Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification.

摘要: Absolute quantification of target proteins within complex biological samples is critical to a wide range research and clinical applications. This protocol provides step-by-step instructions for the development application quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic peptides are identified based on numerous criteria. includes identifying proteotypic peptides, avoiding sites posttranslational modification, analyzing uniqueness peptide protein. Next, crude external standards synthesized used develop SRM assays, resulting perform qualitative analyses samples. Finally, purified, quantified, heavy isotope labeled internal prepared dilution series assays. Analysis all MS data presented. was accurately assay absolute abundance chemotaxis signaling pathway RAW 264.7 cells (a mouse monocyte/macrophage cell line). The Gi2 heterotrimeric G-protein α-subunit) described in detail.