This application note describes a method for high-sensitivity SISCAPA peptide quantitation using Agilent UHPLC and the 6490 QQQ. SISCAPA is an antibody-capture-based quantitative method that allows high-sensitivity, high-specificity peptide quantitation for small molecule peptides, low-concentration peptides, and peptides that are difficult to quantify.
This document describes a method for specific and quantitative measurement of protein biomarkers in plasma using Stable Isotope-Labeled Standards with Capture on Anti-Peptide Antibodies (SISCAPA). The technique enriches target peptides from complex digests to enable detection at low concentrations. The study evaluates the performance of the Agilent 6490 Triple Quadrupole Mass Spectrometer equipped with iFunnel technology in this context.
| Column | Eclipse Plus EC-C18, 1.8 um, 2.1 x 50 mm |
|---|---|
| Column temperature | 35 °C |
| Flow rate | 0.6 mL/min |
| Injection volume | 20 pL |
| Stop time | 5 min |
| Drying gas | 15 L/min, 150 °C |
| Sheath gas | 11 L/min, 200 °C |
| Neblizer pressure | 30 psig |
| Nozzle voltage | 300 V |
| Cycle time | 500 ms |
The analytical run time is 5 minutes.
The method provides approximately 6-fold higher sample throughput than nanoflow methods.
The lower limit of detection is in the neighborhood of 50 amol/10 pL digest.