For sizzling hot spots consisting of two tiling peptides, each with a p value less than 0.05 and having the same direction of fold switch, the probability of random occurrence of a hot spot is 0.05 0.05 0.5 = 0.00125 (X 0.5 accounts for the same direction of fold change). samples and 86 matched normal controls from a high-risk cohort revealed 48 proteins with three or more reactive epitopes in NSCLC samples relative to controls. Indie mass spectrometry analysis identified 40 of the 48 proteins in pre-diagnostic sera from NSCLC samples, of which 21 occurred in the immunoglobulin bound portion. Additionally, 63 and 34 proteins encompassed three or more epitopes that were unique for squamous cell lung malignancy and lung adenocarcinoma, respectively. Collectively, these data show that tiled peptide arrays provide a means to delineate epitopes encoded across the genome that trigger an autoantibody response associated with tumor development. strong class=”kwd-title” Keywords: Pre-diagnostic plasma, Non-small cell lung malignancy, autoantibody signature, peptide array Introduction Substantial evidence points to the occurrence of a humoral immune response to tumor antigens early during tumor development. For some cancers, this is manifest as paraneoplastic neurological symptoms occurring prior to diagnosis due to the production of autoantibodies to neural cell proteins (1,2). For most patients the autoantibody response occurs during tumor development without manifestation of symptoms. Harnessing the immune response to tumor antigens in the form of autoantibodies represents a encouraging approach for malignancy early detection (3C5). Because of a limited immune response to any particular epitope among malignancy subjects, strategies are needed to improve sensitivity and specificity for early detection applications through the identification of multiple antigenic epitopes. Such an effort requires comprehensive methods capable of capturing among a vast number of potential epitopes, those that confer the greatest sensitivity and specificity. Several strategies have been applied to the discovery of circulating autoantibody markers in malignancy, mostly by relying on samples obtained at the time of diagnosis (6C8). Serological screening of cDNA expression libraries (SEREX) has Cd200 been utilized to discover potential immunogenic markers (9). Tumor cell lysate-derived protein arrays have been utilized to define autoantibody Hyperforin (solution in Ethanol) signatures including natural proteins (10C16). Hyperforin (solution in Ethanol) Tumor antigens have also been discovered through the use of recombinant arrays (17C19), phage-display libraries (20,21) and tumor homogenates (22). Peptide arrays have been utilized to determine the optimal amino acid length Hyperforin (solution in Ethanol) for antibody binding, and to interrogate signaling pathways and kinase activity (23,24). Substrates for arraying proteins and peptides have included nitrocellulose, glass, silicon wafers and plastic (25). Chemical synthesis of peptides around the array surface allows incorporation of modifications, including phosphorylation, acetylation or methylation, on amino acids. In this study we have implemented an innovative strategy using tiled peptide arrays encompassing the entire coding sequences in the human genome representing 2,781,902 tiled peptides to determine the occurrence of peptide epitopes in lung malignancy sera collected up to one year prior to diagnosis, and the extent of similarities and differences in peptide epitopes between squamous lung malignancy and lung adenocarcinoma. Materials and Methods Serum samples. Serum samples were collected as part of the Beta-Carotene and Retinol Efficacy Trial (CARET) study, which is a multicenter, randomized, double-blinded, placebo-controlled trial to evaluate the cancer prevention efficacy and security of daily supplementation with 30 mg of beta-carotene and 25,000 IU of retinyl palmitate in 18,314 individuals at high risk of developing lung malignancy (26). In this study, serum from 86 subjects collected up to a year prior to a diagnosis of non-small cell lung malignancy (NSCLC) and from 86 healthy controls matched for age, gender, and smoking status were selected from your CARET cohort for peptide array analysis. Of the 86 subjects subsequently diagnosed with lung malignancy, 32 patients experienced adenocarcinoma, 30 experienced squamous cell carcinoma, and 24 subjects were classified as non-squamous, non-adeno NSCLC. The characteristics of the subjects in the study are summarized in Supplementary Table 1. An independent set of 42 pre-diagnostic NSCLC samples was utilized to determine the occurrence in blood circulation of proteins exhibiting peptide reactivity among cases. All study participants gave written informed consent to participate in the study, and the research was approved by the institutional review boards of all of the participating institutions. All studies were conducted in accordance with the Declaration of Helsinki. Array design and synthesis Peptide arrays were designed based on protein sequences obtained from the Universal Protein Resource (UniProt) (Supplementary Physique 1). 2,781,902 16-mer peptides with 12 amino acids overlap representing the coding sequences in the genome and 4.547 scrambled peptides were randomly distributed on the array. Microarrays were synthesized with a Roche-NimbleGen Maskless Array Synthesizer (MAS) by light-directed solid-phase peptide synthesis using an amino-functionalized plastic support (Greiner Bio-One) coupled with a 6-aminohexanoic acid linker and amino acid derivatives transporting a photosensitive 2-(2-nitrophenyl) propyloxycarbonyl (NPPOC) protection group (Orgentis Chemicals). Amino acids (final concentration 20 mM) were pre-mixed for 10 min in em N,N /em -Dimethylformamide (DMF, Sigma Aldrich) with em N,N,N,N /em -Tetramethyl-O-(1H-benzotriazol-1-yl)uranium-hexafluorophosphate.