AI Article Synopsis
- Protein phosphorylation is a key signaling mechanism, but standard analyses often miss low-abundance phosphorylation events, limiting the detection of important changes in individual proteins.
- The proposed method enhances phospho-mapping by optimizing the enrichment of phosphopeptides from immunoprecipitated samples using a cost-effective Fe-NTA approach, which is compatible with existing protocols.
- This technique allows for better coverage of phosphorylation sites, improving insights into protein phosphorylation patterns, and complements current phosphoproteomics analyses.
Article Abstract
Protein phosphorylation is a nearly universal signaling mechanism. To date, a number of proteomics tools have been developed to analyze phosphorylation. Phosphoproteome-wide analyses using whole cell extracts suffer from incomplete coverage, often missing phosphorylation events from low-abundance proteins. In order to increase coverage of phosphorylation sites on individual proteins of interest ("phospho-mapping"), immunoprecipitation (IP) followed by phosphoenrichment is necessary. Unfortunately, most commercially available phosphoenrichment kits are not readily scalable to the low-microgram quantities of protein present in IP eluates. Here, we describe a simple method specifically optimized for the enrichment of phosphopeptides from IP samples using an Fe-NTA based method. This method can be added downstream of any standard immunoprecipitation protocol and upstream of any MS analysis pipeline. The protocol described herein is cost effective, uses commonly available laboratory reagents, and can be used to obtain deep coverage of individual protein phosphorylation patterns, supplementary to phosphoproteomics data. Graphical abstract: Phospho-mapping workflow for a hypothetical protein of interest.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8376508 | PMC |
| http://dx.doi.org/10.21769/BioProtoc.4113 | DOI Listing |
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