Application Note: High-Throughput SISCAPA-based Peptide Quantitation Using an Agilent RapidFire High-Throughput Mass Spectrometry System
2 January 2013
Mass spectrometry is increasingly used to measure proteins in biological fluids and provide an effective method for reproducible quantitation of biomarkers. However, the major restrictions are limited sensitivity and low throughput associated with peptide chromatography. This application note, demonstrates how to overcome these limitations using Agilent RapidFire/MS technology to enable sensitive quantitation of peptides from plasma digests thus creating a high-throughput stable isotope standards and capture by antipeptide antibodies (SISCAPA) workflow. This workflow has been developed to enrich low abundance peptides from complex mixtures such as trypsin-digested human plasma or serum. Ultilizing the optimized SISCAPA procedure generated eluted peptides with sufficient purity to be analyzed by the RapidFire platform without chromatography upstream of MS, thus reducing the cycle time to just 10 seconds per sample. This method can be multiplexed to facilitate analysis of large sample sets for biomarker analysis in a fraction of the time of other methods.