Apr 30, 2026
Peptide-Centric Local Stability Assay
This protocol is a draft, published without a DOI.
- 1University of Edinburgh
Protocol Citation: Ayush Regmi Bagale 2026. Peptide-Centric Local Stability Assay. protocols.io https://dx.doi.org/
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: April 28, 2026
Last Modified: April 30, 2026
Protocol Integer ID: 315861
Keywords: wide limited proteolysis assay, reproducible profiling of proteome, changes in protease susceptibility, level analysis of protein stability, protein stability, pelsa to mammalian cell lysate, proteolysi, protease inhibitor, detecting protein, peptide, normalization of protein concentration, protease susceptibility, throughput proteome, protein concentration, mammalian cell lysate, native protein conformation, preserving native protein conformation, proteome, centric local stability assay, format compatible with downstream liquid chromatography, mass spectrometry, trypsin digestion, resolved trypsin digestion, downstream liquid chromatography
Abstract
High-throughput Proteome-wide Limited Proteolysis Assay (HT-PELSA) is a mass spectrometry–based method for detecting protein–ligand interactions through ligand-induced changes in protease susceptibility. This protocol describes an optimized workflow for applying HT-PELSA to mammalian cell lysates, based on implementation in K562 cells. Cells are lysed under mild, detergent-free conditions using repeated freeze–thaw cycles in phosphate-buffered saline supplemented with protease inhibitors, minimizing viscosity and preserving native protein conformations. Following normalization of protein concentration, lysates are incubated with small-molecule ligands under controlled conditions.
The workflow is adapted for high-throughput processing in a 96-well format, where samples are subjected to rapid, time-resolved trypsin digestion. Proteolysis is quenched with denaturant, followed by reduction, alkylation, and acidification. Peptides are subsequently purified using C18 solid-phase extraction in a plate-based format compatible with downstream liquid chromatography–mass spectrometry (LC–MS) analysis. Inclusion of matched vehicle controls and post-quench ligand supplementation enables correction for potential ionization artifacts.
This protocol enables scalable and reproducible profiling of proteome-wide ligand binding events in mammalian systems. The streamlined format reduces handling time and sample variability, making it suitable for compound screening, target engagement studies, and systems-level analysis of protein stability and conformational changes.
Peptide-Centric Local Stability Assay
HT-PELSA for Mammalian Cells (K562 Lysate)
1. Prepare mammalian cell lysate
Harvest cells
Pellet cells at 1,000 × g for 5 min
Wash cells 3× with ice-cold PBS
Store pellet at −80 °C until use
Lysis
Prepare lysis buffer: PBS + 1% (v/v) protease inhibitor cocktail (Sigma P8340)
Resuspend cells in ice-cold lysis buffer
Snap-freeze in liquid nitrogen
Thaw in 37 °C water bath
Repeat freeze–thaw cycle 3× total
Lysate handling
Option A (cleared lysate): centrifuge at 20,000 × g for 10 min at 4 °C
Option B (crude lysate): skip centrifugation and use directly
Normalization
Measure protein concentration (e.g., Rapid Gold BCA)
Adjust lysate to 1.2 mg/mL using lysis buffer
2. Ligand incubation (in lysate)
Ligand prep
Prepare ligand stocks at 100× in DMSO
Prepare control samples with matched DMSO volume
Incubation
Add ligand to lysate
Incubate at 25 °C for 30 min
3. High-throughput PELSA digestion setup (96-well)
Replicates
Split each sample into four 50 µL replicates in a 96-well plate
Trypsin digestion
Prepare second 96-well plate with 5 µL trypsin (5 mg/mL; Sigma T1426) per well
Keep trypsin on ice until use
Transfer 50 µL sample into trypsin-containing wells
Mix by pipetting up/down for 30 s
Digest at 22 °C for 3 min 30 s
4. Quench + reduction/alkylation
Quench
Add 165 µL of 8 M guanidine HCl + 60 mM HEPES pH 8.2 (3× volume)
Ionization control
Add ligand to control samples to match treated samples
Reduction/alkylation
Add TCEP to 10 mM final concentration
Add chloroacetamide (CAA) to 40 mM final concentration
Heat at 95 °C for 5 min
Acidification
Add TFA to 1% final concentration
5. 96-well C18 cleanup (Sep-Pak tC18 100 mg plate)
Plate conditioning
Spin empty plate at 3,000 × g for 5 min
Wash 2× with 1 mL 100% ACN (20 × g, 1 min each)
Wash 2× with 1 mL 0.1% TFA (100 × g, 1 min, accel 5/9)
Sample loading
Load samples (100 × g, 1 min, accel 5/9)
Wash
Wash 2× with 1 mL 0.1% TFA (same spin settings)
Elution
Elute 2× with 100 µL 50% ACN + 0.1% TFA (20 × g, 1 min each)
Spin at 500 × g for 1 min
Collection
Collect eluate into MS-compatible 96-well plate
Dry samples
Notes
Use spacer to prevent tips touching collection plate during elution
Inject small fraction of eluate (e.g., ~4%)
Adaptation note (RPE1 cells)
Wash and scrape or pellet adherent cells
Apply same PBS + 1% protease inhibitor lysis
Perform 3× liquid nitrogen freeze–thaw cycles
Proceed with protocol unchanged after normalization to 1.2 mg/mL