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Mitchell J Frederick,
Amy J VanMeter,
Mayur A Gadhikar,
Ying C Henderson,
Hui Yao,
Curtis C Pickering,
Michelle D Williams,
Adel K El-Naggar,
Vlad Sandulache,
Emily Tarco,
Jeffrey N Myers,
Gary L Clayman,
Lance A Liotta,
Emanuel F Petricoin, Valerie S Calvert,
Valentina Fodale,
Jing Wang,
Randal S Weber
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ABSTRACT: Molecular targeted therapy represents a promising new strategy for treating cancers because many small-molecule inhibitors targeting protein kinases have recently become available. Reverse-phase protein microarrays (RPPAs) are a useful platform for identifying dysregulated signaling pathways in tumors and can provide insight into patient-specific differences. In the present study, RPPAs were used to examine 60 protein end points (predominantly phosphoproteins) in matched tumor and nonmalignant biopsy specimens from 23 patients with head and neck squamous cell carcinoma to characterize the cancer phosphoproteome. RPPA identified 18 of 60 analytes globally elevated in tumors versus healthy tissue and 17 of 60 analytes that were decreased. The most significantly elevated analytes in tumor were checkpoint kinase (Chk) 1 serine 345 (S345), Chk 2 S33/35, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) S65, protein kinase C (PKC) ζ/ι threonine 410/412 (T410/T412), LKB1 S334, inhibitor of kappaB alpha (IκB-α) S32, eukaryotic translation initiation factor 4E (eIF4E) S209, Smad2 S465/67, insulin receptor substrate 1 (IRS-1) S612, mitogen-activated ERK kinase 1/2 (MEK1/2) S217/221, and total PKC ι. To our knowledge, this is the first report of elevated PKC ι in head and neck squamous cell carcinoma that may have significance because PKC ι is an oncogene in several other tumor types, including lung cancer. The feasibility of using RPPA for developing theranostic tests to guide personalized therapy is discussed in the context of these data.
American Journal Of Pathology 02/2011; 178(2):548-71. · 4.89 Impact Factor
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ABSTRACT: The progression of nonalcoholic fatty liver disease (NAFLD) has been linked to deregulated exchange of the endocrine signaling between adipose and liver tissue. Proteomic assays for the phosphorylation events that characterize the activated or deactivated state of the kinase-driven signaling cascades in visceral adipose tissue (VAT) could shed light on the pathogenesis of nonalcoholic steatohepatitis (NASH) and related fibrosis. Reverse-phase protein microarrays (RPMA) were used to develop biomarkers for NASH and fibrosis using VAT collected from 167 NAFLD patients (training cohort, N = 117; testing cohort, N = 50). Three types of models were developed for NASH and advanced fibrosis: clinical models, proteomics models, and combination models. NASH was predicted by a model that included measurements of two components of the insulin signaling pathway: AKT kinase and insulin receptor substrate 1 (IRS1). The models for fibrosis were less reliable when predictions were based on phosphoproteomic, clinical, or the combination data. The best performing model relied on levels of the phosphorylation of GSK3 as well as on two subunits of cyclic AMP regulated protein kinase A (PKA). Phosphoproteomics technology could potentially be used to provide pathogenic information about NASH and NASH-related fibrosis. This information can lead to a clinically relevant diagnostic/prognostic biomarker for NASH.
Journal of Proteome Research 05/2010; 9(6):3218-24. · 5.11 Impact Factor
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Alessandra Silvestri,
Alfonso Colombatti, Valerie S Calvert,
Jianghong Deng,
Enzo Mammano,
Claudio Belluco,
Francesco De Marchi,
Donato Nitti,
Lance A Liotta,
Emanuel F Petricoin,
Mariaelena Pierobon
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ABSTRACT: Tissues are complex structures composed of different cell types, each of which present specific functions and characteristics. To better understand and measure the effect of tumor cell enrichment on protein pathway profiling and drug target activation measurements, the signaling activation portraits of laser capture microdissected (LCM) cancer epithelium and tumor stroma were compared with patient-matched whole-tissue specimens from 53 primary colorectal cancer samples. Microdissected material and whole-tissue lysate from contiguous cryostat sections were subjected to reverse-phase protein microarray analysis to determine the level of phopshorylation and expression of 75 different proteins known to be involved in cancer progression. The results revealed distinct differences in the protein activation portraits of cancer epithelium and stroma. Moreover, we found that the signaling activation profiles of the undissected whole-tissue specimens are profoundly different from the matched LCM material. Attempts to rescale the undissected pathway information based on percent endogenous tumor epithelium content were unsuccessful in recapitulating the LCM tumor epithelial signatures. Analysis of epidermal growth factor receptor phosphorylation and COX2 expression in these same sample sets revealed wholesale differences in the rank ordering of patient determination when LCM was compared with undissected samples. On the basis of these data, we conclude that accurate protein pathway activation status, which is under evaluation as a basis for patient selection and stratification for personalized therapy, must include upfront cellular-enrichment techniques such as LCM to generate accurate drug target activation status.
