A Direct Comparison of Sample Preparation Formats used for Immunoaffinity-Mass Spectrometry Based Quantification of Protein Biomarkers

摘要

1. Antibody loading of 1 μg is sufficient for both formats (4 μg used to ensure excess). 2. When using protein A/G coupling, DSS cross-linking was necessary for both formats. 3. On-substrate digestion is used as our default, unless target protein needs to be reduced/alkylated. 4. Beads yielded approximately 3-fold greater signal for both analytes which was attributed to greater recovery, however, beads also exhibited greater background (～2-fold), which scaled with sample volume used. 5. Similar precision was obtained by beads and plates and was not affected by on- versus off-substrate digestion. 6. Plates are limited to sample volumes of ≤200 μL. In contrast, beads allow linear scaling to 1 mL enabling increased sample pre-concentration to occur. 7. Plates offer vastly improved speed and flexibility owing to compatibility with semi-automated methods (multi-channel pipettors, plate washers, etc.)