Protein microarrays using liquid phase fractionation of cell lysates

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Esophageal adenocarcinoma, currently the seventh leading cause of cancer-related death, has been associated with the presence of Barrett metaplasia. The malignant potential of Barrett metaplasia is evidenced by ultimate progression of this condition to invasive adenocarcinoma. We utilized liquid phase separation of proteins with chromatofocusing in the first dimension and nonporous reverse phase HPLC in the second dimension followed by ESI-TOF mass spectrometry to identify proteins differentially expressed in six Barrett metaplasia samples as compared with six esophageal adenocarcinoma samples; all six Barrett samples were obtained from the identical six patients from whom we obtained the esophageal adenocarcinoma tissue. Approximately 300 protein bands were detected by mass mappings, and 38 differentially expressed proteins were identified by LC-MS/MS. The false positive rates of the peptide identifications were evaluated by reversed database searching. Among the proteins that were identified, Rho GDP dissociation inhibitor 2, ␣-enolase, Lamin A/C, and nucleoside-diphosphate kinase A were demonstrated to be up-regulated in both mRNA and protein expression in esophageal adenocarcinomas relative to Barrett metaplasia. Candidate proteins were examined at the mRNA level using high density oligonucleotide microarrays. The cellular expression patterns were verified in both esophageal adenocarcinomas and in Barrett metaplasia by immunohistochemistry. These differentially expressed proteins may have utility as useful candidate markers of esophageal adenocarcinoma. Molecular & Cellular Proteomics 6:987-999, 2007.Esophageal adenocarcinoma is increasing rapidly in Western countries and is currently the seventh leading cause of cancer-related death (1). Esophageal adenocarcinoma has been associated with the presence of Barrett metaplasia, a condition in which the normal squamous epithelium of the esophagus is replaced by columnar epithelium. The malignant potential of this condition is evidenced by the progression of Barrett metaplasia to low grade dysplasia, high grade dysplasia, and ultimately to invasive adenocarcinoma. The risk of developing adenocarcinoma is 30 -125 times higher in people who have Barrett metaplasia than people who do not. The prognosis of patients with esophageal adenocarcinoma remains poor with overall 5-year survival rates of only 5-15% (1). Unfortunately patients often present with regionally advanced disease (2). Given the poor prognosis associated with esophageal adenocarcinoma, it is imperative to improve our understanding of the tumorigenesis and the factors associated with increased risk. It is possible that therapeutic targets or protein markers can be identified that will ultimately facilitate improved patient survival.Proteomics technologies have been used for the identification of candidate markers for early cancer detection (3). The global analysis of protein expression complements genomics analyses. For example, proteomics analysis may provide further insight into post-translational mod...

Section: Methodsmentioning

confidence: 99%

Comparative Proteomics Analysis of Barrett Metaplasia and Esophageal Adenocarcinoma Using Two-dimensional Liquid Mass Mapping

Zhao

Chang

et al. 2007

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“…cellular processes emphasizes the importance of using a screening platform containing proteins extracted from tumor, itself reflecting a physiologically realistic swath of the prostate cancer proteome. Such correlations to tumor function would be difficult to recapitulate using proteins fractionated from cell lines (35,36) or using phage array platforms (15,37). In addition, the multidimensional protein fractionation-coupled microarray retains post-translational modifications that are most indicative of the cellular phenotype and in most cases better reflects the reality of humoral response to cancer antigens.…”

