Limited proteolysis (LiP) involves controlled enzymatic digestion of proteins to generate smaller peptide fragments while maintaining the overall protein structure. By using specific proteases, researchers can selectively cleave peptide bonds at specific sites, generating fragments with specific lengths and sequences, providing valuable insights into protein conformation, dynamics, and interactions.
LiP is achieved by specific proteases cleaving proteins at specific sites, resulting in a defined set of peptide fragments. In LiP-MS, the selected proteases are typically enzymes with specific cleavage properties, such as trypsin or pepsin. These enzymes recognize specific amino acid sequences or structural domains and cleave the protein chain nearby.
After limited proteolysis, the generated peptide fragments need to undergo mass spectrometry analysis to determine their molecular mass and composition. This is a crucial step in LiP-MS. Liquid chromatography-mass spectrometry (LC-MS) is a commonly used technique for mass spectrometry analysis. Firstly, the produced peptide fragments are separated by liquid chromatography and introduced into the mass spectrometer. Then, the mass spectrometer ionizes the peptide fragments, giving them charges, and accelerates them to the mass analyzer. In the mass spectrometry analysis, the peptide fragments are fragmented, and their mass-to-charge ratio (m/z) is measured to determine their molecular mass. These mass spectrometry data are recorded, resulting in a mass spectrum. By interpreting and analyzing the mass spectrum, the composition and sequence of the peptide fragments can be determined. By comparing and matching against known protein databases, the corresponding proteins for the peptide fragments can be identified.
Schematic of LiP–MS (Shuken et al., 2022)
a) Prepare samples under different treatment conditions, such as Condition 1, Condition 2, Condition 3, and so on.
b) Extract proteins under non-denaturing conditions.
c) Add a broad-spectrum protease to the protein samples. For example, protease K (commonly used), thermolysin (thermostable protease), subtilisin (Bacillus protease), papain (papaya protease), chymotrypsin, or elastase. These proteases tend to cleave regions of unfolded proteins.
d) Denature the proteins to terminate the proteolytic reaction.
e) Digest the proteins with trypsin.
f) Terminate the enzymatic digestion. Add formic acid to the sample to achieve pH<3, desalt and dry the peptide fragments, and proceed to mass spectrometry analysis.
Depending on the experimental goals, various mass spectrometry techniques can be employed to analyze the peptide fragments obtained from LiP. These techniques include data-dependent acquisition (DDA), data-independent acquisition (DIA), selected reaction monitoring (SRM), or parallel reaction monitoring (PRM). Liquid chromatography separation is typically performed using a conventional 2-3 hour gradient, and mass spectrometry parameters are set according to standard DDA, DIA, SRM, or PRM modes. Database search software and parameters are also set as per standard practices. It is important to note that the enzyme digestion in the search parameters should be set to semi-tryptic mode. For quantification, select semi-tryptic unique peptide fragments without missed cleavages, with at least a 2-fold difference and a q-value less than 0.02. Usually, peptide fragments showing conformational changes identified through DDA/DIA screening require subsequent targeted validation.
LiP-SRM workflow (Feng et al., 2014)
Cells, blood, and tissues are just a few examples of complicated biological samples that the approach can be used.
Samples don't need to be purified, labeled, or enhanced. Human cells currently have about 3,500 LiP cleavage sites, which correspond to about 1,200 proteins.
It can be used with various targeted approaches (PRM, SRM), qualitative and quantitative procedures (DDA, DIA), and other mass spectrometry methods.
It allows for both high-throughput investigations of conformational changes in protein networks as well as the research of conformational changes in particular proteins.
The method's great sensitivity enables the identification of both large and subtle structural alterations. Longer chromatographic gradients can be used to identify conformational changes in proteins with low abundance.
Analysis of protein conformational changes caused by post-translational modifications.
Investigation of changes in protein enzymatic activity (conformational changes can impact enzyme activity).
Analysis of protein-protein interactions and networks.
Examination of protein-(metabolite/drug) small molecule interactions and networks.
Discovery of disease biomarkers (e.g., in neurodegenerative diseases, the ratio of amyloid/soluble proteins).