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Updated: May 27, 2026
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Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry
Published on: July 31, 2011
Optimized conditions for a quantitative SELDI TOF MS protein assay
Lee Lomas1, Charlotte H Clarke, Vanitha Thulasiraman
1Laboratory Separations Division, Bio-Rad Laboratories, Hercules, CA, USA. lee_lomas@bio-rad.com
Methods in Molecular Biology (Clifton, N.J.)
|November 16, 2011
Summary
This study introduces a novel method for reproducible peptide and protein diagnostic assays using surface-enhanced laser desorption/ionization (SELDI) mass spectrometry. By incorporating a complex protein matrix, the assay achieves high quantitative reproducibility with less than 10% coefficient of variation.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Biotechnology
Background:
- Diagnostic test development relies on factors like sample availability and quantitative reproducibility.
- Laser Desorption/Ionization Mass Spectrometry (LDI-MS) is suitable for peptide and protein analysis.
- Surface-Enhanced Laser Desorption/Ionization (SELDI)-biochips enhance reproducibility and relative quantitation in MS.
Purpose of the Study:
- To develop reproducible SELDI-based assays for peptide and protein diagnostics.
- To establish a method for improved quantitative reproducibility in SELDI assays.
- To present a strategy for internal normalization and reproducible ionization background.
Main Methods:
- Incorporating a complex artificial protein matrix into the sample for analysis.
- Utilizing the protein matrix to create a common and reproducible ionization background.
- Employing internal normalization standards within the SELDI-MS assay.
Main Results:
- Achieved quantitative assays with coefficients of variation (CVs) typically less than 10% across assays and days.
- Demonstrated the successful implementation of the method using an E. coli protein matrix.
- Outlined criteria for selecting suitable complex protein matrices for SELDI assay development.
Conclusions:
- The developed SELDI assay method significantly enhances quantitative reproducibility.
- The incorporation of a complex protein matrix is key to achieving reliable diagnostic assays.
- The method is adaptable to various protein matrix sources provided specific criteria are met.
