12
15
18
9
12
15
18

become prohibitive. While shorter peptide length leads to more

peptide sequences to synthesize (Table 2), shorter peptides are
also more economical to synthesize. The number of peptides in
the library will depend on the length of the protein sequence. The
obvious goal is to select the minimum number of peptides that can
yield the best results. The common practice is to use 8 to 20 residues,
preferably in the 12- to16-residue range, and use an offset number
that is roughly 1/3 of the peptide length. Thus, common examples
would be 12 residues long with an offset of 4, or 15 residues with
an offset of 5. This is to ensure that at least three sequences overlap
(Figure 3).

Amino Acid
Offset Number
3
4
5
6
3
4
5
6
3
4
5
6
3
4
5
6
3
4
5
6
3
4
5
6

Table 2
Relationship between the length of the protein, the length of peptides in the library, amino acid offset number and the number of peptide sequences.

Number of Peptides
in Complete Libraries
32
23
18
15
65
48
38
32
132
98
78
65
198
148
118
98
265
198
158
132
331
248
198
165

wherebiobegins.com/pepscreen

Suggested Strategies for Selecting Peptide Library

A C D E F G H

I K L M N P Q R S T V W Y A C D E F G H

A C D E F G H

I K

E F G H
H

I K L M N P Q R S T V W Y

I K L M N
I K L M N P Q R

Length = 9
Offset = 3
No. of peptides = 12

L M N P Q R S T V
P Q R S T V W Y A
S T V W Y A C D E

W Y A C D E F G H
C D E F G H
F G H

I K L
I K L M N P
I K L M N P Q R S
M N P Q R S T V W
Q R S T V W Y

A C D E F G H
F G H

I K L M N
I K L M N P Q R S

Length = 12
Offset = 4
No. of peptides = 8

K L M N P Q R S T V W Y
P Q R S T V W Y A C D E

T V W Y A C D E F G H
A C D E F G H
F G H

I
I K L M N
I K L M N P Q R S
K L M N P Q R S T V W Y

A C D E F G H
G H

I K L M N P Q R
I K L M N P Q R S T V W Y
M N P Q R S T V W Y A C D E F

Length = 15
Offset = 5
No. of peptides = 6

S T V W Y A C D E F G H
A C D E F G H
G H

A C D E F G H
H

I K L M N P Q R S T V
I K L M N P Q R S T V W Y A C D E

I K L
I K L M N P Q R
I K L M N P Q R S T V W Y

Length = 18
Offset = 6
No. of peptides = 5

P Q R S T V W Y A C D E F G H
W Y A C D E F G H
F G H

I K L M
I K L M N P Q R S T
I K L M N P Q R S T V W Y

Figure 3
Examples of peptide libraries. Note that this 40-mer
protein is considered a very short protein. Shorter
libraries may be chosen for shorter proteins, while
longer libraries are chosen for longer proteins. Again,
note the relationship between peptide length and the
number of peptides to synthesize. The residues inside
the dotted box constitute the hypothetical epitope.

biomolecules

PEPscreen
An Enabling Technology for Peptide-Based Drug Discovery

Sequence Optimization
Once an epitope is identified the next step is to perform studies to
demonstrate structure and function relationships. These studies are
usually composed of two phases: peptide sequence optimization,
followed by structure stabilization. Ideally, sequence optimization
should be performed by synthesizing all possible sequence
combinations for a given number of residues that constitute an
epitope. Unfortunately, this is impractical because the number
of peptide sequence permutations increase exponentially with
the length of the peptide (Table 3) and it would be impossible to
individually synthesize (at least for now) all the sequences in the
peptide library. Instead, there are four practical strategies that are
used to generate alternative combinatorial libraries.
1. Alanine scanning library (Figure 4A) Alanine is systematically
substituted into each amino acid position in the previously
identified epitope. The purpose of this strategy is to identify
the amino acids in the native sequence that are essential for
activity. Thus, substitution of the essential amino acids with
alanine would be reflected as a significant reduction in activity,
and the degree of reduction in activity is usually taken as a
relative measure of the importance of the particular amino acid
being substituted. Alanine is the amino acid of choice for library
scanning because it is the smallest amino acid that maintains
chirality.
2. Truncation library (Figure 4B) This is a series of peptide
sequences representing the systematic truncation of the
flanking residues to determine the minimum length required
for optimum peptide activity. If the essential amino acids have
already been identified, the direction of truncation can be
selected around these essential amino acids, as opposed to
systematic truncation from both ends of the peptide sequence.
Peptide Length
2
3
4
5
10
15
20

