Workflow to Quantify Protein Biomarkers By Liquid Chromatograph-Tandem Mass Spectrometry

Recent advances in LC-MS/MS technology and the use of hybrid sample preparation techniques where immunoprecipitation is used to isolate proteins from biological matrices prior to LC-MS/MS analysis, have resulted in the increased use of LC-MS/MS assays to quantify protein biomarkers. However, there remains a lack of industry-standard guidance on method development and validation of hybrid LC-MS/MS assays, particularly regarding the assessment of parallelism. For hybrid LC-MS/MS assays, parallelism needs to be established between the surrogate and authentic matrices, and also between the recombinant and authentic forms of the protein. This paradigm is unlike the one generally used in small molecule LC-MS/MS biomarker assays where parallelism need only be established between one of the pairs above. In this presentation, we will use an endogenous ~80 kDa protein as an exemplar biomarker, and human and mouse plasma as authentic matrices, and demonstrate a three-step workflow that collectively: 1) identifies a suitable surrogate matrix for quantification of the protein, 2) establishes parallelism between the surrogate matrix and authentic matrix, and 3) assesses parallelism between the recombinant and authentic forms of the protein. We will also describe a method qualification step that tests whether both forms of parallelism have been achieved and/or accounted for. Additionally, we will demonstrate how non-specific binding can greatly impact surrogate matrix selection, and will introduce the audience to the concept of the matrix:antibody ratio (“MAR”), which can be used to establish parallelism between the surrogate and authentic matrices. The MAR is analogous to the concept of minimum required dilution (“MRD”) used frequently in ligand-binding assays (“LBAs”), and we will discuss the comparability of MAR and MRD and the pros and cons of each method. Finally, we will offer strategies to adjust for a lack of parallelism at any step in the workflow, to enhance the accuracy of the measurement of the endogenous proteins. Overall, we will present a tool that can be used in future bioanalytical studies to develop effective hybrid LC-MS/MS methods for endogenous protein biomarkers of interest.