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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

Validation of affinity partner for phage clones.A) Western blot of the protein that binds to clone 8, probed using anti-pyruvate kinase M2 antibody. B) ELISA of clone 8 incubated with purified pyruvate kinase M2, or with BSA or recombinant annexin A2 as negative controls. C) Western blot of cell fractionation using anti-pyruvate kinase M2 antibody D) ELISA on intact, non-permeabilized L3.6pl cells with anti-pyruvate kinase M2 antibody. E) Western blot of clone 15 associated protein probed with anti annexin A2 antibody. E) ELISA showing binding of clone 15 to annexin A2 protein. F) Western blot of cell fractionation using anti annexin A2 antibody. G) ELISA on intact, non-permeabilized L3.6pl cells with anti-Annexin A2 antibody.
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pone-0022471-g003: Validation of affinity partner for phage clones.A) Western blot of the protein that binds to clone 8, probed using anti-pyruvate kinase M2 antibody. B) ELISA of clone 8 incubated with purified pyruvate kinase M2, or with BSA or recombinant annexin A2 as negative controls. C) Western blot of cell fractionation using anti-pyruvate kinase M2 antibody D) ELISA on intact, non-permeabilized L3.6pl cells with anti-pyruvate kinase M2 antibody. E) Western blot of clone 15 associated protein probed with anti annexin A2 antibody. E) ELISA showing binding of clone 15 to annexin A2 protein. F) Western blot of cell fractionation using anti annexin A2 antibody. G) ELISA on intact, non-permeabilized L3.6pl cells with anti-Annexin A2 antibody.

Mentions: The methodology for identifying the cell-surface binding partners of the phage is very similar to an immunoprecipitation. Phage were labeled with biotin and sulfo-SAED, a photoactive crosslinker. Loading of sulfo-SAED was characterized by fluorescence spectroscopy to be 660 photolinkers/plaque forming unit. These labeled phage clones were incubated with the cells then photolysed to activate the sulfo-SAED; crosslinking the phage with the protein they bind to. The cells were lysed, the phage and protein extracted with streptavidin beads and the cross linked protein released by disulfide cleavage, then analyzed by SDS/PAGE. Gel analysis demonstrated that Clone 8 has one major band at 60 KDa (Fig. 2A). Tryptic digest of the band followed by mass spectroscopic analysis identified pyruvate kinase M2 as the protein present in the analyzed band (Fig. 2B). In contrast Clone 15 yielded a band at 37 KDa (Fig. 2C) that upon analysis was revealed to be annexin A2 (Fig. 2D). To confirm the mass spectroscopy data, the samples from the pulldowns were examined by western blotting (Fig. 3A and 3E). As a further independent confirmation of the protein binding partner of the phage clones, ELISA was used to examine the phage binding to recombinant proteins (Fig. 3B and 3F). For example, phage clone 8 had a 9-fold higher binding to pyruvate kinase M2 when compared with BSA. In addition, clone 15 was specific to Annexin A2 as it had 5 fold higher binding when compared with BSA. A few phage clones were cross examined against proteins other than BSA that did not match their binding partner to control against general non-specific binding to other proteins (Fig. 3B).


A functional proteomic method for biomarker discovery.

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

Validation of affinity partner for phage clones.A) Western blot of the protein that binds to clone 8, probed using anti-pyruvate kinase M2 antibody. B) ELISA of clone 8 incubated with purified pyruvate kinase M2, or with BSA or recombinant annexin A2 as negative controls. C) Western blot of cell fractionation using anti-pyruvate kinase M2 antibody D) ELISA on intact, non-permeabilized L3.6pl cells with anti-pyruvate kinase M2 antibody. E) Western blot of clone 15 associated protein probed with anti annexin A2 antibody. E) ELISA showing binding of clone 15 to annexin A2 protein. F) Western blot of cell fractionation using anti annexin A2 antibody. G) ELISA on intact, non-permeabilized L3.6pl cells with anti-Annexin A2 antibody.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022471-g003: Validation of affinity partner for phage clones.A) Western blot of the protein that binds to clone 8, probed using anti-pyruvate kinase M2 antibody. B) ELISA of clone 8 incubated with purified pyruvate kinase M2, or with BSA or recombinant annexin A2 as negative controls. C) Western blot of cell fractionation using anti-pyruvate kinase M2 antibody D) ELISA on intact, non-permeabilized L3.6pl cells with anti-pyruvate kinase M2 antibody. E) Western blot of clone 15 associated protein probed with anti annexin A2 antibody. E) ELISA showing binding of clone 15 to annexin A2 protein. F) Western blot of cell fractionation using anti annexin A2 antibody. G) ELISA on intact, non-permeabilized L3.6pl cells with anti-Annexin A2 antibody.
Mentions: The methodology for identifying the cell-surface binding partners of the phage is very similar to an immunoprecipitation. Phage were labeled with biotin and sulfo-SAED, a photoactive crosslinker. Loading of sulfo-SAED was characterized by fluorescence spectroscopy to be 660 photolinkers/plaque forming unit. These labeled phage clones were incubated with the cells then photolysed to activate the sulfo-SAED; crosslinking the phage with the protein they bind to. The cells were lysed, the phage and protein extracted with streptavidin beads and the cross linked protein released by disulfide cleavage, then analyzed by SDS/PAGE. Gel analysis demonstrated that Clone 8 has one major band at 60 KDa (Fig. 2A). Tryptic digest of the band followed by mass spectroscopic analysis identified pyruvate kinase M2 as the protein present in the analyzed band (Fig. 2B). In contrast Clone 15 yielded a band at 37 KDa (Fig. 2C) that upon analysis was revealed to be annexin A2 (Fig. 2D). To confirm the mass spectroscopy data, the samples from the pulldowns were examined by western blotting (Fig. 3A and 3E). As a further independent confirmation of the protein binding partner of the phage clones, ELISA was used to examine the phage binding to recombinant proteins (Fig. 3B and 3F). For example, phage clone 8 had a 9-fold higher binding to pyruvate kinase M2 when compared with BSA. In addition, clone 15 was specific to Annexin A2 as it had 5 fold higher binding when compared with BSA. A few phage clones were cross examined against proteins other than BSA that did not match their binding partner to control against general non-specific binding to other proteins (Fig. 3B).

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