Section: Discussionmentioning

confidence: 99%

Humoral Response Profiling Reveals Pathways to Prostate Cancer Progression

Taylor

Pal

et al. 2008

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There is considerable evidence for an association between prostate cancer development and inflammation, which results in autoantibody generation against tumor proteins. This immune system-driven amplification of the autoantibody response to intracellular antigens can serve as a sensitive tool to detect low abundance serum proteomic tumor markers for prostate cancer as well as provide insight into biological processes perturbed during cancer development. Here we examine serum humoral responses in a cohort of 34 patients with either benign prostatic hyperplasia or clinically localized prostate cancer (PCa). The experimental strategy couples multidimensional liquid-phase protein fractionation of localized and metastatic prostate cancer tissue lysates to protein microarrays and subsequent mass spectrometry. A supervised learning analysis of the humoral response arrays generated a parsimonious predictor having 78% sensitivity and 75% specificity in distinguishing PCa from benign prostatic hyperplasia in a cohort of American males with elevated prostate-specific antigen. Enrichment analysis of the PCa-specific humoral signature revealed large scale immune reprogramming mediated by STAT transcription factors and the generation of autoantibodies to enzymes involved in nitrogen metabolism. Meta-analysis of independent prostate cancer gene expression data validated the presence of STAT-induced immunomodulation. Concomitant validation of elevated levels of the nitrogen metabolism pathway was obtained by direct measurement of metabolic levels of glutamate and aspartate in prostate cancer tissues. Thus, in addition to functioning as markers in prostate cancer detection, humoral response profiles can serve as powerful tools revealing pathway dysregulation that might otherwise be suppressed by the complexity of the cancer proteome. Molecular & Cellular Proteomics 7:600 -611, 2008.Although prostate carcinoma is the leading cancer diagnosis in American men, its early detection facilitates effective treatment modalities and improved mortality (1). Although the advent of prostate-specific antigen (PSA) 1 screening has led to earlier detection of prostate cancers (2), its lack of specificity for neoplasm has resulted in a dramatic increase in the number of subsequent prostate needle biopsies (3). As the population of men 65 years and older is expected to increase from 14 million in the year 2000 to 31 million by 2030 (4), it will be increasingly important to distinguish men with benign prostatic hyperplasia from those having neoplastic disease warranting clinical intervention. Thus, there is a compelling need to define additional clinical markers for accurate detection of prostate cancers.This situation has spawned a wide range of serum-based early detection methodologies, including protein microarrays (5). Complicating this approach is the fact that potentially viable tumor biomarkers are embedded among a bounty of proteomic noise. This noise includes housekeeping and other highly abundant proteins, whereas the relatively low abun...

“…Several approaches have been used to identify TAAs in cancer, including natural protein arrays prepared with fractions obtained from two-dimensional LC separations of tumoral samples (29,30) or protein extracts from cancer cells or tissue (9,31) probed by Western blot with patient sera, cancer tissue peptide libraries expressed as cDNA expression libraries for serological screening (serological analysis of recombinant cDNA expression libraries (SEREX)) (22,32), or peptides expressed on the surface of phages in combination with microarrays (17,18,33,34). However, these approaches suffer from several drawbacks.…”

Section: Colorectal Cancer (Crc)mentioning

confidence: 99%

Identification of Tumor-associated Autoantigens for the Diagnosis of Colorectal Cancer in Serum Using High Density Protein Microarrays

Babel

Barderas

Dı́az-Uriarte

et al. 2009

147

143

There is a mounting evidence of the existence of autoantibodies associated to cancer progression. Antibodies are the target of choice for serum screening because of their stability and suitability for sensitive immunoassays. By using commercial protein microarrays containing 8000 human proteins, we examined 20 sera from colorectal cancer (CRC) patients and healthy subjects to identify autoantibody patterns and associated antigens. Fortythree proteins were differentially recognized by tumoral and reference sera (p value <0.04) in the protein microarrays. Five immunoreactive antigens, PIM1, MAPKAPK3, STK4, SRC, and FGFR4, showed the highest prevalence in cancer samples, whereas ACVR2B was more abundant in normal sera. Three of them, PIM1, MAPKAPK3, and ACVR2B, were used for further validation. A significant increase in the expression level of these antigens on CRC cell lines and colonic mucosa was confirmed by immunoblotting and immunohistochemistry on tissue microarrays. A diagnostic ELISA based on the combination of MAPKAPK3 and ACVR2B proteins yielded specificity and sensitivity values of 73.9 and 83.3% (area under the curve, 0.85), respectively, for CRC discrimination after using an independent sample set containing 94 sera representative of different stages of progression and control subjects. In summary, these studies confirmed the presence of specific autoantibodies for CRC and revealed new individual markers of disease (PIM1, MAPKAPK3, and ACVR2B) with the potential to diagnose CRC with higher specificity and sensitivity than previously reported serum biomarkers. Molecular & Cellular Proteomics 8:2382-2395, 2009.