A new approach using the H-SRM assay on a TSQ Quantum Ultra triple quadrupole mass spectrometer for simultaneous qualitative and quantitative targeted protein analysis was evaluated. This approach gave excellent sensitivity, analytical assay precision and quantitative accuracy for targeted protein quantitation in whole human serum by dramatically reducing non-specific interference from serum background ions. The H-SRM assay greatly improves assay specificity and detection limits thereby offering significant advantages for biomarker verification and validation studies.

1. The proposed H-SRM assay for peptide detection and confirmation with time alignment of multiple fragment ions from each peptide had lower detection limits than the traditional approach for peptide detection and identification using SRM-triggered MS/MS data acquisition and provided wider quantitative dynamic range.
All 50 targeted peptides were identified using the H-SRM methodology. Traditional approaches identified only 36 peptides (QED-MS/MS on TSQ Quantum Ultra) and 32 peptides (EPI MS/MS on 4000 Q TRAP). The new approach also allowed for efficient simultaneous targeted peptide peak identification and quantitation in a single LC run.

2. The H-SRM assay provided significant improvement of S/N ratios resulting in unambiguous detection of targeted peptides. Excellent analytical precision was obtained for all selected dwell times (5 ms, 10 ms, and 20 ms) on the TSQ Quantum Ultra. Approximately 95% of the peptides gave CVs <15% for all three dwell times. No significant signal loss was observed when using shorter dwell times.

3. The H-SRM assay provided the widest dynamic range for targeted peptide quantitation. The dynamic range for detecting proteins in this study was over four
orders of magnitude.

4. The quantitative accuracy of the H-SRM assay was excellent. The average relative quantitation error was ±4% 5. Aided by higher resolution precursor ion isolation and faster cycle time, the TSQ Quantum Ultra provided high quality SRM-triggered QED-MS/MS data and identified more targeted peptides by a Mascot search compared with the 4000 Q TRAP.

6. Monitoring multiple y-type product ions (four or more) provided the most selective and sensitive means of further method refinement to determine proteotypic peptides and corresponding transitions including fragment
ion ratios, which could subsequently be used for validated methods.

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