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Intracellular Protein Crosslinking Detection Methods

Author: Creative Proteomics
by Creative Proteomics
Posted: Feb 02, 2023

What is Intracellular Protein Crosslinking?

Intracellular protein crosslinking usually refers to the linking of two amino acids between intracellular protein molecules or within protein molecules by covalent bonds other than peptide bonds. Intracellular crosslinking is currently considered a post-translational modification of proteins. It plays an important role in the structure and function of proteins.

It has been found that intracellular protein crosslinks include disulfide bonds, isopeptide bonds, tryptophan crosslinks, tyrosine crosslinks, formaldehyde crosslinks, methylglyoxal (MG) crosslinks, NOS bridges, etc. Studies on disulfide bonds at the histological level have shown that disulfide bonds in proteins can be divided into two major categories, structural and functional, with functional disulfide bonds involved in functions such as catalysis and regulation. Similar analytical work is required for other intracellular crosslinks in proteins. The discovery of these intracellular crosslinking sites will contribute to the study of the regulatory functions of proteins, apoptosis, and disease mechanisms.

Mass spectrometry, with its high throughput and high sensitivity, is one of the tools for large-scale identification of intracellular protein crosslinks. A preliminary set of mass spectrometry-based protein cross-linking analysis method has been developed, including sample pretreatment, data acquisition, qualitative analysis, quantitative analysis and bioinformatics analysis.

Intracellular Protein Crosslinking Assay

  • Sample pre-treatment

Sample pretreatment refers to the processing of samples before they are sent to mass spectrometry for data collection. The main purpose is to make crosslinks easier to be collected and accurately identified by mass spectrometry, including enzymatic digestion, separation and enrichment, and amino acid blocking. The specific treatments need to be designed according to the characteristics of the crosslinks.

The purpose of enzymatic digestion is to digest the cross-linked proteins into cross-linked peptides and use them for mass spectrometry data acquisition. Cross-linked peptides can be divided into three basic forms: mono-link, loop-link and cross-link.

Due to the specificity of the enzymatic cleavage site and cross-linking site, and the uneven distribution of amino acids in the protein, not all the cross-linked peptides obtained from the enzymatic digestion of complex samples can be obtained in the above three simple cross-linked forms. Therefore, more diverse complex cross-linked forms need to be considered for large-scale cross-linking identification.

The low content and abundance of crosslinked peptides will result in many crosslinks not being collected and identified. Therefore, performing isolation enrichment is also essential in sample pretreatment. Affinity chromatography, strong cation exchange (SCX) and size exclusion chromatography (SEC) are often used in the separation process. The enrichment at the protein level is performed one step before the enzymatic digestion because the proteins have been digested into peptides after the enzymatic digestion. Enrichment of low abundance proteins can also be achieved by removing high peak proteins during data acquisition using methods such as quadrupole and ion trap.

Amino acid blocking is mainly used to avoid breakage and rearrangement of crosslinks during data acquisition. Most protein crosslinks rely on chemical covalent bonds to directly link amino acids on proteins. The formation process of partial crosslinks is reversible. The crosslinks are easily altered by the added reagents and the environment of the system. These altered cross-links, if incorrectly considered as natural cross-links, will lead to subsequent studies in the wrong direction.

  • Data acquisition

Cross-linked peptide identification is performed by comparing the theoretical fragmentation ions generated by the candidate peptide with the actual fragmentation ions collected in the spectrum. There are several ways to perform peptide fragmentation. Different fragmentation methods will result in different fragment ions from the peptide.

Partial cross-linking is the direct joining of two amino acid side chains by chemical bonds, and the fragmentation process breaks the chemical bonds in the peptide segment. Therefore, whether the cross-linked portion is broken during fragmentation requires experiments to demonstrate. Usually, the synthesized standard peptide fragments are subjected to cross-linking experiments, and then they are subjected to data acquisition using mass spectrometry, and the pattern of fragment ions during fragmentation is probed according to the fragment ions in the spectra.

  • Qualitative analysis

Theoretical enzymatic cleavage of protein sequences in the database is performed to obtain candidate peptides, and theoretical spectra are generated to match with actual spectra for peptide spectra thus enabling qualitative analysis of protein crosslinks.

  • Quantitative analysis

The quantitative analysis of cross-linking is mainly performed on cross-linked peptides and includes labeled and label-free quantification. In labeled quantification, the cross-linked content is reflected by the intensity of the reporter ion. In label-free quantification, the parent ion chromatographic curve corresponding to the cross-linked peptide is reconstructed based on the qualitative results of protein cross-linking. The peak area enclosed by the chromatographic curve reflects the protein cross-linking content.

  • Bioinformatics analysis

Bioinformatics analysis can elucidate the possible relationship between crosslinks and protein structure and function. The cross-linked sites are usually mapped to protein sequences based on the identified results, and the protein cross-linkage network is mapped to reflect the distribution of cross-linkages in the protein. The identified cross-linked peptide sites can then be analyzed by motif analysis to identify the hotspots of cross-link formation. The crosslinking of proteins with significant differences may be a potential factor leading to altered protein function and may be an important biomarker.

About the Author

Creative Proteomics has gradually grown into an integrated service provider with targeted lipidomics and untargeted lipidomics analysis services for researchers in pharmaceutical, biotechnology, agriculture, and nutrition industries.

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Author: Creative Proteomics

Creative Proteomics

Member since: Oct 25, 2021
Published articles: 40

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