In-Depth, Reproducible Analysis of Human Plasma Using IgY 14 and SuperMix Immunodepletion
| Autor: | David W. Speicher, Kurt T. Barnhart, Lynn A. Beer, Bonnie Ky |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
Proteomics
0301 basic medicine Proteome Immunoglobulins Mass spectrometry High-performance liquid chromatography Article Plasma 03 medical and health sciences Tandem Mass Spectrometry medicine Humans Polyacrylamide gel electrophoresis Chromatography 030102 biochemistry & molecular biology biology Chemistry Trypsin Blood proteins 030104 developmental biology biology.protein Electrophoresis Polyacrylamide Gel Antibody Biomarkers Chromatography Liquid medicine.drug |
| Zdroj: | Methods in Molecular Biology ISBN: 9781493970568 |
| DOI: | 10.1007/978-1-4939-7057-5_7 |
| Popis: | Identification of cancer and other disease biomarkers in human plasma has been exceptionally challenging due to the complex nature of plasma and the presence of a moderate number of high- and medium-abundance proteins which mask low-abundance proteins of interest. As a result, immunoaffinity depletion formats combining multiple antibodies to target the most abundant plasma proteins have become the first stage in most plasma proteome discovery schemes. This protocol describes the use of tandem IgY 14 and SuperMix immunoaffinity depletion to reproducibly remove >99% of total plasma protein. This greatly increases the depth of analysis of human plasma proteomes. Depleted plasma samples can then be analyzed in a single high-resolution LC-MS/MS run on a Q Exactive Plus mass spectrometer, followed by label-free quantitation. If greater depth of analysis is desired, the depleted plasma can be further fractionated by separating the sample for a short distance on a 1D SDS gel and cutting the gel into uniform slices prior to trypsin digestion. Alternatively, the depleted plasma can be reduced, alkylated, and digested with trypsin followed by high-pH reversed-phase HPLC separation. |
| Databáze: | OpenAIRE |
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