There is considerable evidence that the presence of cancer can elicit a humoral immune response to specific proteins in the host, and these resulting autoantibodies may have potential as noninvasive biomarkers. To characterize the autoantibody repertoire present in the sera of patients with lung adenocarcinoma, we developed a high-density peptide microarray derived from biopanning a lung cancer phage display library. Using a 2,304-element microarray, we interrogated a total of 250 sera from Michigan lung cancer patients and noncancer controls to develop an "autoantibody profile" of lung adenocarcinoma. A set of 22 discriminating peptides derived from a training set of 125 serum samples from lung adenocarcinoma patients and control subjects was found to predict cancer status with 85% sensitivity and 86% specificity in an independent test set of 125 sera. Sequencing of the immunoreactive phage-peptide clones identified candidate humoral immune response targets in lung adenocarcinoma, including ubiquilin I, a protein that regulates the degradation of several ubiquitin-dependent proteasome substrates. An independent validation set of 122 serum samples from Pittsburgh was examined using two overlapping clones of ubiquilin 1 that showed 0.79 and 0.74 of the area under the receiver operating characteristics curve, respectively. Significantly increased levels of both ubiquilin 1 mRNA and protein, as well as reduced levels of the phosphorylated form of this protein, were detected in lung tumors. Immunofluorescence using anti-ubiquilin 1 antibodies confirmed intracellular expression within tumors cells. These studies indicate that autoantibody profiles, as well as individual candidates, maybe useful for the noninvasive detection of lung adenocarcinoma. ©2007 American Association for Cancer Research.