Laboratory Investigation 03/2010; 90(5):787-96. · 3.64 Impact Factor
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ABSTRACT: Adipose tissue has been suggested to contribute to the pathogenesis of various disease states, including prostate cancer. We investigated the association of cytokines and growth factors secreted by periprostatic adipose tissue with pathological features of aggressive prostate cancer.
Periprostatic adipose tissue was harvested from patients undergoing radical prostatectomy and cultured for 24 hours to generate conditioned medium or snap frozen immediately for functional signaling profiling. Multiplex analysis of the periprostatic adipose tissue conditioned medium was used to detect cytokine levels and compared to patient matched serum from 7 patients. Interleukin-6 in serum and periprostatic adipose tissue conditioned medium was further analyzed by enzyme-linked immunosorbent assay and correlated with clinical variables, such as age, body mass index and Gleason score, in 45 patients. Interleukin-6 expression in periprostatic adipose tissue was determined by immunohistochemistry. Reverse phase protein microarray technology was used to analyze cell signaling networks in periprostatic adipose tissue.
Interleukin-6 in periprostatic adipose tissue conditioned medium was approximately 375 times greater than that in patient matched serum and levels correlated with pathological grade. This finding was further extended by cell signaling analysis of periprostatic adipose tissue, which showed greater phosphorylation on Stat3 with high grade tumors (any component of Gleason score 4 or 5).
Higher Gleason score correlated with high levels of conditioned medium derived interleukin-6. Moreover, cell signaling analysis of periprostatic adipose tissue identified activated signaling molecules, including STAT3, that correlated with Gleason score. Since STAT3 is interleukin-6 regulated, these findings suggest that periprostatic adipose tissue may have a role in modulating prostate cancer aggressiveness by serving as a source of interleukin-6. Also, we found low numbers of inflammatory cells in the fat, suggesting that adipocytes are the major secretors of interleukin-6.
The Journal of urology 09/2009; 182(4):1621-7. · 4.02 Impact Factor
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Amy J VanMeter,
Adrianna S Rodriguez,
Elise D Bowman,
Jin Jen,
Curtis C Harris,
Jianghong Deng, Valerie S Calvert,
Alessandra Silvestri,
Claudia Fredolini,
Vikas Chandhoke,
Emanuel F Petricoin,
Lance A Liotta,
Virginia Espina
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ABSTRACT: Little is known about lung carcinoma epidermal growth factor (EGF) kinase pathway signaling within the context of the tissue microenvironment. We quantitatively profiled the phosphorylation and abundance of signal pathway proteins relevant to the EGF receptor within laser capture microdissected untreated, human non-small cell lung cancer (NSCLC) (n = 25) of known epidermal growth factor receptor (EGFR) tyrosine kinase domain mutation status. We measured six phosphorylation sites on EGFR to evaluate whether EGFR mutation status in vivo was associated with the coordinated phosphorylation of specific multiple phosphorylation sites on the EGFR and downstream proteins. Reverse phase protein array quantitation of NSCLC revealed simultaneous increased phosphorylation of EGFR residues Tyr-1148 (p < 0.044) and Tyr-1068 (p < 0.026) and decreased phosphorylation of EGFR Tyr-1045 (p < 0.002), HER2 Tyr-1248 (p < 0.015), IRS-1 Ser-612 (p < 0.001), and SMAD Ser-465/467 (p < 0.011) across all classes of mutated EGFR patient samples compared with wild type. To explore which subset of correlations was influenced by ligand induction versus an intrinsic phenotype of the EGFR mutants, we profiled the time course of 115 cellular signal proteins for EGF ligand-stimulated (three dosages) NSCLC mutant and wild type cultured cell lines. EGFR mutant cell lines (H1975 L858R) displayed a pattern of EGFR Tyr-1045 and HER2 Tyr-1248 phosphorylation similar to that found in tissue. Persistence of phosphorylation for AKT Ser-473 following ligand stimulation was found for the mutant. These data suggest that a higher proportion of the EGFR mutant carcinoma cells may exhibit activation of the phosphatidylinositol 3-kinase/protein kinase B (AKT)/mammalian target of rapamycin (MTOR) pathway through Tyr-1148 and Tyr-1068 and suppression of IRS-1 Ser-612, altered heterodimerization with ERBB2, reduced response to transforming growth factor beta suppression, and reduced ubiquitination/degradation of the EGFR through EGFR Tyr-1045, thus providing a survival advantage. This is the first comparison of multiple, site-specific phosphoproteins with the EGFR tyrosine kinase domain mutation status in vivo.
