# Protocol for label-free quantitative lysine lactylproteome profiling

## TL;DR

This paper describes a detailed protocol for analyzing lactylation, a protein modification linked to L-lactate, using label-free mass spectrometry techniques.

## Contribution

The paper introduces a comprehensive, step-by-step protocol for profiling and quantifying the lactylproteome without the need for labeling.

## Key findings

- The protocol includes cell preparation, protein extraction, and lactylpeptide enrichment using antibody-conjugated agarose beads.
- LC-MS/MS parameters and MaxQuant settings are provided for label-free quantification of lactylated peptides.
- The method enables global profiling of lactylation, a PTM involved in cellular signaling and metabolism.

## Abstract

L-lactate has been recognized as an essential molecule for signaling and metabolic balance. Lactylation, a post-translational modification (PTM) derived from L-lactate, is commonly observed on various proteins and plays essential roles in cellular processes. Here, we present a protocol to globally profile the lactylation proteome and perform label-free quantification. We provide steps for cell preparation, protein extraction, digestion, peptide desalting, and lactylpeptide enrichment. Additionally, we outline the parameters for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.

For complete details on the use and execution of this protocol, please refer to Li et al.1

•Instructions for the preparation of cell culture, transfection, and lactate treatment•Steps for enriching lactylpeptide based on antibody-conjugated agarose beads•Guidance on LC-MS setup and MaxQuant parameters for label-free quantification analysis

Instructions for the preparation of cell culture, transfection, and lactate treatment

Steps for enriching lactylpeptide based on antibody-conjugated agarose beads

Guidance on LC-MS setup and MaxQuant parameters for label-free quantification analysis

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

L-lactate has been recognized as an essential molecule for signaling and metabolic balance. Lactylation, a post-translational modification (PTM) derived from L-lactate, is commonly observed on various proteins and plays essential roles in cellular processes. Here, we present a protocol to globally profile the lactylation proteome and perform label-free quantification. We provide steps for cell preparation, protein extraction, digestion, peptide desalting, and lactylpeptide enrichment. Additionally, we outline the parameters for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.