In silico design and performance of peptide microarrays for breast cancer tumour-auto-antibody testing

Parvez Syed, István Gyurján, Albert Kriegner, Klemens Vierlinger, Christian F. Singer, Christine Rappaport-Fürhauser, Johannes Zerweck, Johannes Söllner, Andreas Weinhäusel


The simplicity and potential of minimally invasive testing using sera from patients makes auto-antibody based biomarkers a very promising tool for use in cancer diagnostics. Protein microarrays have been used for the identification of such auto-antibody signatures. Because high throughput protein expression and purification is laborious, synthetic peptides might be a good alternative for microarray generation and multiplexed analyses.
In this study, we designed 1185 antigenic peptides, deduced from proteins expressed by 642 cDNA expression clones found to be sero-reactive in both breast tumour patients and controls. The sero-reactive proteins and the corresponding peptides were used for the production of protein and peptide microarrays. Serum samples from females with benign and malignant breast tumours and healthy control sera (n=16 per group) were then analysed. Correct classification of the serum samples on peptide microarrays were 78% for discrimination of ‘malignant versus healthy controls’, 72% for ‘benign versus malignant’ and 94% for ‘benign versus controls’. On protein arrays, correct classification for these contrasts was 69%, 59% and 59%, respectively.
The over-representation analysis of the classifiers derived from class prediction showed enrichment of genes associated with ribosomes, spliceosomes, endocytosis and the pentose phosphate pathway. Sequence analyses of the peptides with the highest sero-reactivity demonstrated enrichment of the zinc-finger domain. Peptides’ sero-reactivities were found negatively correlated with hydrophobicity and positively correlated with positive charge, high inter-residue protein contact energies and a secondary structure propensity bias. This study hints at the possibility of using in silico designed antigenic peptide microarrays as an alternative to protein microarrays for the improvement of tumour auto-antibody based diagnostics.


Peptide microarrays, auto-antibody, protein microarray, antigenic peptide design, Antigenic motif enrichment


Andresen H, Grötzinger C, Zarse K, Kreuzer OJ, Ehrentreich-Förster E & Bier FF 2006 Functional peptide microarrays for specific and sensitive antibody diagnostics. Proteomics 6 1376-1384.

Backes C, Ludwig N, Leidinger P, Harz C, Hoffmann J, Keller A, Meese E & Lenhof HP 2011 Immunogenicity of autoantigens. BMC.Genomics 12 340.

Baneyx F 1999 Recombinant protein expression in Escherichia coli. Curr.Opin.Biotechnol 10 411-421.

Baneyx F & Mujacic M 2004 Recombinant protein folding and misfolding in Escherichia coli. Nat.Biotechnol 22 1399-1408.

Boros LG, Lee PW, Brandes JL, Cascante M, Muscarella P, Schirmer WJ, Melvin WS & Ellison EC 1998 Nonoxidative pentose phosphate pathways and their direct role in ribose synthesis in tumors: is cancer a disease of cellular glucose metabolism? Med.Hypotheses 50 55-59.

Bussow K, Nordhoff E, Lubbert C, Lehrach H & Walter G 2000 A human cDNA library for high-throughput protein expression screening. Genomics 65 1-8.

Carter D, Dillon DC, Reynolds LD, Retter MW, Fanger G, Molesh DA, Sleath PR, McNeill PD, Vedvick TS, Reed SG, Persing DH & Houghton RL 2003 Serum antibodies to lipophilin B detected in late stage breast cancer patients. Clin.Cancer Res 9 749-754.

Chapman C, Murray A, Chakrabarti J, Thorpe A, Woolston C, Sahin U, Barnes A & Robertson J 2007 Autoantibodies in breast cancer: their use as an aid to early diagnosis. Ann.Oncol 18 868-873.

Conroy SE, Gibson SL, Brunstrom G, Isenberg D, Luqmani Y & Latchman DS 1995 Autoantibodies to 90 kD heat-shock protein in sera of breast cancer patients. Lancet 345 126.

Cretich M, Damin F, Pirri G & Chiari M 2006 Protein and peptide arrays: recent trends and new directions. Biomol Eng 23 77-88.

Disis ML, Calenoff E, McLaughlin G, Murphy AE, Chen W, Groner B, Jeschke M, Lydon N, McGlynn E, Livingston RB & . 1994 Existent T-cell and antibody immunity to HER-2/neu protein in patients with breast cancer. Cancer Res 54 16-20.