Molecular & Cellular Proteomics 08/2008; 7(10):1902-24. · 7.40 Impact Factor
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04/2008: pages 165 - 175; , ISBN: 9783527622153
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ABSTRACT: Although the genome provides information about the somatic genetic changes existing in the tissue and underpins pathology, it is the proteins that do the work of the cell and are functionally responsible for almost all disease processes. Moreover, many diseases such as cancer are a manifestation of deranged cellular protein molecular networks and cell-signaling pathways. These pathways contain a large and growing collection of drug targets, governing cellular survival, proliferation, invasion, and cell death. Thus, the promise of proteomics resides in the study of molecules that extend beyond correlation to causality. The clinical utility of reverse phase protein microarrays, a new technology invented in our laboratory, lies in its ability to generate a functional map of known cell-signaling networks or pathways for an individual patient obtained directly from a biopsy specimen. This patient-specific circuit diagram provides key information that identifies critical nodes or pathways that may serve as drug targets for individualized or combinatorial therapy through the quantification of phosphorylation states of proteins. Using this technique, the entire cellular proteome is immobilized on a substratum with subsequent immunodetection of the phosphorylated, or activated, state of cell-signaling proteins. The results of which pathways are "in use" can then be correlated with biological and clinical information and serve as both a diagnostic and a therapeutic guide: thus providing a "theranostic" endpoint.
Methods in molecular biology (Clifton, N.J.) 02/2008; 441:113-28.
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ABSTRACT: Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease. Omental adipose tissue, a biologically active organ secreting adipokines and cytokines, may play a role in the development of NAFLD. We tested this hypothesis with reverse-phase protein microarrays (RPA) for multiplexed cell signaling analysis of adipose tissue from patients with NAFLD. Omental adipose tissue was obtained from 99 obese patients. Liver biopsies obtained at the time of surgery were all read by the same hepatopathologist. Adipose tissue was exposed to rapid pressure cycles to extract protein lysates. RPA was used to investigate intracellular signaling. Analysis of 54 different kinase substrates and cell signaling endpoints showed that an insulin signaling pathway is deranged in different locations in NAFLD patients. Furthermore, components of insulin receptor-mediated signaling differentiate most of the conditions on the NAFLD spectrum. For example, PKA (protein kinase A) and AKT/mTOR (protein kinase B/mammalian target of rapamycin) pathway derangement accurately discriminates patients with NASH from those with the non-progressive forms of NAFLD. PKC (protein kinase C) delta, AKT, and SHC phosphorylation changes occur in patients with simple steatosis. Amounts of the FKHR (forkhead factor Foxo1)phosphorylated at S256 residue were significantly correlated with AST/ALT ratio in all morbidly obese patients. Furthermore, amounts of cleaved caspase 9 and pp90RSK S380 were positively correlated in patients with NASH. Specific insulin pathway signaling events are altered in the adipose tissue of patients with NASH compared with patients with nonprogressive forms of NAFLD. CONCLUSION: These findings provide evidence for the role of omental fat in the pathogenesis, and potentially, the progression of NAFLD.
Hepatology 08/2007; 46(1):166-72. · 11.66 Impact Factor
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ABSTRACT: Cancer has a genomic and proteomic basis. Genomic information provides information about the somatic genetic changes existing in the tumor that provides a survival advantage driving neoplastic progression. On the other hand, proteomics aids in the identification of dysregulated cellular proteins, including known or novel drug targets, governing cellular survival, proliferation, invasion, and cell death. The clinical utility of reverse phase protein microarrays lies in their ability to generate a map of known cell signaling networks or pathways for an individual patient. This protein network map aids in identifying critical nodes or pathways that may serve as drug targets for individualized or combinatorial therapy. Reverse phase protein microarrays are one of the tools available for profiling the protein molecular pathways in a given cellular sample. This type of microarray can uniquely quantify phosphorylation states of proteins. An entire cellular proteome is immobilized on a substratum with subsequent immunodetection of total and activated forms of cell signaling proteins. The pattern of signal intensity generated by the protein spots can be correlated with biological and clinical information as diagnostic and prognostic indicators.
