Structure and cross link proteomics

Amino acids

Charged Amino acids

Polar Amino acids

Non-polar amino acids

Hydrophobic and hydrophilic

Special cases

Polar

Non-polar

Non-polar

Cysteine can form disulfide bond which is important for protein folding

Glycine is the smallest amino acid. Due to its size, it is rare to be replaced by other amino acids. Thus, it is usually located at constant position.

Proline is a circular amino acid. Due to its special formation, it affects protein structure. It can terminate the alpha helix.

Amino acid, peptide and protein

Secondary structure of protein

Tertiary structure of protein

• Three dimensional shape of a protein
• The tertiary structure will have a single polypeptide chain "backbone" with one or more protein secondary structures, the protein domains.
• Amino acid side chains may interact and bond in a number of ways.

Protein domain

• A region of the protein's polypeptide chain that is self-stabilizing
• Folds independently from the rest.
• Many proteins consist of several domains
• One domain may appear in a variety of different proteins
• Domains often form functional units

Protein shape and protein function

• Protein shape is normally related to its function -> family

Contact sites of proteins

• If the locations of amino acids of a protein or between two proteins are close, they may have an interaction and may assist to form the protein conformation.

Structure stability

Antibody-antigen interaction

Protein complex

Protein-protein interaction

2^nd structure formation

Protein-protein interaction (PPI)

• The interactions include
• Electrostatic forces
• Hydrogen bonding
• Hydrophobic effect
• Shape plays a key role

The ways to study protein structure or PPIs

X-ray and NMR

• The conventional experimental methods to study protein structures or know how protein-protein interactions perform are X-ray and NMR.
• X-ray
• Useful for large structures: Not limited by size or atomic weight
• High atomic resolution
• The sample must be crystallizable
• NMR
• Dynamic technique
• Non-destructive and non-invasive
• Can provide unique insights into dynamics and intramolecular interactions
• Size limited
• Noise level is high
• Low atomic resolution

They can only study one protein at once

Cross-linking proteomics

• Cross-linking proteomics is a technique used in proteomics research to study protein-protein interactions and protein structures.
• It can systematically study protein structure and interaction by checking the contact site of the residues.
• It involves the use of cross-linking reagents to covalently link interacting or neighboring proteins within a complex or protein structure.
• By cross-linked protein complexes, cross-linking proteomics provides valuable information about protein-protein interactions, the architecture of protein complexes, and the arrangement of protein domains. It can help elucidate protein interaction networks, validate protein-protein interaction models, and contribute to understanding the three-dimensional structures of proteins and protein complexes.

Cross linker

• Crosslinkers, also known as bifunctional crosslinkers, are reagents that contain two or more reactive groups which covalently attach via a spacer to functional groups on proteins or other biomolecules.
• Three types of crosslinkers are available: homobifunctional crosslinkers, heterobifunctional crosslinkers, and photoreactive crosslinkers.
• Homobifunctional crosslinkers
• Have identical reactive groups, primarily amine-to-amine or sulfhydryl-to-sulfhydryl. They are typically used to form intramolecular crosslinks or to prepare polymers from monomers.
• Heterobifunctional crosslinkers
• Have different reactive groups such as amine-to-sulfhydryl, carboxyl-to-amine, or sulfhydryl-to-carboxyl. They are useful for preparing conjugates between two different biomolecules.
• Photoreactive crosslinkers
• Part of heterobifunctional crosslinkers, it reacts with nucleophiles or form C-H insertion sites after exposure to UV light.

A crosslinker has certain characteristics

functionality

length

MS feature

A common crosslinker example 1: disuccinimidyl suberate (DSS)

a single carbon chain known as �‘a spacer arm’ or ‘a linker region’

A common crosslinker example 2: disuccinimidyl sulfoxide (DSSO)

A common crosslinker example 3: NHS-ester/diazirine

NHS-ester

NHS-diazirine

UV cross linking

• UV cross-linking is a technique used in molecular biology and protein-protein interaction studies. Here are some advantages and disadvantages of UV cross-linking:
• Advantages of UV cross-linking:
• Simplicity: UV cross-linking is a relatively simple and straightforward technique, requiring minimal specialized equipment.
• Speed: UV cross-linking reactions can be performed rapidly, allowing for efficient experimental workflows.
• In situ cross-linking: UV cross-linking can be performed directly on cells or tissues, preserving the native context of protein interactions.
• Non-specific cross-linking: UV cross-linking can capture both specific and non-specific protein interactions.
• Preservation of weak interactions
• Disadvantages of UV cross-linking:
• Non-specificity: leading to false-positive interactions.
• Limited distance constraints: UV cross-linking primarily captures proximal interactions within a short distance.
• Protein damage
• Low cross-linking efficiency
• Experimental optimization: UV cross-linking conditions, such as UV intensity, exposure time, and cross-linking reagent concentration, may require optimization for each specific application to achieve optimal results.

Structural information is lost during typical shotgun proteomics experiments

MS/MS spectra

proteins

peptides

No interacted peptides are remained

Chemical crosslinking makes spatially close residues remain together

chemical crosslinking combined with mass spectrometry

XL-MS, CX-MS or CLMS

crosslinked proteins

proteins

crosslink products

MS/MS spectra

Obtained peptide mixture is more complex

single or

ordinary peptide

Obtained peptide mixture is rather more complex

Obtained peptide mixture is more complex

the most abundant

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e.g. strong cation exchange �or size exclusion chromatography to enrich crosslinked peptides

crosslink products

single or

ordinary peptide

Obtained peptide mixture is more complex

monolinked peptide or

deadend or

Type 0

crosslink products

e.g.�DSS mass shift ∼156 Da

Obtained peptide mixture is more complex

looplinked peptide or

cyclic peptide or

intra-peptide or

Type 1

crosslink products

e.g.�DSS mass shift:∼138Da

Obtained peptide mixture is more complex

crosslinked peptide or

inter-peptide

Type 2

crosslink products

Obtained peptide mixture is more complex

high order crosslink or

multiple modifications

Type 3

crosslink products

Type 2, 1

Type 2, 2

Specialized and automated software are required �to analyze complex crosslink samples

MS/MS data set

ID XL-peptides Spectrum title Score�1 ASEGKK-DKERR SpecA 126�2 ASEGKK-DKERR SpecB 84

3 ASEGKK-ADKLEEQIR SpecC 32

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Cross-linked peptide-to-spectrum match (CSM)

Workflow of cross linking proteomics

Identification of non-cleavable or MS-cleavable crosslinkers

Identification of non-cleavable or MS-cleavable crosslinkers

Identification of non-cleavable or MS-cleavable crosslinkers

Example

Structural modeling

Visualization

List of identified crosslinks

• A crosslinker imposes a certain distance constraint

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• Euclidean distance between two central carbon (Cα−Cα)

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