Colloidal Silica as a Platform for Trace Protein Analysis and Recovery

Persistent Link:
http://hdl.handle.net/10150/202703
Title:
Colloidal Silica as a Platform for Trace Protein Analysis and Recovery
Author:
Egas Proaño, David Alexis
Issue Date:
2011
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Early intervention in cancer and other illnesses is highly desired. The evolution of the proteomics field has benefited the possibility of getting to this goal by allowing researchers to look at different biomarkers. However, the high complexity of biological samples and the low levels at which biomarkers are found in these fluids make the analyses even more complicated.Protein microarrays have arisen as alternatives to traditional methods to look at multiple protein levels simultaneously with the benefit of high specificity, low limits of detection and the requirement of small samples. In this work, a significant improvement in net signals obtained with fluorescent detection (using three-dimensional scaffolds based on silica colloidal crystals -SCC-) is presented in contrast to commercially available flat substrates.A novel approach to extract trace proteins in solution and more complex matrices by using sub-micrometer silica particles as support for antibodies in affinity capture experiments is presented. Bovine Serum Albumin, Ephrin-receptor A2, Alpha-fetoprotein, and Prostate Specific Antigen have been used as model proteins. Recoveries of 90% or more are obtained with this method and reusability of the particles was achieved. MALDI-MS detection was successfully performed with the protein extracts which opens up the opportunity of further analysis such as determining post-translational modifications which is relevant when dealing with biomarker candidates.Last we present the use of our substrates as alternatives to conventional targets in mass spectrometry (MS). Traditional Matrix-assisted Laser Desorption Ionization MS (MALDI-MS) of proteins presents the problem of adduct formation with clusters from the matrix used in the process. Those adducts can affect the accurate determination of the molecular weight for a given protein and when could potentially mask slight differences in molecular weight of very similar proteins in mixtures. We present the alternative of using SCC on silicon wafers as a target for MALDI-MS samples. Our peak widths are extremely narrow and approach the one of the isotopic envelops. At the same time, porosity of our material seems to prevent the formation of adducts, which enables the differentiation of proteins with small molecular weight differences like mutants or same proteins from different sources.
Type:
text; Electronic Dissertation
Keywords:
Recovery; Silica; Trace; Chemistry; Colloidal; Protein
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Chemistry
Degree Grantor:
University of Arizona
Advisor:
Wirth, Mary J.; Wysocki, Vicky H.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleColloidal Silica as a Platform for Trace Protein Analysis and Recoveryen_US
dc.creatorEgas Proaño, David Alexisen_US
dc.contributor.authorEgas Proaño, David Alexisen_US
dc.date.issued2011-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractEarly intervention in cancer and other illnesses is highly desired. The evolution of the proteomics field has benefited the possibility of getting to this goal by allowing researchers to look at different biomarkers. However, the high complexity of biological samples and the low levels at which biomarkers are found in these fluids make the analyses even more complicated.Protein microarrays have arisen as alternatives to traditional methods to look at multiple protein levels simultaneously with the benefit of high specificity, low limits of detection and the requirement of small samples. In this work, a significant improvement in net signals obtained with fluorescent detection (using three-dimensional scaffolds based on silica colloidal crystals -SCC-) is presented in contrast to commercially available flat substrates.A novel approach to extract trace proteins in solution and more complex matrices by using sub-micrometer silica particles as support for antibodies in affinity capture experiments is presented. Bovine Serum Albumin, Ephrin-receptor A2, Alpha-fetoprotein, and Prostate Specific Antigen have been used as model proteins. Recoveries of 90% or more are obtained with this method and reusability of the particles was achieved. MALDI-MS detection was successfully performed with the protein extracts which opens up the opportunity of further analysis such as determining post-translational modifications which is relevant when dealing with biomarker candidates.Last we present the use of our substrates as alternatives to conventional targets in mass spectrometry (MS). Traditional Matrix-assisted Laser Desorption Ionization MS (MALDI-MS) of proteins presents the problem of adduct formation with clusters from the matrix used in the process. Those adducts can affect the accurate determination of the molecular weight for a given protein and when could potentially mask slight differences in molecular weight of very similar proteins in mixtures. We present the alternative of using SCC on silicon wafers as a target for MALDI-MS samples. Our peak widths are extremely narrow and approach the one of the isotopic envelops. At the same time, porosity of our material seems to prevent the formation of adducts, which enables the differentiation of proteins with small molecular weight differences like mutants or same proteins from different sources.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectRecoveryen_US
dc.subjectSilicaen_US
dc.subjectTraceen_US
dc.subjectChemistryen_US
dc.subjectColloidalen_US
dc.subjectProteinen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorWirth, Mary J.en_US
dc.contributor.advisorWysocki, Vicky H.en_US
dc.contributor.committeememberPemberton, Jeanne E.en_US
dc.contributor.committeememberWalker, F. Annen_US
dc.contributor.committeememberGuzmán, Roberto Z.en_US
dc.contributor.committeememberWirth, Mary J.en_US
dc.contributor.committeememberWysocki, Vicky H.en_US
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