Phospho-Proteomics Method Optimization and Application to Stimulated Jurkat Cells

TL;DR

This study optimizes phospho-proteomics methods for low-input samples, exploring lysis conditions and applying the approach to stimulated Jurkat cells to identify key phospho-sites involved in T-cell activation.

Contribution

It introduces optimized protocols for low-input phospho-proteomics and demonstrates their application in identifying phospho-sites in stimulated Jurkat cells.

Findings

Higher phospho-peptide identification with phospho-bulk titration.

Optimal decrosslinking achieved with boiling in Tris-SDS for 60 min.

Identification of 7 phospho-sites related to T-cell stimulation.

Abstract

In clinical proteomics, available input is often limited. In addition, phospho-proteomics is of particular interest since the dysregulation of these post-translational modifications (PTMs) has been implicated in various diseases such as cancer. We therefore assessed the feasibility of low input phospho-proteomics via phospho-bulk titration and low-input starting material. We found that there was identification of more phospho-peptides through phospho-bulk titration because of sample loss during preparation of low input starting material. Additionally, we explored various lysis buffers and boiling times for efficiency of decrosslinking formalin-fixed cells since cells and tissues are often fixed for preservation and sorting via FACS. We found that boiling in 0.05M Tris pH 7.6 with 5% SDS for 60 min yielded the highest number of phospho-peptides. Lastly, we applied Evotips Pure and phospho-bulk titration to treated Jurkat cells and identified 7 phospho-sites involved in T-cell stimulation.