Methods in molecular biology (Clifton, N.J.) 02/2007; 383:321-36.
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ABSTRACT: Deciphering the cellular and molecular interactions that drive disease within the tissue microenvironment holds promise for discovering drug targets of the future. In order to recapitulate the in vivo interactions thorough molecular analysis, one must be able to analyze specific cell populations within the context of their heterogeneous tissue microecology. Laser-capture microdissection (LCM) is a method to procure subpopulations of tissue cells under direct microscopic visualization. LCM technology can harvest the cells of interest directly or can isolate specific cells by cutting away unwanted cells to give histologically pure enriched cell populations. A variety of downstream applications exist: DNA genotyping and loss-of-heterozygosity (LOH) analysis, RNA transcript profiling, cDNA library generation, proteomics discovery and signal-pathway profiling. Herein we provide a thorough description of LCM techniques, with an emphasis on tips and troubleshooting advice derived from LCM users. The total time required to carry out this protocol is typically 1-1.5 h.
Nature Protocol 02/2006; 1(2):586-603. · 8.36 Impact Factor
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11/2005; , ISBN: 9780470011539
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Puneeth Iyengar,
Virginia Espina,
Terence W Williams,
Ying Lin,
David Berry,
Linda A Jelicks,
Hyangkyu Lee,
Karla Temple,
Reed Graves,
Jeffrey Pollard, [......],
Bruce J Trock,
Marc Lippman, Valerie S Calvert,
Emanuel F Petricoin,
Lance Liotta,
Ekaterina Dadachova,
Richard G Pestell,
Michael P Lisanti,
Paolo Bonaldo,
Philipp E Scherer
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ABSTRACT: The interactions of transformed cells with the surrounding stromal cells are of importance for tumor progression and metastasis. The relevance of adipocyte-derived factors to breast cancer cell survival and growth is well established. However, it remains unknown which specific adipocyte-derived factors are most critical in this process. Collagen VI is abundantly expressed in adipocytes. Collagen(-/-) mice in the background of the mouse mammary tumor virus/polyoma virus middle T oncogene (MMTV-PyMT) mammary cancer model demonstrate dramatically reduced rates of early hyperplasia and primary tumor growth. Collagen VI promotes its growth-stimulatory and pro-survival effects in part by signaling through the NG2/chondroitin sulfate proteoglycan receptor expressed on the surface of malignant ductal epithelial cells to sequentially activate Akt and beta-catenin and stabilize cyclin D1. Levels of the carboxyterminal domain of collagen VIalpha3, a proteolytic product of the full-length molecule, are dramatically upregulated in murine and human breast cancer lesions. The same fragment exerts potent growth-stimulatory effects on MCF-7 cells in vitro. Therefore, adipocytes play a vital role in defining the ECM environment for normal and tumor-derived ductal epithelial cells and contribute significantly to tumor growth at early stages through secretion and processing of collagen VI.
Journal of Clinical Investigation 06/2005; 115(5):1163-76. · 15.39 Impact Factor
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Emanuel F Petricoin,
Verena E Bichsel, Valerie S Calvert,
Virginia Espina,
Mary Winters,
Lynn Young,
Claudio Belluco,
Bruce J Trock,
Marc Lippman,
David A Fishman,
Dennis C Sgroi,
Peter J Munson,
Laura J Esserman,
Lance A Liotta
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ABSTRACT: Mapping tumor cell protein networks in vivo will be critical for realizing the promise of patient-tailored molecular therapy. Cancer can be defined as a dysregulation or hyperactivity in the network of intracellular and extracellular signaling cascades. These protein signaling circuits are the ultimate targets of molecular therapy. Each patient's tumor may be driven by a distinct series of molecular pathogenic defects. Thus, for any single molecular targeted therapy, only a subset of cancer patients may respond. Individualization of therapy, which tailors a therapeutic regimen to a tumor molecular portrait, may be the solution to this dilemma. Until recently, the field lacked the technology for molecular profiling at the genomic and proteomic level. Emerging proteomic technology, used concomitantly with genomic analysis, promises to meet this need and bring to reality the clinical adoption of molecular stratification. The activation state of kinase-driven signal networks contains important information relative to cancer pathogenesis and therapeutic target selection. Proteomic technology offers a means to quantify the state of kinase pathways, and provides post-translational phosphorylation data not obtainable by gene arrays. Case studies using clinical research specimens are provided to show the feasibility of generating the critical information needed to individualize therapy. Such technology can reveal potential new pathway interconnections, including differences between primary and metastatic lesions. We provide a vision for individualized combinatorial therapy based on proteomic mapping of phosphorylation end points in clinical tissue material.
