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Combined proteomic approaches for the identification of specific amino acid residues modified by 4-hydroxy-2-nonenal under physiological conditions
- Source :
- Journal of proteome research. 9(11)
- Publication Year :
- 2010
-
Abstract
- Proteins modified by 4-hydroxy-2-nonenal (HNE) are cellular markers of oxidative stress in health and disease. HNE is generated by free radical chain reactions during oxidative stress as a major end-product of the oxidative fatty acid metabolism. Identification and quantitative analysis of HNE-modified proteins are readily performed by using specific antibodies raised against them. Further on, the identification of the amino acid residues involved in the HNE-modification is an additional step in proteomic post-transcriptional modification analysis to explain the nature of the specificity underlying oxidative stress mechanisms. For this purpose, a combined protocol of immune-detection, peptide enrichment, mass spectrometry, and de novo protein sequencing has been developed. The methodology was first examined in the model protein bovine serum albumin (BSA), allowing the comparison of matrix-assisted laser desorption/ionization-tandem time of flight (MALDI-TOF/TOF) mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS) performance and sensitivity. Peptide enrichment was optimized by affinity chromatography on HNE-BSA resulting in increased sensitivity. Identification of amino acid residues modified by HNE was finally ascertained by de novo sequencing analysis. The improved methodology was demonstrated on human erythrocyte membrane proteins allowing the identification of HNE-lysine and HNE-histidine Michael adducts in the β-spectrin under physiological conditions.
- Subjects :
- Proteomics
Peptide
medicine.disease_cause
Mass spectrometry
Biochemistry
Protein sequencing
Affinity chromatography
medicine
Animals
Humans
Histidine
Bovine serum albumin
Amino Acids
chemistry.chemical_classification
Aldehydes
biology
Lysine
Erythrocyte Membrane
Membrane Proteins
Spectrin
Serum Albumin, Bovine
General Chemistry
Oxidative Stress
chemistry
biology.protein
Cattle
Post-translational protein modification
Quantitative analysis (chemistry)
Protein Processing, Post-Translational
Oxidative stress
Biomarkers
Subjects
Details
- ISSN :
- 15353907
- Volume :
- 9
- Issue :
- 11
- Database :
- OpenAIRE
- Journal :
- Journal of proteome research
- Accession number :
- edsair.doi.dedup.....fc05ad93246e2389b2e7cbd5d65e3afc