Number of Sequence Permutations

400
8,000
160,000
3,200,000
10,240,000,000,000
32,768,000,000,000,000,000
104,857,600,000,000,000,000,000,000

Table 3
Relationship between peptide length and the number of possible sequence permutations for
the 20 naturally occurring amino acids.

3. Random library (Figure 4C) This is a shotgun approach where

selected residues in the peptide sequence (called the wobble
sequence) are simultaneously substituted with a mixture of all
20 amino acids, or a mixture of pre-determined amino acids. The
mixtures of random libraries are then assayed. The advantage is
synthesizing fewer peptides and, theoretically, all possible amino
acid combinations in the wobble are covered. However, these
types of libraries are only applicable in assays that involve binding
to an immobilized ligand to form a complex that can be isolated,
and where the peptides in the complex can then be identified
by sequencing. Another disadvantage is that the sequences in
the library are synthesized and processed at different efficiencies,
resulting in disproportionate abundance of each peptide in the
mixture. In practice, this strategy is usually used for preliminary
identification of a group of active sequences that can then be
re-synthesized to validate the preliminary results.
4. Positional scanning library (Figure 4D) A selected position
or positions in a peptide sequence are each systematically
replaced with different amino acids in order to determine the
preferred amino acid residues at these positions, as manifested
by corresponding increases in activity. There are variations on
how this library is constructed and the choice of the specific
strategy is usually dependent on prior information. The major
advantage is that each member of the library can be produced in
pure form and tested individually. The disadvantage is that, even
though it appears that more peptides are already synthesized,
the sequences still do not cover all possible permutations in
the wobble, since only one position is changed at a time while
the rest of the residues are held constant. However, this is still a
powerful strategy that produces high quality data, albeit a lot of
experimental work.

wherebiobegins.com/pepscreen

Sequence Stabilization
For some applications, the identification of the epitope and
optimization of the epitope sequence may constitute the end of
the investigation. However, for peptide-based drug development
the optimum peptide sequence usually does not constitute the
final drug candidate. Peptide drugs are usually chemically and
conformationally unstable in circulation and, therefore, their
structures must be stabilized to maintain their potency over time.
The most common strategy to stabilize peptide structures is to
substitute selected amino acids with non-standard amino acids.
Some examples of non-standard amino acids are either homologs of
natural amino acids such as ornithine, homolysine, norleucine, and
norvaline, or the chiral analogs (D-forms) of the naturally-occuring
amino acids (L-forms). Incorporation of these non-standard amino
acids bring about at least three different effects:

A. Alanine Scanning
Library

C. Random
Library

B. Truncation
Library

K L M N P Q

K L M N P Q

K L

X20 X20 X20

A L M N P Q

L M N P Q

K L

X20 X20

K A M N P Q

M N P Q

K L

X20

K L A N P Q

N P Q

K L M

N P Q
X20 X20

K L M A P Q

K L M N P

K L M N

K L M N A Q

K L M N

K L M

K L M N P A

K L M

X20

X20

M N P
L M N

D. Positional Scanning
Library
K L

3. Non-standard amino acids can be used to lock a preferred

structural conformation of a peptide drug candidate.
Another stabilization strategy is to incorporate intramolecular
bridges to form cyclic structures. These can be in the form of disulfide
bonds or lactam bridges of different lengths. Cyclic peptide analogs
serve at least two purposes. One is to lock the active conformation
in place, and the other is to prevent (or at least minimize) proteolytic
degradation.
Finally, peptide sequences can be stabilized through chemical
modification of the N- and C-termini. This is particularly important
in peptide sequences that are susceptible to degradation by
exopeptidases. Chemical modifications of the N- and C-termini are
commonly accomplished by acetylation and amidation, respectively.
Alternately, chemical modifications by adding more bulky groups
such as polyethylene glycols and lipids have also been used.