Journal of Clinical Oncology 06/2005; 23(15):3614-21. · 18.37 Impact Factor
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Katherine M Sheehan, Valerie S Calvert,
Elaine W Kay,
Yiling Lu,
David Fishman,
Virginia Espina,
Joy Aquino,
Runa Speer,
Robyn Araujo,
Gordon B Mills,
Lance A Liotta,
Emanuel F Petricoin,
Julia D Wulfkuhle
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ABSTRACT: Cancer can be defined as a deregulation or hyperactivity in the ongoing network of intracellular and extracellular signaling events. Reverse phase protein microarray technology may offer a new opportunity to measure and profile these signaling pathways, providing data on post-translational phosphorylation events not obtainable by gene microarray analysis. Treatment of ovarian epithelial carcinoma almost always takes place in a metastatic setting since unfortunately the disease is often not detected until later stages. Thus, in addition to elucidation of the molecular network within a tumor specimen, critical questions are to what extent do signaling changes occur upon metastasis and are there common pathway elements that arise in the metastatic microenvironment. For individualized combinatorial therapy, ideal therapeutic selection based on proteomic mapping of phosphorylation end points may require evaluation of the patient's metastatic tissue. Extending these findings to the bedside will require the development of optimized protocols and reference standards. We have developed a reference standard based on a mixture of phosphorylated peptides to begin to address this challenge.
Molecular & Cellular Proteomics 05/2005; 4(4):346-55. · 7.40 Impact Factor
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ABSTRACT: Defects in cell signaling pathways play a central role in cancer cell growth, survival, invasion and metastasis. An important goal of proteomics is to characterize and develop "circuit maps" of these signaling pathways in normal and diseased cells. We have used reverse-phase protein array technology coupled with laser capture microdissection and phospho-specific antibodies to examine the activation status of several key molecular "gates" involved in cell survival and proliferation signaling in human ovarian tumor tissue. The levels of activated extracellular-regulated kinase (ERK1/2) varied considerably in tumors of the same histotype, but no significant differences between histotypes were observed. Advanced stage tumors had slightly higher levels of phosphorylated ERK1/2 compared to early stage tumors. The activation status of Akt and glycogen synthase kinase 3beta, key proteins and indicators of the state of the phosphatidylinositol 3-kinase/Akt pro-survival pathway also showed more variation within each histotype than between the histotypes studied. Our results demonstrate the utility of reverse phase protein microarrays for the multiplexed analysis of signal transduction from discreet cell populations of cells procured directly from human ovarian tumor specimens and suggest that patterns in signal pathway activation in ovarian tumors may be patient-specific rather than type or stage specific.
PROTEOMICS 12/2003; 3(11):2085-90. · 4.51 Impact Factor
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Robert L Grubb, Valerie S Calvert,
Julia D Wulkuhle,
Cloud P Paweletz,
W Marston Linehan,
John L Phillips,
Rodrigo Chuaqui,
Alfredo Valasco,
John Gillespie,
Michael Emmert-Buck,
Lance A Liotta,
Emanuel F Petricoin
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ABSTRACT: Reverse phase protein arrays represent a new proteomics microarray technology with which to study the fluctuating state of the proteome in minute quantities of cells. The activation status of cell signaling pathways controls cellular fate and deregulation of these pathways underpins carcinogenesis. Changes in pathway activation that occur between early stage prostatic epithelial lesions, prostatic stroma and the extracellular matrix can be analyzed by obtaining pure populations of cell types by laser capture microdissection (LCM) and analyzing the relative states of several key phosphorylation points within the cellular circuitry. We have applied reverse phase protein array technology to analyze the status of key points in cell signaling involved in pro-survival, mitogenic, apoptotic and growth regulation pathways in the progression from normal prostate epithelium to invasive prostate cancer. Using multiplexed reverse phase protein arrays coupled with LCM, the states of signaling changes during disease progression from prostate cancer study sets were analyzed. Focused analysis of phospho-specific endpoints revealed changes in cellular signaling events through disease progression and between patients. We have used a new protein array technology to study specific molecular pathways believed to be important in cell survival and progression from normal epithelium to invasive carcinoma directly from human tissue specimens. With the advent of molecular targeted therapeutics, the identification, characterization and monitoring of the signaling events within actual human biopsies will be critical for patient-tailored therapy.
