Wuhan YanGeneBio Wuhan YanGeneBio is an innovative biotechnology service company dedicated to drug target discovery and drug mechanism validation. We provide solutions spanning from target discovery to mechanism validation for novel drug development, the modernization of traditional Chinese medicine, and natural product research. By integrating core technologies—including drug target fishing approaches such as ABPP, TPP, and Lip-MS; drug–target interaction site identification via crosslinking mass spectrometry and Lip-MS combined with molecular docking; affinity detection platforms (SPR/MST/BLI/ITC/DSF) for drug–target binding; high-throughput screening platforms such as protein microarrays and Olink multiplex assays; as well as siRNA drug synthesis and organoid-based drug screening—we deliver high-precision services in compound target identification, binding site analysis, affinity validation, and functional verification for our clients. Limited Proteolysis coupled with Mass Spectrometry (LiP-MS) is a label-free, high-throughput, and in situ conformation-resolving core technology in the field of structural proteomics. Through mild proteolysis with proteinase K and high-resolution mass spectrometry-based quantification, it enables precise characterization of protein conformational dynamics and interactions within complex systems. This article systematically describes the principles of LiP-MS, the standardized experimental workflow, methods for result and data analysis, core technical advantages, representative application cases, and the experimental service support system. It provides reproducible control data and scientific evidence materials to offer standardized technical solutions for drug development, disease mechanisms, and biomarker development. Principles of LiP-MS LiP-MS is centered on the principle that protein conformation determines proteolytic accessibility: in the native state, the flexible surface regions of proteins are readily and rapidly cleaved by the broad-specificity, non-specific protease proteinase K (PK); when a protein binds a small molecule, undergoes post-translational modification, or engages in protein–protein interactions, the local conformation tightens/relaxes, altering the exposure of cleavage sites and generating characteristic peptide abundance differences. Technical workflow: mild limited proteolysis → complete digestion → peptide enrichment → LC-MS/MS quantification → screening of conformation-differential peptides → target and site resolution. Without relying on chemical modification or protein purification, it preserves physiological conformation in situ and enables structural dynamics monitoring at the whole-proteome scale. Standardized Experimental Workflow 1. Sample Preparation Cell lysate / tissue homogenate / body fluid samples, quantified by BCA; set a treatment group (drug/perturbation) and a control group (solvent), with n = 3 biological replicates. 2. Limited Proteolysis (LiP) PK enzyme-to-substrate ratio 1:100 (wt/wt), incubated at 25 °C for 5 min, inactivated at 99 °C for 5 min; simultaneously set up a total proteolysis group (TrP) for protein abundance normalization. 3. Peptide Preparation DTT reduction, IAA alkylation, overnight trypsin digestion; C18 SPE desalting, lyophilization, and reconstitution in 0.1% formic acid. 4. MS Acquisition Orbitrap high-resolution mass spectrometry in DIA mode; chromatography: C18 column, 60 min gradient; parameters: resolution 120K, mass accuracy < 3 ppm. 5. Quality Control and Reproducibility RSD < 15%, peptide identification rate > 85%, no spurious peaks in blanks, ensuring data reliability. Results and Data Analysis 1. Data Processing Pipeline MaxQuant database search → MSstatsLiP correction → normalization by TrP abundance → screening of conformational differential peptides with |FC| ≥ 2 and P < 0.05. 2. Control Data (Core Evidence) Throughput: ~3,500 proteins and ~40,000 peptides identified per run, with 3–5× broader coverage than traditional structural methods; Sensitivity: conformational response detection limit down to the nM level, with sample consumption only 1/10 of labeling-based methods; Specificity: false-positive rate < 5%, with site localization precision at the amino-acid level; Reproducibility: correlation coefficient of biological replicates R² > 0.95, technical replicates R² > 0.98. 3. Key Conclusion LiP-MS can simultaneously distinguish conformational changes from abundance changes, eliminating expression-level interference and achieving true structural dynamics resolution. Core Advantages of LiP-MS Label-free in situ resolution: no modification, no purification, preserving physiological conformation and avoiding activity interference; High-throughput panoramic coverage: parallel monitoring of thousands of protein conformations in a single experiment, without bias; High sensitivity and micro-sample compatibility: suitable for clinical micro-samples (cerebrospinal fluid, biopsy tissue); Site-level precise localization: directly reveals binding/allosteric regions, supporting mechanism elucidation and molecular design; Broad applicability: applicable to prokaryotic/eukaryotic systems, purified proteins/complex omics, and in vitro/in situ systems. Representative Application Cases 1. Drug Target Discovery and Validation After treating cells with a small molecule, LiP-MS screened out 12 proteins with significant conformational changes; co-purification, SPR, and molecular docking verification confirmed 2 novel targets, improving the hit rate by 40% and shortening the cycle by 60%. 2. Disease Biomarker Development In clinical tissue samples, 23 disease-specific conformational peptides were identified and used to build a diagnostic model with AUC = 0.92, outperforming traditional abundance-based biomarkers (AUC = 0.78). 3. Mechanistic Elucidation of Protein Post-Translational Modification and Interactions Conformational rearrangements induced by modifications were resolved, and 8 key allosteric sites were localized, revealing the regulatory mechanism of signaling pathways and providing a structural basis for targeted drug design. LiP-MS Support Experiments and Service Advantages 1. Standard Support Experiments Protein quantification and quality control (BCA, SDS-PAGE); Orthogonal optimization of digestion conditions (time, enzyme amount, temperature); DIA/PRM targeted verification; Bioinformatics analysis (differential screening, clustering, site annotation, PPI network, molecular docking). 2. Core Service Advantages Fully standardized workflow: one-stop from sample to report, reproducible and traceable; Data rigor: dual-group controls + triple biological replicates + multiple statistical corrections; Fast delivery: routine samples yield data within 3–5 working days; In-depth interpretation: provides mechanistic conclusions, figures, and writing materials to support papers and grant applications.
LiP-MS Platform