2. The non-standard amino acids can be substituted for amino acids

that are prone to oxidation and post-translational modification.

X20 X20 X20

K L A N P Q

K L M A P Q

K L M N A Q

K L C N P Q

K L M C P Q

K L M N C Q

K L D N P Q

K L M D P Q

K L M N D Q

K L E N P Q

K L M E P Q

K L M N E Q

K L

K L M F

P Q

K L M N F Q

K L G N P Q

K L M G P Q

K L M N G Q

K L H N P Q

K L M H P Q

K L M N H Q

K L

K L M I

K L M N

F N P Q

N P Q

P Q

I Q

K L K N P Q

K L M K P Q

K L M N K Q

K L

K L M L

P Q

K L M N L Q

K L M N P Q

L N P Q

K L M M P Q

K L M N M Q

K L N N P Q

K L M N P Q

K L M N N Q

K L P N P Q

K L M P P Q

K L M N P Q

K L Q N P Q

K L M Q P Q

K L M N Q Q

K L R N P Q

K L M R P Q

K L M N R Q

K L S N P Q

K L M S P Q

K L M N S Q

K L M
T N P Q

K L M T
N P Q

K L M N T
P Q

K L M
V N P Q

K L M V
N P Q

K L M N V
P Q

K L W
M N P Q

K L M W
N P Q

K L M N W
P Q

K L M
Y N P Q

K L M Y
N P Q

K L M N Y
P Q

Q
Q

P Q

L M N P

1. The non-standard amino acids make the peptide resistant to

proteolytic degradation since they are usually not recognized
by proteolytic enzymes.

P Q

Figure 4
Schematic representation of the different strategies in constructing peptide
libraries for sequence optimization. The presumed essential positions are enclosed
in the dotted box.

biomolecules

PEPscreen
Interest panel header
An
Enabling
for Peptide-Based Drug Discovery
Interest
panelTechnology
text

PEPscreen Custom
Peptide Libraries

PEPscreen Service
Specifications

Design your own custom peptide library for:

Library Size
Peptide Length
Quantity

Drug discovery
Vaccine development
Protein interaction studies

24 peptide minimum
6 to 20 amino acids
0.5 - 2mg or 2 - 5mg
Dried film at the bottom
of individual tubes
Free amine or acetylated
Free acid or amidated
Any commercially available
non-standard amino acid
Any mixture of commercially
available amino acids

Peptide Form
N-Terminal
C-Termini

Benefits of Using PEPscreen

PEPscreen utilizes a proprietary, state-of-the-art robotic platform
for high-throughput peptide synthesis. Using this technology
coupled with our optimized Fmoc chemistry (Figure 5), Sigma can
provide high-quality peptide libraries with:

Unsurpassed flexibility: Include various peptide lengths

and designs in a single library

Low variability: Completely automated synthesis

Non-standard Amino Acids

Random Library

Cyclization, phosphorylation,
biotinylation, PEGylation, acylation, etc.
Flc, FITC, Dansyl, Dabcyl, Dabsyl,
TAMRA, Lissamine, etc.

Chemical Modifications
Dye Labeling

eliminates errors

P2

Easy-to-use formatting: Individual tubes arrayed in 96-well

format eliminates cross-contamination and facilitates highthroughput robotic assays. Individual tubes are triple-labeled
for maximum user flexibility

P1
O

Fmoc

N
H

AA2

Fast delivery: Receive your library in less than 7 business days!

O
OH

NH AA1

Fmoc

Fmoc

Activation

Deblocking
P1

P2

O
Fmoc

N
H

AA2

H2N AA1

O
AA2

C
Fmoc

NH AA1

Resin

Final deblock
P1

P2

O
H2N

AA2

NH AA1

Resin

P2
Resin

Figure 5
Schematic representation of solid phase
peptide synthesis using Fmoc chemistry.

