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Updated: Jun 5, 2026
09:04
Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification
Published on: August 17, 2015
Evaluation of large scale quantitative proteomic assay development using peptide affinity-based mass spectrometry
Jeffrey R Whiteaker1, Lei Zhao, Susan E Abbatiello
1Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, WA 98109, USA.
Molecular & Cellular Proteomics : MCP
|January 20, 2011
Summary
Developing antibodies for stable isotope standards and capture by antipeptide antibodies (SISCAPA) assays is feasible. Multiplex immunization strategies enable high success rates for generating quantitative protein assays from human plasma.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Immunology
Background:
- Stable isotope standards and capture by antipeptide antibodies (SISCAPA) is a method for quantitative peptide measurement.
- Antibody production is crucial for developing robust SISCAPA assays.
- Large-scale development requires understanding antibody production success rates.
Purpose of the Study:
- To assess the feasibility and success rate of producing suitable antipeptide antibodies for SISCAPA assays.
- To inform strategies for large-scale development of SISCAPA assays.
- To evaluate a multiplex immunization strategy for antibody generation.
Main Methods:
- A multiplex immunization strategy using up to five proteotypic peptides per protein target in rabbits.
- Enzyme-linked immunosorbent assay (ELISA) for measuring antipeptide antibody titers.
- Characterization of affinity-purified antibodies in SISCAPA-multiple reaction monitoring assays using human plasma.
Main Results:
- 403 proteotypic peptides from 89 protein targets were used for immunization.
- 220 antipeptide antibodies were selected for affinity purification.
- Over 50% of assays detected target peptides at <0.5 fmol/μl in human plasma.
- 100% assay success rate for targeted proteins using multiplex immunization.
Conclusions:
- The multiplex immunization strategy is effective for generating antipeptide antibodies for SISCAPA assays.
- It is feasible for a single laboratory to develop hundreds of SISCAPA assays annually.
- This approach supports cost-effective generation of numerous quantitative protein assays.
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