Dohlman JG, Lupas A & Carson M 1993 Long charge-rich alpha-helices in systemic autoantigens. Biochem Biophys Res Commun 195 686-696.

Duburcq X, Olivier C, Malingue F, Desmet R, Bouzidi A, Zhou F, Auriault C, Gras-Masse H & Melnyk O 2004 Peptide-protein microarrays for the simultaneous detection of pathogen infections. Bioconjug Chem 15 301-316.

Floyd S & De CP 1998 Endocytosis proteins and cancer: a potential link? Trends Cell Biol 8 299-301.

Fodor SP, Read JL, Pirrung MC, Stryer L, Lu AT & Solas D 1991 Light-directed, spatially addressable parallel chemical synthesis. Science 251 767-773.

Frank R 2002 The SPOT-synthesis technique. Synthetic peptide arrays on membrane supports--principles and applications. J Immunol Methods 267 13-26.

Geysen HM, Barteling SJ & Meloen RH 1985 Small peptides induce antibodies with a sequence and structural requirement for binding antigen comparable to antibodies raised against the native protein. Proc Natl Acad Sci U.S.A 82 178-182.

Hamrita B, Chahed K, Kabbage M, Guillier CL, Trimeche M, Chaieb A & Chouchane L 2008 Identification of tumor antigens that elicit a humoral immune response in breast cancer patients' sera by serological proteome analysis (SERPA). Clin Chim Acta 393 95-102.

Hardin JA 1986 The lupus autoantigens and the pathogenesis of systemic lupus erythematosus. Arthritis Rheum 29 457-460.

Heller G, Geradts J, Ziegler B, Newsham I, Filipits M, Markis-Ritzinger EM, Kandioler D, Berger W, Stiglbauer W, Depisch D, Pirker R, Zielinski CC & Zochbauer-Muller S 2007 Downregulation of TSLC1 and DAL-1 expression occurs frequently in breast cancer. Breast Cancer Res Treat 103 283-291.

Iuchi S 2001 Three classes of C2H2 zinc finger proteins. Cell Mol.Life Sci 58 625-635.

Keller A, Backes C, Al-Awadhi M, Gerasch A, Kuntzer J, Kohlbacher O, Kaufmann M & Lenhof HP 2008 GeneTrailExpress: a web-based pipeline for the statistical evaluation of microarray experiments. BMC.Bioinformatics 9 552.

Keşmir C, Nussbaum AK, Schild H, Detours V & Brunak S 2002 Prediction of proteasome cleavage motifs by neural networks. Protein Eng 15 287-296

Kolaskar AS & Tongaonkar PC 1990 A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett 276 172-174.

Li YX, Yue W, Wang Y, Zhang L, Gu M & Xu S 2010 Detecting EGFR autoantibodies in serums of NSCLC patients with peptide array. Zhongguo Fei Ai Za Zhi 13 727-730.

Lu H, Goodell V & Disis ML 2008 Humoral immunity directed against tumor-associated antigens as potential biomarkers for the early diagnosis of cancer. J Proteome Res 7 1388-1394.

Ludwig N, Keller A, Leidinger P, Harz C, Backes C, Lenhof HP & Meese E 2012 Is there a general autoantibody signature for cancer? Eur J Cancer doi:10.1016/j.ejca.2012.01.017

Lundegaard C, Lamberth K, Harndahl M, Buus S, Lund O & Nielsen M 2008 NetMHC-3.0: accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8-11. Nucleic Acids Res 36 W509-512.

Min DH & Mrksich M 2004 Peptide arrays: towards routine implementation. Curr.Opin.Chem.Biol 8 554-558.

Montenarh M 2000 Chapter 20 - Humoral Immune Response Against the Growth Suppressor p53 in Human Malignancies. In Cancer and Autoimmunity, pp 193-203. Eds S Yehuda & MG Eric. Amsterdam: Elsevier.

Pisani P, Bray F & Parkin DM 2002 Estimates of the world-wide prevalence of cancer for 25 sites in the adult population. Int.J.Cancer 97 72-81.

Piura E & Piura B 2011 Autoantibodies to tailor-made panels of tumor-associated antigens in breast carcinoma. J Oncol. doi:10.1155/2011/982425.

Preininger C, Bodrossy L, Sauer U, Pichler R & Weilharter A 2004 ARChip epoxy and ARChip UV for covalent on-chip immobilization of pmoA gene-specific oligonucleotides. Anal Biochem 330 29-36.