P1

Cleavage and
deprotection
O

O
H2N

AA2

Repeat steps
for each amino
acid addition

Resin

P1

P2

N
H

Coupling

Fmoc

Resin

NH AA1

Free peptide (Final Product)

OH

Resin

Resin

AA1

Amino Acid 1

AA2

Amino Acid 2

Fmoc

Fmoc protecting
group

Activator

P1

Side-chain
protecting group 1

P2

Side-chain
protecting group 2

wherebiobegins.com/pepscreen

Plate Format and Labeling

The peptides are dried as thin films at the bottom of individual
tubes. This is to prevent the peptide from smearing throughout the
inside of the tube during transit, which makes re-suspension in small
volumes difficult. The tubes are individually capped and arranged in
a standard 8 x 12 tube plate (Figure 6) for compatibility with high
throughput assays. This format also allows the flexibility to select
only the tubes or peptides of interest and rearrange them into a
convenient assay format.

Each tube is triple labeled with alpha-numeric, barcode, and 2D

barcode system. The alpha-numeric label is for convenient visual
identification of individual tubes. The standard barcode and 2D
barcode labels are to facilitate automated tracking, prevent
human errors, and facilitate re-sorting in case the tubes are
accidentally mixed.

Individual Tubes and Lids

r
"MMPX
JOEJWJEVBM
PS
HSPVQ
IBOEMJOH
r
1SFWFOU
DSPTTDPOUBNJOBUJPO
Individual Tubes Labels
r
"MQIBOVNFSJD
MBCFMT
BMMPX
WJTVBM



SFBEJOH
PG
JOEJWJEVBM
MBCFMT
r
#BSDPEFT
GBDJMJUBUF
BVUPNBUFE
EBUB



QSPDFTTJOH
BOE
QSFWFOU
IVNBO
FSSPST
Plate Lid Locks
r
&BTZ
PQFOJOH
BOE
DMPTJOH
r
1SFWFOU
TQJMMJOH
PVU
PG
UVCFT
Plate Label
r
"MQIBOVNFSJD
MBCFMT
BMMPX
WJTVBM



JEFOUJDBUJPO
PG
JOEJWJEVBM
QMBUFT
r
#BSDPEFT
GBDJMJUBUF
BVUPNBUFE
EBUB



QSPDFTTJOH
BOE
QSFWFOU
IVNBO
FSSPST

Individual 2D Barcodes
r
'BDJMJUBUFT
BVUPNBUFE
XIPMF
QMBUF



TDBOOJOH
UP
WFSJGZ
UVCF
QPTJUJPOT
r
&OBCMFT
JEFOUJDBUJPO
PG
UVCFT
XJUIPVU



SFNPWJOH
UIF
UVCFT
GSPN
UIF
QMBUF
SBDL

Figure 6
Packaging and labeling system used for PEPscreen peptides.

biomolecules

PEPscreen
Interest panel header
An
Enabling
for Peptide-Based Drug Discovery
Interest
panelTechnology
text

Quality Control of Peptides

of an MS-QC data is shown in Figure 7. All MS data are burned onto
a CD and supplied with the final product. HPLC analysis can also be
performed for a reasonable fee by prior arrangement.

Each peptide must pass both the MALDI-TOF Mass Spectrometry

analysis and the final gross weight criteria before they are shipped.
For a peptide to pass the MS criterion, the desired molecular mass
must be one of the three major ions by MALDI-TOF MS. An example

Mode of operation:
Extraction mode:
Polarity:
Acquisition control:

Spec #1[BP = 1600.2, 14797]

100

1600.11

Actual MW of
peptide product
(1600.11)

Total ion
count

Accelerating voltage:
Grid voltage:
1.5E+4 Guide wire 0:
Extraction delay time:

Polarity
(Positive mode)

20000 V
94.5%
0.04%
100 nsec

Acquisition mass range:

Number of laser shots:
Laser intensity:
Laser Rep Rate:
Calibration type:
Calibration matrix:
Low mass gate:

Mass
500 - 6000 Da
15/spectrum
window
1550
3.0 Hz
External - D:\Data\CHEMISTRY
a-Cyano-4-hydroxycinnamic acid
300 Da

Digitizer start time:

Bin size:
Number of data points:
Vertical scale:
Vertical offset:
Input bandwidth:

14.634
2 nsec
17859
200 mV
0%
150 MHz

Laser control:
Sample positioning:
Search pattern file:
Auto storage mode:

