Mass spectrometry-based proteomics has transformed the study of cell signalling, enabling global studies of dynamic protein phosphorylation. A widespread post-translational modification, phosphorylation regulates almost all cellular processes, with two-thirds of cellular proteins containing phosphorylation sites.
The authors in this leading proteomics lab recently published their EasyPhos workflow, which simplified measurement of large numbers of phosphoproteomes without compromised coverage and addressed practical challenges posed by proteome-wide analysis of such complex phosphorylation events. They eliminated the need for fractionation, creating an inherent sensitivity advantage and performed the entire protocol in parallel 96-well format. The increased sample throughput enabled analysis of hundreds of phosphoproteomes in minimal time at impressive depth (>10,000 quantified phosphorylation sites), in single LC–MS runs. A key limitation: being optimized for ~1 mg of protein starting material, the protocol fell short when presented with smaller starting quantities.
Here, they detail their updated protocol to address this, including troubleshooting:
First, they eliminate protein precipitation steps, allowing the entire protocol (including digestion) to be performed in a single 96-well plate. This greatly minimises opportunities for sample loss or variability and reduces sample preparation time by a whole day. Further, they re-evaluate all buffers used, to maximize performance with reduced quantities of starting material.
The nanospray columns used for ultra-high-performance liquid chromatography (UHPLC)–MS/MS analysis were initially self-pulled and packed in-house, but in a further timesaver, the authors recommend IonOpticks Aurora columns as an equally effective alternative.
Applying the modified protocol to glioblastoma cells treated with epidermal growth factor (EGF), EasyPhos quantified 20,132 distinct phosphopeptides from 200μg of protein in under one day of measurement time, revealing thousands of EGF-regulated phosphorylation events.
Read the full paper
High-throughput and high-sensitivity phosphoproteomics with the EasyPhos platform.
Nat Protoc 13, 1897–1916 (6 Sept 2018). doi: https://doi.org/10.1038/s41596-018-0014-9
Humphrey SJ, Karayel O, James DE, Mann M.
Commentary by Jarrod Sandow, PhD.
About the author
Jarrod has a background in biotechnology and completed his PhD at the Institute of Medical and Veterinary Science in Adelaide. He is a co-inventor of IonOpticks’ core technology and is driven towards developing innovative solutions for the global proteomics research community that will enable scientists and clinicians to discover more from their samples to accelerate advances in biological and medical research.