Spectral libraries contain years of confirmed peptide IDs, but most remain unused after discovery work wraps. Moving those findings into routine quantitation demands new standards, fresh optimization, and long validation cycles that slow method development.
This application note shows how Agilent scientists built a 1,000-protein targeted panel on the 6495D triple quadrupole using an existing K562 library and Skyline. The workflow avoids synthetic peptide costs, achieves single-cell-equivalent loads, and delivers a median CV of 7.5 percent with 98.5 percent retention time agreement against high-resolution MS. Integrated nanoflow LC, a Newomics ion source, and automated sample loading make the method practical for routine studies.
Download the white paper to:
- Learn the method development workflow that bypasses isotope-labeled standards
- Understand how multi-ion confirmation and retention time prediction enable high-confidence peptide identification
- See the validation approach comparing MRM and data-dependent acquisition across 456 peptides
