As epitope mimics, mimotopes have been widely utilized in the study of epitope prediction and the development of new diagnostics, therapeutics, and vaccines. Screening the random peptide libraries constructed with phage display or any other surface display technologies provides an efficient and convenient approach to acquire mimotopes. However, target-unrelated peptides creep into mimotopes from time to time through binding to contaminants or other components of the screening system. In this study, we present SAROTUP, a free web tool for scanning, reporting and excluding possible target-unrelated peptides from real mimotopes. Preliminary tests show that SAROTUP is efficient and capable of improving the accuracy of mimotope-based epitope mapping. It is also helpful for the development of mimotope-based diagnostics, therapeutics, and vaccines.
In 1985, Smith pioneered phage display technology, an in vitro methodology and system for presenting, selecting and evolving proteins and peptides displayed on the surface of phage virion [
Powered by phage display technology, mimotopes can be acquired in a relatively cheap, efficient and convenient way, that is, screening phage-displayed random peptides libraries with a given target. However, not all phages selected out are target-specific, because the target itself is only one component of the screening system [
The results from phage display technology might be a mixture of target-unrelated peptides and mimotopes, and it can be difficult to discriminate TUP from mimotopes since the binding assays used to confirm the affinity of peptides for the target often employ the same components as the initial panning experiment [
In this study, we describe a web server named SAROTUP, which is an acronym for “Scanner And Reporter Of Target-Unrelated Peptides”. SAROTUP was coded with Perl as a CGI program and can be freely accessed and used to scan peptides acquired from phage display technology. It is capable of finding, reporting, and precluding possible target-unrelated peptides, which is very helpful for the development of mimotope-based diagnostics, therapeutics, and vaccines. The power and efficiency of SAROTUP was also demonstrated by preliminary tests in the present study.
Recently, Menendez and Scott reviewed a collection of target-unrelated peptides recovered in the screening of phage-displayed random peptide libraries with antibodies [
Known patterns of target-unrelated peptides.
TUP Category | TUP Pattern | Mechanism in brief |
---|---|---|
Capturing agents | H-P-[QM], G-D-[WF]-x-F, W-x-W-L, E-P- | Binding to streptavidin |
D-W-[FY], D-V-E-x-W-[LIV] | ||
W-x-P-P-F-[RK] | Binding to biotin | |
W-[TS]-[LI]-x(2)-H-[RK] | Binding to Protein A | |
R-T-[LI]-[TS]-K-P, [LFW]-x-F-Q, W-I-S- | Binding to secondary antibody | |
x(2)-D-W, Q-[LV]-[LV]-Q, RTYK | ||
Constant region of antibody (the target) | S-S-[IL], GELVW, G-[LI]-T-D-[WY], | Binding to the Fc fragment |
[RHK]-P-S-P, P-S-P-[RK] | ||
Screening solid phase | W-x(2)-W, WHWRLPS, F-H-x(2)-W | Binding to plastic |
Contaminants in the target sample | F-H-E-x-W-P-[ST] | Binding to contaminant bovine serum albumin |
QSYP | Binding to contaminant bovine IgG | |
Phage mutation | HAIYPRH | Growing faster than other phages |
The SAROTUP was implemented as a free online service, powered by Apache and Perl. Three pages are designed and integrated into a tabbed web interface with cascading style sheets codes. The core program of SAROTUP was
We constructed two-test data sets from [
A summary of the first test data set for SAROTUP.
Target | Template | Complex | Peptides | Source |
---|---|---|---|---|
17b | HIV gp120 envelope glycoprotein (gp120) | 1GC1 | 11 | [ |
trastuzumab | human receptor tyrosine-protein kinase erbB-2 (HER2) | 1N8Z | 5 | [ |
82D6A3 | human von Willebrand factor (vWF) | 2ADF | 5 | [ |
13b5 | HIV-1 capsid protein p24 | 1E6J | 14 | [ |
BO2C11 | human coagulation factor VIII | 1IQD | 27 | [ |
cetuximab | human epidermal growth factor receptor | 1YY9 | 4 | [ |
80R | SARS-coronavirus spike protein S1 | 2GHW | 42 + 18 | [ |
b12 | HIV gp120 envelope glycoprotein (gp120) | 2NY7 | 2 + 32 + 19 | [ |
The second data set is composed of 100 peptides in raw sequence format. It has two groups. The first group has 77 sequences compiled from the first data set without any known TUP motifs; the second group has 23 sequences sourced from [
As a free online service, the web interface of SAROTUP has successfully been implemented as a tabbed web page. The left tab is the default page, providing a brief introduction to this web service. The right tab is a more detailed help page. Click the middle tab will display a web form. The upper section of the form is for basic input (Figure
Snapshot of the upper section of SAROTUP.
The lower section of the form has a series of options (Figure
Snapshot of the lower section of SAROTUP.
After the users submit their request, the scanning results of SAROTUP will be displayed on the middle tabbed page. If any target-unrelated peptides are found, they will be reported in a table. At the same time, a new panel of peptides excluding target-unrelated peptides is produced and can be downloaded from the hyperlink created by the SAROTUP server (Figure
Snapshot of SAROTUP result page. Target-unrelated peptides in the b12 test case are reported in the table. The new panel of peptides excluding the target-unrelated peptides can be downloaded from the hyperlink.
We have tested SAROTUP on the Internet Explorer (version 6.0), Mozilla Firefox (version 3.5.2), and Google Chrome (version 3.0). Although SAROTUP looks a little bit different among different browsers, it works normally on all browsers tested.
