Proteomic Analysis of Lung Adenocarcinoma: Identification of a Highly Expressed Set of Proteins in Tumors 1 Guoan Chen, Tarek G. Gharib, Chiang-Ching Huang, Dafydd G. Thomas, Kerby A. Shedden, Jeremy M. G. Taylor, Sharon L. R. Kardia, David E. Misek, Thomas J. Giordano, Mark D. Iannettoni, Mark B. Orringer, Samir M. Hanash, and David G. Beer 2 Section of General Thoracic Surgery, Departments of Surgery [G. C., T. G. G., M. D. I., M. B. O., D. G. B.], Biostatistics [C-C. H., K. A. S., J. M. G. T.], Pathology [T. J. G., D. G. T.], Pediatrics [D. E. M., S. M. H.], and Epidemiology [S. L. R. K.], University of Michigan, Ann Arbor, Michigan 48109 ABSTRACT Purpose: The goal of this study was to identify potential protein markers in lung adenocarcinomas. Experimental Design: A series of 93 lung adenocarcino- mas (64 stage I and 29 stage III) and 10 uninvolved lung samples were examined for quantitative differences in pro- tein expression using two-dimensional PAGE. Candidate proteins were identified using matrix-assisted laser desorp- tion/ionization mass spectrometry or peptide sequencing. The levels of the individual isoforms of nine proteins found to be overexpressed in the lung tumors were examined. Potential mechanisms for overexpression were examined by comparing mRNA expression levels, assessed using oligonu- cleotide arrays, to the protein values in the same samples. Results: Antioxidant enzyme AOE372, ATP synthase subunit d (ATP5D), 1,4-galactosyltransferase, cytosolic in- organic pyrophosphatase, glucose-regulated M r 58,000 pro- tein, glutathione-S-transferase M4, prolyl 4-hydroxylase  subunit, triosephosphate isomerase, and ubiquitin thioles- terase (UCHL1) were identified as being significantly over- expressed in lung adenocarcinomas. The expression of these proteins was increased from 1.4- to 10.6-fold as compared with uninvolved lung tissue. The expression of the individual protein isoforms was correlated with 10 clinicopathological variables as well as with each gene’s mRNA level in the same sample. Both isoforms of glucose-regulated M r 58,000 pro- tein were found to be significantly correlated with their mRNA expression profiles (P < 0.05), indicating that in- creased transcription likely underlies the increased expres- sion of these proteins. Conclusions: Two-dimensional PAGE and mass spec- trometry can identify proteins showing increased expression in lung adenocarcinoma. The association of specific isoforms of these proteins with clinical variables and understanding the regulation of their expression will aid in determination of their potential use as biomarkers in this cancer. INTRODUCTION Adenocarcinomas constitute a biologically heterogeneous group of lung tumors and are now the most common type of lung cancer (1). Although many insights into the molecular pathology of lung tumors have been achieved, additional infor- mation is critical to both our understanding of the development and progression of these tumors as well as to aid in early diagnosis. The analysis of genes overexpressed in lung cancer, and that they may serve as tumor markers, has been the subject of extensive research. The most commonly evaluated markers include neuron-specific enolase, carcinoembryonic antigen, cy- tokeratin 19 fragments (CYFRA 21-1), squamous cell carci- noma antigen, cancer antigen CA 125, and tissue polypeptide antigen (2). Although the analysis of multiple biological mark- ers may be more informative than the use of a single marker (3), very few markers have been accepted for routine clinical use, either because of conflicting reports or because associations are insufficient for formulating clinical treatment plans (4). The detection of new candidate markers is complex because of the known heterogeneity of lung cancers. 2D-PAGE 3 is a powerful research technique, which makes it possible to simultaneously examine hundreds of polypeptides in a tissue sample. It has been widely used for the detection and identification of potential tumor markers (5). This study ana- lyzed 93 lung adenocarcinomas and 10 uninvolved lung samples for protein expression using 2D-PAGE. Analysis software were used to obtain quantitative measures for individual protein spots. Proteins of interest were identified using MALDI-MS or peptide sequencing. Associations between the proteins that were over- expressed in the lung adenocarcinomas and clinicopathological features of the tumor were determined. Evaluation of the same tumor samples for mRNA expression using oligonucleotide Received 12/18/01; revised 4/15/02; accepted 4/15/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NCI Grant U19 CA-85953. 2 To whom requests for reprints should be addressed, at General Tho- racic Surgery, MSRB II, B560, Box 0686, University of Michigan Medical School, Ann Arbor, MI 48109-0686. E-mail: dgbeer@ umich.edu. 3 The abbreviations used are: 2D-PAGE, two-dimensional PAGE; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrom- etry; ISH, in situ hybridization; AOE372, antioxidant enzyme; ATP5D, ATP synthase subunit d; B4GALT, 1,4-galactosyltransferase; PPase, cytosolic inorganic pyrophosphatase; GRP58, glucose-regulated M r 58,000 protein; GSTM4, glutathione-S-transferase M4; P4HB, prolyl 4-hydroxylase,  subunit; TPI, triosephosphate isomerase; UCHL1, ubiquitin thiolesterase (PGP9.5). 2298 Vol. 8, 2298 –2305, July 2002 Clinical Cancer Research Research. on December 11, 2021. © 2002 American Association for Cancer clincancerres.aacrjournals.org Downloaded from