High-Throughput SISCAPA Quantitation of Peptides from Human Plasma Digests by Ultrafast, Liquid Chromatography-Free Mass Spectrometry
Leigh Anderson, PhD
Friday, March 8, 2013
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Chapters
Introduction
Protein Quantitation by Immunoassay is Subject to Serious Weaknesses in Specificity
Mass Spectrometry
MRM of Proteotypic Tryptic Peptides Provides Highly Specific Assays for Proteins > 1ug/ml in Plasma
Plasma Proteins Measureable by Direct MRM Measurement
Addressing MRM Limitations via Specific Enrichment of Analyte Peptides: SISCAPA
SISCAPA Process Schematic Diagram
Combining the Best Features of Mass Spectrometry and Immunoassay: SISCAPA
SISCAPA Peptide Enrichment
Characterizing a SISCAPA Assay: Soluble Mesothelin Peptide LLGPHVEGLK in Plasma
Practical Issues in Large Scale Peptide-MS Assays: Robustness & Automation
Automated SISCAPA-MRM Platform
Automated SISCAPA Work␣ow in 96-well Plates
Tryptic Digestion: A Previously Unoptiomized Workflow Step
Reproducibility of Automated Digestion
Inter-assay Variation: Small and Correctable
Day to Day Variation for Real Samples
Sample Volume Determined by MS Detection Sensitivity
Peptide Signal Scales with Plasma Sample Volume
Increasing MS Detection Throughput: Faster LC - or No LC?
First Step Towards High-Throughput MRM: Move From Nano␣ow to Normal Flow Chromatography
Normal Flow Allows Reduced LC-MS Cycle Time for Peptide Quantitation
High Speed QQQ-MS Peptide Quantitation Without Chromatography: RapidFire
High Throughput Peptide Quantitation by Direct Injection (RapidFire)
Medium-to-High Abundance Proteins Measured Without Chromatography via SISCAPA-RapidFire
Comparison of 7sec vs 3min MS Methods
Summary
Acknowledgments
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