Automated
Automated
D:\Data\CHEMISTRY\BIC and Ca
Save best

50

Min. intensity:
Max. intensity:
Resolution:
Signal-to-noise:

4000
40000
0
20

40

Sample well:
Plate ID:
Serial number:
Instrument name:
Plate type filename:
Lab name:

A12_b
TITAN 96 x 2
1105
PE Biosystems
C:\96 well X2 plate.plt
PE Biosystems

Absolute x-position:
Absolute y-position:
Relative x-position:
Relative y-position:
Shots in spectrum:
Source pressure:
Mirror pressure:
TC2 pressure:
TIS gate width:
TIS flight length:

37428
36892
-37.0153
49.288
15
8.588e-007
0
0.07063
30
940

90

80

70

% Intensity

Linear
Delayed
Positive
Automatic

60

Relative
Intensity
30

20

10

1503.02

Product with
Val deletion
(1503.02)

0
499.0

0
1599.4

2699.8

3800.2

4900.6

Mass (m/z)

Mass-to-charge
ratio

Peptide Number
(93)

6001.0

C-term
[OH]

Acquired: 18:53:00, July 10, 2010

Sample Description: OrderNo: YYYYY; PlateNo: 1; Plate Position: 93; Seq: XXXAAAYYYBBBCCC; TheorMW: 1600; C:[OH]; N:[H], Comments: QC_CODE(Plate Label: 2VNYUY, E
\\Houclst01\Groups\Peptides\VOYAGER\TITAN\2VNYUY\93_A12_b_0001.dat

Order No.
(YYYYY)

Peptide Expected MW
Plate Position
(1600)
Sequence
Plate ID
(A12)
(XXXAAAYYYBBBCCC)
(2VNYUY)

Figure 7
Typical pdf image of MALDI-TOF MS-QC data provided. The more relevant information is highlighted. For a peptide to be deemed
correct, the molecular mass corresponding to the main peak must be within 1 part per thousand relative to the theoretical
molecular mass. Note that since MALDI-TOF generally yields P+H ion, the molecular mass is generally synonymous to the
mass-to-charge ratio (m/z).

N-term
[H]

wherebiobegins.com/pepscreen

Handling and Storage of PEPscreen Peptides

Complete solubilization of peptides is important for successful
screening of peptide activities. Peptides can be fully active only if
they are completely solubilized and are able to assume the correct
conformation for binding to their receptors. As the number of
peptides in a set increase, so does the potential solubility variation of
the peptides within the set. Therefore, in order to obtain accurate and
reliable peptide activity data, careful attention should be devoted to
the process of dissolving peptide sets.
The strategy for dissolving the PEPscreen peptide set is different from
dissolving individual peptides. For individual peptides, conditions are
chosen for optimum solubility based on the given peptide sequence.
However, for peptide sets (or peptide libraries) conditions are chosen

to dissolve as many of the peptides in the set as possible in the

first solubilization attempt. This usually involves the initial use of a
stronger solvent such as DMSO, DMF, acetic acid, etc., followed by
sonication of the peptide sample for several minutes. Depending on
how many peptides remain insoluble after this first attempt, another
solvent system may be added either to the whole set or only to the
insoluble peptides. Either way, the peptide solutions are further
diluted with more benign buffers in order to reduce or minimize
the potential effects of the strong solvents used for the initial
solubilization. Suggested solubilization strategies are schematically
described in Figure 8.

PEPscreen Peptide Sets

Option 11

Option 21

1. Add 200 ML of 50% HOAc/water

2. Sonicate for 5 minutes3

1. Add 200 ML of DMSO or DMF

2. Sonicate for 5 minutes

<90% Dissolved
1. Add 200 ML of 50% HOAc in water to all peptides4
2. Sonicate for 5 minutes

>90% Dissolved
1. Add 100 ML of ACN to
insoluble peptides5
2. Sonicate for 5 minutes

Insoluble
1. Lyophilize insoluble peptides or peptide set6
2. Dissolve in 100 ML DMSO
3. Sonicate for 5 minutes

Peptide Solution
Normalize volume or concentration
with working buffer7

Peptide Stock Solution8

Peptide Storage9

Figure 8
Flow diagram highlighting the
optimized stategies for solubilizing
peptide sets.

11

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