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A functional proteomic method for biomarker discovery.

Reynolds F, Panneer N, Tutino CM, Wu M, Skrabal WR, Moskaluk C, Kelly KA - PLoS ONE (2011)

Bottom Line: Proteomic strategies that allow unbiased identification of proteins and their post-transcriptional and -translation modifications are an essential complement to genomic strategies.Methods are therefore needed that allow rational identification of targets based on function and relevance to disease.For proof of principle, the method successfully identified molecular binding partners, three of them novel, for 15 peptides specific for pancreatic cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia, United States of America.

ABSTRACT
The sequencing of the human genome holds out the hope for personalized medicine, but it is clear that analysis of DNA or RNA content alone is not sufficient to understand most disease processes. Proteomic strategies that allow unbiased identification of proteins and their post-transcriptional and -translation modifications are an essential complement to genomic strategies. However, the enormity of the proteome and limitations in proteomic methods make it difficult to determine the targets that are particularly relevant to human disease. Methods are therefore needed that allow rational identification of targets based on function and relevance to disease. Screening methodologies such as phage display, SELEX, and small-molecule combinatorial chemistry have been widely used to discover specific ligands for cells or tissues of interest, such as tumors. Those ligands can be used in turn as affinity probes to identify their cognate molecular targets when they are not known in advance. Here we report an easy, robust and generally applicable approach in which phage particles bearing cell- or tissue-specific peptides serve directly as the affinity probes for their molecular targets. For proof of principle, the method successfully identified molecular binding partners, three of them novel, for 15 peptides specific for pancreatic cancer.

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Related in: MedlinePlus

Tissue microarray data.Values are pathologist's scoring of number of cells stained (0–3) and intensity of staining (0–3) multiplied together. A) Representative tumor section stained for plectin. Note the membrane staining. B) Pathologist's scoring of human cancer biopsy specimens stained for plectin. C) Representative PDAC tumor biopsy section stained for pyruvate kinase M2. D) Pathologist's scoring of pyruvate kinase M2 stained human cancer biopsy tissue sections.
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pone-0022471-g004: Tissue microarray data.Values are pathologist's scoring of number of cells stained (0–3) and intensity of staining (0–3) multiplied together. A) Representative tumor section stained for plectin. Note the membrane staining. B) Pathologist's scoring of human cancer biopsy specimens stained for plectin. C) Representative PDAC tumor biopsy section stained for pyruvate kinase M2. D) Pathologist's scoring of pyruvate kinase M2 stained human cancer biopsy tissue sections.

Mentions: While these proteins were verified to be associated with the surface of the L3.6pl cell line, we sought to determine the potential relevance to pancreatic cancer of selected proteins through immunohistochemistry. Antibodies to either pyruvate kinase M2 or plectin were used to determine the expression levels and extent of staining in a series of human biopsy specimens (Fig. 4). These were chosen as neither have been previously reported in pancreatic cancer whereas Annexin A2 is a known protein expressed in pancreatic cancer. As the kRAS mutation that initiates pancreatic cancer [17] is common to many other cancers [18], the immunohistochemistry was conducted in a tissue microarray format with 20 of the most common lethal human cancers (n = 3–12 patients for each cancer). A pathologist ranked the stained tissues for percentage of cells stained (0–3) and intensity of staining (0–3), with the average product of these shown in Fig. 4. Plectin shows strong membrane staining in pancreatic cancer (Fig. 4A) and is significant in other cancers including bile duct cholangiocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, ovarian cancer, and intestinal type stomach cancer (Fig. 4B). Closer examination of a representative pancreatic cancer sample (Fig. 4A) shows cytoplasmic and membrane staining, consistent with the cell fractionation data [15]. Pyruvate kinase M2 also showed strong staining in pancreatic cancer and almost all other cancer types (Fig. 4D), with both cytoplasmic and membrane staining (Fig. 4C). These data suggest that both of these may be potential markers of pancreatic cancer and other cancers as well.


