Large-scale implementation of sequential protein and peptide immunoaffinity enrichment LC/nanoLC-MS/MS for human β-nerve growth factor

Background: A previously described immunoaffinity (IA)-LC-MS/MS assay for human beta-nerve growth factor (beta-NGF) was implemented to support large-scale sample testing for multiple clinical trials. Methodology & results: The procedure was modified to increase throughput by simultaneous preparation of two 96-well plates and LC duty-cycle reduction. Robustness of the LC method and nano-ESI was ensured during large-scale assay execution by closely monitoring and, if needed, replacing system components prior to failure. Following validation, the assay was used to analyze approximately 19,000 samples from multiple clinical studies over several years. Conclusion: Routine implementation of the beta-NGF IA-LC-MS/MS assay supported drug development programs. This optimized assay format now serves as a template for other clinical protein biomarker assays.