As shown in Table
Target-unrelated peptides in the first test data set.
Target | Target-unrelated peptides | Mechanism |
---|---|---|
cetuximab | V | Binding to plastic |
80R | CE | Binding to the Fc fragment |
YSTP | Binding to the Fc fragment | |
b12 | NLRSTSFFEL | Binding to plastic |
N | Binding to plastic | |
N | Binding to plastic | |
ICFPFNTRYCIFAMMV | Binding to the Fc fragment |
For the above 3 cases, the genuine epitopes recognized by cetuximab, 80R, and b12 monoclonal antibodies are compiled according to the CED records [
Mimotope-based epitope prediction with or without SAROTUP procedure.
Target | Prediction without SAROTUP procedure | Genuine epitope | Prediction with SAROTUP procedure |
---|---|---|---|
cetuximab | N134, E136, S137, I138, Q139, W140, R141, Q164, K185, L186, T187, K188 | P349, R353, L382, Q384, Q408, H409, Q411, F412, V417, S418, I438, S440, G441, K443, K465, I467, S468, N469, G471, N473 | K375, I401, R403, R405, T406, K407, |
80R | H445, V458, P459, F460, S461, P462, D463, G464, K465, P466, C467, T468, | R426, S432, Y436, K439, Y440, Y442, P469, P470, A471, L472, C474, Y475, W476, L478, N479, D480, G482, Y484, T485, T486, T487, G488,Y491, Q492 | L443, R444, H445, I455, S456, N457, V458, P459, F460, S461, P462, D463, G464, K465, P466, C467, T468, |
b12 | I108, C109, S110, L111, D113, Q114, S115, L116, K117, P118, C119, V120, P206, K207, V208, S209, F210, E211, P212, I213, P214, I251, R252, P253, I424, N425, M426, W427, C428, K429, | N280, A281, S365, G366, G367, D368, P369, I371, V372, T373, Y384, N386, P417, R419, V430, G431, K432, T455, R456, G472, G473, D474, M475 | W95, T232, F233, N234, T236, S257, L260, N262, G263, S264, L265, A266, E267, E268, E269, V270, V271, T290, S291, S364, |
As shown in Table
We also scanned the second data set to evaluate the efficiency of SAROTUP. The second data set has 100 peptides, varying from 6 to 22 residues long. Suppose that matching each pattern to each peptide manually costs 10 seconds, then it would take a researcher more than 6 hours (23,000 seconds) to look through the second data set for target-unrelated peptides, even if he is as prompt during the whole period. However, it took only one second for SAROTUP to complete this work. Besides, a table of target-unrelated peptides and a new panel of peptides excluding TUP was produced at the same time by SAROTUP. It is true that some target-unrelated peptides can be identified through control and binding competition experiments. However, using SAROTUP first will certainly save a lot of labor, money, and time for researchers in this area.
Although the target of all tests described previously were monoclonal antibodies, SAROTUP can be customized and used in scanning the results from phage display technology using other targets such as enzymes and receptors. This is because their screening systems are similar. For the same reason, we can also expect that SAROTUP will extend its use to other similar in vitro evolution techniques, such as ribosome display [
Furthermore, SAROTUP will not only benefit the mimotope-based epitope mapping, but also the development of new diagnostics, therapeutics, and vaccines. Target-unrelated peptides are not appropriate candidates for mimotope based diagnostics, therapeutics, and vaccines, since they are mimics to components or contaminants of the screening system rather than target. Therefore, it is reasonable to find and exclude possible target-unrelated peptides from the candidate list of new diagnostics, therapeutics, and vaccines. Take the cetuximab as an example. Riemer et al. screened a phage-displayed random peptides library with the cetuximab and got four different peptides, that is, QFDLSTRRLK, QYNLSSRALK, VWQRWQKSYV, and MWDRFSRWYK [
SAROTUP must be used with caution since it is a tool only based on pattern matching at present. There are a lot of target-unrelated peptides bearing no known motifs [
It is also possible that a SAROTUP predicted target unrelated peptide is actually target-specific. To decrease this kind of false positives, the users should customize the scan according to their experiment at the section of advance options. For example, the user should select “antibody without Fc fragment” as the target if Fab was used in biopanning; this will prevent SAROTUP from reporting peptides bearing the Fc-binding motifs as TUP. As described above, SAROTUP in future will also provide an exact match tool based on database search. In this way, a match might mean that different research groups have isolated the same peptide with a variety of targets. It is obvious that this peptide can hardly be a true target binder. Thus, the false positive rate of SAROTUP can be decreased further when its new feature become available.
At last, we must point out that the controlled experiment is still the gold standard to distinguish TUPs from the specific mimotopes. The report of SAROTUP should be verified with experiment.
SAROTUP, a web application for scanning, reporting and excluding target-unrelated peptides has been coded with Perl. It helps researchers to predict epitope more accurately based on mimotopes. It is also useful in the development of diagnostics, therapeutics, and vaccines. To our knowledge, SAROTUP is the first web tool for TUP detecting and data cleaning. It is very convenient for the community to access SAROTUP through
The authors are grateful to the anonymous reviewers for their valuable suggestions and comments, which have led to the improvement of this paper. This work was supported in part by the National Natural Science Foundation of China under the Grant 30600138 and the Scientific Research Foundation of UESTC for Youth under the Grant JX0769.