Skip to main content
U.S. flag

An official website of the United States government

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you’ve safely connected to the .gov website. Share sensitive information only on official, secure websites.

A multipathway phosphopeptide standard for rapid phosphoproteomics assay development

Published

Author(s)

Brian Searle, Allis Chien, Antonius Koller, David Hawke, Anthony Herren, Jenny Kim, Kimberly Lee, Ryan Leib, Alissa Nelson, Jian Min Ren, Paul Stemmer, Yiying Zhu, Ben Neely, Bhavin Patel

Abstract

Recent advances in methodology have made phosphopeptide analysis a tractable problem for many proteomics researchers. There are now a wide variety of robust and accessible enrichment strategies to generate phosphoproteomes while free or inexpensive software tools for quantitation and site localization have simplified phosphoproteome analysis workflow tremendously. As a research group under the Association for Biomolecular Resource Facilities umbrella, the Proteomics Standards Research Group has worked to develop a multipathway phosphopeptide standard based on a mixture of heavy-labeled phosphopeptides designed to enable researchers to rapidly develop assays. This mixture contains 131 mass spectrometry vetted phosphopeptides specifically chosen to cover as many known biologically interesting phosphosites as possible from seven different signaling networks: AMPK signaling, death and apoptosis signaling, ErbB signaling, insulin/insulin-like growth factor-1 signaling, mTOR signaling, PI3K/AKT signaling, and stress (p38/SAPK/JNK) signaling. Here, we describe a characterization of this mixture spiked into a HeLa tryptic digest stimulated with both epidermal growth factor and insulin-like growth factor-1 to activate the MAPK and PI3K/AKT/mTOR pathways. We further demonstrate a comparison of phosphoproteomic profiling of HeLa performed independently in five labs using this phosphopeptide mixture with data-independent acquisition. Despite different experimental and instrumentation processes, we found that labs could produce reproducible, harmonized datasets by reporting measurements as ratios to the standard, while intensity measurements showed lower consistency between labs even after normalization. Our results suggest that widely available, biologically relevant phosphopeptide standards can act as a quantitative "yardstick" across laboratories and sample preparations enabling experimental designs larger than a single laboratory can perform. Raw data files are publicly available in the MassIVE dataset MSV000090564.
Citation
Molecular and Cellular Proteomics
Volume
22
Issue
10

Keywords

proteomics, phosphorylation, phosphopeptide, mass spectrometry, targeted, data independent acquisition, stable isotope label

Citation

Searle, B. , Chien, A. , Koller, A. , Hawke, D. , Herren, A. , Kim, J. , Lee, K. , Leib, R. , Nelson, A. , Ren, J. , Stemmer, P. , Zhu, Y. , Neely, B. and Patel, B. (2023), A multipathway phosphopeptide standard for rapid phosphoproteomics assay development, Molecular and Cellular Proteomics, [online], https://doi.org/10.1016/j.mcpro.2023.100639, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=935990 (Accessed June 16, 2024)

Issues

If you have any questions about this publication or are having problems accessing it, please contact reflib@nist.gov.

Created August 30, 2023, Updated September 28, 2023