Qian F, Odunsi K, Blatt LM, Scanlan MJ, Mannan M, Shah N, Montgomery J, Haddad F & Taylor M 2005 Tumor associated antigen recognition by autologous serum in patients with breast cancer. Int J Mol Med 15 137-144.

R Development Core Team 2005 R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing.

Reineke U & Sabat R 2009 Antibody epitope mapping using SPOT peptide arrays. Methods Mol Biol 524 145-167.

Ron D & Kazanietz MG 1999 New insights into the regulation of protein kinase C and novel phorbol ester receptors. FASEB J 13 1658-1676.

Selak S, Mahler M, Miyachi K, Fritzler ML & Fritzler MJ 2003 Identification of the B-cell epitopes of the early endosome antigen 1 (EEA1). Clin Immunol 109 154-164.

Shin DS, Kim DH, Chung WJ & Lee YS 2005 Combinatorial solid phase peptide synthesis and bioassays. J Biochem Mol Biol 38 517-525.

Simon R, Lam A, Li MC, Ngan M, Menenzes S & Zhao Y 2007 Analysis of gene expression data using BRB-ArrayTools. Cancer Inform 3 11-17.

Simon R, Radmacher MD, Dobbin K & McShane LM 2003 Pitfalls in the use of DNA microarray data for diagnostic and prognostic classification. J.Natl.Cancer Inst. 95 14-18.

Singh SM & Panda AK 2005 Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 99 303-310.

Sollner J, Grohmann R, Rapberger R, Perco P, Lukas A & Mayer B 2008 Analysis and prediction of protective continuous B-cell epitopes on pathogen proteins. Immunome Res 4 1.

Southern EM 1988 Analysing polynucleotide sequences. Great Britain patent application GB 8810400.5

Stempfer R, Syed P, Vierlinger K, Pichler R, Meese E, Leidinger P, Ludwig N, Kriegner A, Nohammer C & Weinhausel A 2010 Tumour auto-antibody screening: performance of protein microarrays using SEREX derived antigens. BMC.Cancer 10 627.

Strieter RM, Belperio JA, Phillips RJ & Keane MP 2004 CXC chemokines in angiogenesis of cancer. Semin Cancer Biol 14 195-200.

Sturgeon CM, Duffy MJ, Stenman UH, Lilja H, Brunner N, Chan DW, Babaian R, Bast RC, Jr., Dowell B, Esteva FJ, Haglund C, Harbeck N, Hayes DF, Holten-Andersen M, Klee GG, Lamerz R, Looijenga LH, Molina R, Nielsen HJ, Rittenhouse H, Semjonow A, Shih I, Sibley P, Soletormos G, Stephan C, Sokoll L, Hoffman BR & Diamandis EP 2008 National Academy of Clinical Biochemistry laboratory medicine practice guidelines for use of tumor markers in testicular, prostate, colorectal, breast, and ovarian cancers. Clin Chem 54 e11-e79.

Söllner J 2006 Selection and combination of machine learning classifiers for prediction of linear B-cell epitopes on proteins. J Mol Recognit 19 209-214.

Tan EM 1989 Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. Adv Immunol 44 93-151


Ullah MF & Aatif M 2009 The footprints of cancer development: Cancer biomarkers. Cancer Treat Rev 35 193-200.

Uttamchandani M & Yao SQ 2008 Peptide microarrays: next generation biochips for detection, diagnostics and high-throughput screening. Curr Pharm Des 14 2428-2438.

van Alphen RJ, Wiemer EA, Burger H & Eskens FA 2009 The spliceosome as target for anticancer treatment. Br J Cancer 100 228-232.

Wilce MCJ, Aguilar M & Hearn MTW 1995 Physicochemical Basis of Amino Acid Hydrophobicity Scales: Evaluation of Four New Scales of Amino Acid Hydrophobicity Coefficients Derived from RP-HPLC of Peptides. Anal Chem 67 1210-1219.

Wright GW & Simon RM 2003 A random variance model for detection of differential gene expression in small microarray experiments. Bioinformatics 19 2448-2455.

Zhu Y, Lin H, Li Z, Wang M & Luo J 2001 Modulation of expression of ribosomal protein L7a (rpL7a) by ethanol in human breast cancer cells. Breast Cancer Res Treat 69 29-38.

Full Text: PDF


  • There are currently no refbacks.

Copyright © 2021 Journal of Molecular Biochemistry