A functional proteomic method for biomarker discovery.

Reynolds F, Panneer N, Tutino CM, Wu M, Skrabal WR, Moskaluk C, Kelly KA - PLoS ONE (2011)

Tissue microarray data.Values are pathologist's scoring of number of cells stained (0–3) and intensity of staining (0–3) multiplied together. A) Representative tumor section stained for plectin. Note the membrane staining. B) Pathologist's scoring of human cancer biopsy specimens stained for plectin. C) Representative PDAC tumor biopsy section stained for pyruvate kinase M2. D) Pathologist's scoring of pyruvate kinase M2 stained human cancer biopsy tissue sections.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139652&req=5

pone-0022471-g004: Tissue microarray data.Values are pathologist's scoring of number of cells stained (0–3) and intensity of staining (0–3) multiplied together. A) Representative tumor section stained for plectin. Note the membrane staining. B) Pathologist's scoring of human cancer biopsy specimens stained for plectin. C) Representative PDAC tumor biopsy section stained for pyruvate kinase M2. D) Pathologist's scoring of pyruvate kinase M2 stained human cancer biopsy tissue sections.
Mentions: While these proteins were verified to be associated with the surface of the L3.6pl cell line, we sought to determine the potential relevance to pancreatic cancer of selected proteins through immunohistochemistry. Antibodies to either pyruvate kinase M2 or plectin were used to determine the expression levels and extent of staining in a series of human biopsy specimens (Fig. 4). These were chosen as neither have been previously reported in pancreatic cancer whereas Annexin A2 is a known protein expressed in pancreatic cancer. As the kRAS mutation that initiates pancreatic cancer [17] is common to many other cancers [18], the immunohistochemistry was conducted in a tissue microarray format with 20 of the most common lethal human cancers (n = 3–12 patients for each cancer). A pathologist ranked the stained tissues for percentage of cells stained (0–3) and intensity of staining (0–3), with the average product of these shown in Fig. 4. Plectin shows strong membrane staining in pancreatic cancer (Fig. 4A) and is significant in other cancers including bile duct cholangiocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, ovarian cancer, and intestinal type stomach cancer (Fig. 4B). Closer examination of a representative pancreatic cancer sample (Fig. 4A) shows cytoplasmic and membrane staining, consistent with the cell fractionation data [15]. Pyruvate kinase M2 also showed strong staining in pancreatic cancer and almost all other cancer types (Fig. 4D), with both cytoplasmic and membrane staining (Fig. 4C). These data suggest that both of these may be potential markers of pancreatic cancer and other cancers as well.

Bottom Line: Proteomic strategies that allow unbiased identification of proteins and their post-transcriptional and -translation modifications are an essential complement to genomic strategies.Methods are therefore needed that allow rational identification of targets based on function and relevance to disease.For proof of principle, the method successfully identified molecular binding partners, three of them novel, for 15 peptides specific for pancreatic cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia, United States of America.

ABSTRACT
The sequencing of the human genome holds out the hope for personalized medicine, but it is clear that analysis of DNA or RNA content alone is not sufficient to understand most disease processes. Proteomic strategies that allow unbiased identification of proteins and their post-transcriptional and -translation modifications are an essential complement to genomic strategies. However, the enormity of the proteome and limitations in proteomic methods make it difficult to determine the targets that are particularly relevant to human disease. Methods are therefore needed that allow rational identification of targets based on function and relevance to disease. Screening methodologies such as phage display, SELEX, and small-molecule combinatorial chemistry have been widely used to discover specific ligands for cells or tissues of interest, such as tumors. Those ligands can be used in turn as affinity probes to identify their cognate molecular targets when they are not known in advance. Here we report an easy, robust and generally applicable approach in which phage particles bearing cell- or tissue-specific peptides serve directly as the affinity probes for their molecular targets. For proof of principle, the method successfully identified molecular binding partners, three of them novel, for 15 peptides specific for pancreatic cancer.

Show MeSH
Related in: MedlinePlus