Peptide Arrays for Screening Cancer Specific Peptides

Abstract

In this paper, we describe a novel method to screen peptides for specific recognition by cancer cells. Seventy peptides were synthesized on a cellulose membrane in an array format, and a direct method to study the peptide-whole cell interaction was developed. The relative binding affinity of the cells for different peptides with respect to a lead 12-mer p160 peptide, identified by phage display, was evaluated using the CyQUANT fluorescence of the bound cells. Screening allowed identification of at least five new peptides that displayed higher affinity (up to 3-fold) for MDA-MB-435 and MCF-7 human cancer cells compared to the p160 peptide. These peptides showed very little binding to the control (noncancerous) human umbilical vein endothelial cells (HUVECs). Three of these peptides were synthesized separately and labeled with fluorescein isothiocyanate (FITC) to study their uptake and interaction with the cancer and control cells using confocal laser scanning microscopy and flow cytometry. The results confirmed the high and specific affinity of an 11-mer peptide 11 (RGDPAYQGRFL) and a 10-mer peptide 18 (WXEAAYQRFL) for the cancer cells versus HUVECs. Peptide 11 binds different receptors on target cancer cells as its sequence contains multiple recognition motifs, whereas peptide 18 binds mainly to the putative p160 receptor. The peptide array-whole cell binding assay reported here is a complementary method to phage display for further screening and optimization of cancer targeting peptides for cancer therapy and diagnosis.

Binding of the Phage Display Derived Peptide CaIX-P1 on Human Colorectal Carcinoma Cells Correlates with the Expression of Carbonic Anhydrase IX

“…Incubation of the target with the peptide array and analysis of interactions between target and the spotted sequences can lead to identification of molecules with increasing binding affinity. In recent studies, peptide arrays were successfully applied for the identification of peptide-cell interactions [27]. …”

rapmad: Robust analysis of peptide microarray data

“…Peptide microarrays have been primarily used within screening settings with follow-up experiments focusing on a small number of promising candidates to ensure specificity [4]. Unidentified reactive peptides from a peptide microarray experiment cannot be distinguished from the large number of rightfully excluded unreactive peptides and are thus not further regarded.…”

“…Synthesized peptides are spotted in a grid-layout on glass slides which allow the screening of thousands of peptides within a single experiment with requiring only a small quantity of sample. Applications range from studying the humoral response to HIV [1] or food allergens [2] to the detection of cancer biomarkers [3] and antibody signatures [4] to the characterization of protein-protein interactions [5] and of kinase substrates [6,7]. …”

⿿Blind⿿ targeting in action: From phage display to breast cancer cell targeting with peptide-gold nanoconjugates

“…Importantly, no binding of the P1 and P2 was observed to human normal cells derived from different sources, including mammary epithelial cells (HMEC), peripheral blood mononuclear cells (PBMC), human umbilical vein endothelial cells (HUVEC), normal colon fibroblast, and human CD34+ bone marrow cells [19]. RGDPAYQGRFL peptide (P3) and WXEAAYQRFL peptide (P4) were identified by Kaur et al by a peptide arraywhole cell interaction approach [21]. Flow cytometry and confocal microscopy studies showed that the RGD-containing peptide was highly selective to MDA-MB-435 and MCF-7 cancer cells, but had very little affinity for the control HUVEC cells.…”