Optimized Workflow for Proteomics and Phosphoproteomics With Limited Tissue Samples

Proteomics and phosphoproteomics play crucial roles in elucidating the dynamics of post-transcriptional processes. While experimental methods and workflows have been established in this field, a persistent challenge arises when dealing with small samples containing a limited amount of protein. This limitation can significantly impact the recovery of peptides and phosphopeptides. In response to this challenge, we have developed a comprehensive experimental workflow tailored specifically for small-scale samples, with a special emphasis on neuronal tissues like the trigeminal ganglion. Our proposed workflow consists of seven steps aimed at optimizing the preparation of limited tissue samples for both proteomic and phosphoproteomic analyses. One noteworthy innovation in our approach involves the utilization of a dual enrichment strategy for phosphopeptides. Initially, we employ Fe-NTA Magnetic beads, renowned for their specificity and effectiveness in capturing phosphopeptides. Subsequently, we complement this approach with the TiO₂-based method, which offers a broader spectrum of phosphopeptide recovery. This innovative workflow not only overcomes the challenges posed by limited sample sizes but also establishes a new benchmark for precision and efficiency in proteomic investigations. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Protein extraction and digestion Basic Protocol 2: TMT labeling and peptide cleanup Basic Protocol 3: IMAC Fe-NTA magnetic beads phosphopeptide enrichment Basic Protocol 4: TiO2 enrichment Basic Protocol 5: Fe-NTA phosphopeptide Enrichment Basic Protocol 6: High pH peptide fractionation Basic protocol 7: LC-MS/MS analysis and database search.