PROTEOMICS 12/2003; 3(11):2142-6. · 4.51 Impact Factor
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ABSTRACT: Laser capture microdissection was combined with reverse phase protein lysate arrays to quantitatively analyze the ratios of mitochondrial encoded cytochrome c oxidase subunits to nuclear encoded cytochrome c oxidase subunits, and to correlate the ratios with malignant progression in human prostate tissue specimens. Cytochrome c oxidase subunits I-III comprise the catalytic core of the enzyme and are all synthesized from mitochondrial DNA. The remaining subunits (IV-VIII) are synthesized from cellular nuclear DNA. A significant (P < 0.001, 30/30 prostate cases) shift in the relative concentrations of nuclear encoded cytochrome c oxidase subunits IV, Vb, and VIc compared to mitochondrial encoded cytochrome c oxidase subunits I and II was noted during the progression of prostate cancer from normal epithelium through premalignant lesions to invasive carcinoma. Significantly, this shift was discovered to begin even in the premalignant stage. Reverse phase protein lysate array-based observations were corroborated with immunohistochemistry, and extended to a few human carcinomas in addition to prostate. This analysis points to a role for nuclear DNA encoded mitochondrial proteins in carcinogenesis; underscoring their potential as targets for therapy while highlighting the need for full characterization of the mitochondrial proteome.
PROTEOMICS 10/2003; 3(9):1801-10. · 4.51 Impact Factor
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John W Gillespie,
Carolyn J M Best,
Verena E Bichsel,
Kristina A Cole,
Susan F Greenhut,
Stephen M Hewitt,
Mamoun Ahram,
Yvonne B Gathright,
Maria J Merino,
Robert L Strausberg, [......], Valerie S Calvert,
Michael J Flaig,
Rodrigo F Chuaqui,
Judi C Herring,
John Pfeifer,
Emmanuel F Petricoin,
W Marston Linehan,
Paul H Duray,
G Steven Bova,
Michael R Emmert-Buck
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ABSTRACT: Using a general strategy for evaluating clinical tissue specimens, we found that 70% ethanol fixation and paraffin embedding is a useful method for molecular profiling studies. Human prostate and kidney were used as test tissues. The protein content of the samples was analyzed by one-dimensional gel electrophoresis, immunoblot, two-dimensional gel electrophoresis, and layered expression scanning. In each case, the fixed and embedded tissues produced results similar to that obtained from snap-frozen specimens, although the protein quantity was somewhat decreased. Recovery of mRNA was reduced in both quantity and quality in the ethanol-fixed samples, but was superior to that obtained from formalin-fixed samples and sufficient to perform reverse transcription polymerase chain reactions. Recovery of DNA from ethanol-fixed specimens was superior to formalin-fixed samples as determined by one-dimensional gel electrophoresis and polymerase chain reaction. In conclusion, specimens fixed in 70% ethanol and embedded in paraffin produce good histology and permit recovery of DNA, mRNA, and proteins sufficient for several downstream molecular analyses. Complete protocols and additional discussion of relevant issues are available on an accompanying website (http://cgap-mf.nih.gov/).
American Journal Of Pathology 03/2002; 160(2):449-57. · 4.89 Impact Factor
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ABSTRACT: Protein microarrays have been recently employed for signal pathway profiling and high-throughput protein expression analysis. Reverse-phase arrays, where the array consists of immobilized analytes and lysates has especially shown promise in low abundance analyte detec-tion and signal pathway profiling using phos-pho-specific antibodies. A limitation to current reverse phase array methodology is the inabil-ity to multiplex proteomic-based endpoints as each array can only report one analyte end-point. In this study, we report on the use of a dual dye based approach that can effectively double the number of endpoints observed per array allowing, for example, both phosphospe-cific and total protein levels to be measured and analyzed at once. The method utilizes antibody bound dyes that emit in the infrared spectral region as a means of sensitive and specific detection.
