Post translational modification (PTM)

Understand Biological Regulation by Proteomics

Post translational modification

• Post-translational modification (PTM) is the covalent and generally enzymatic modification of proteins following protein biosynthesis.
• This process occurs in the endoplasmic reticulum and the golgi apparatus.
• Proteins are synthesized by ribosomes translating mRNA into polypeptide chains, which may then undergo PTM to form the mature protein product.
• PTMs are important components in cell signaling, as for example when prohormones are converted to hormones.

Post translational modification

• Post-translational modifications can occur on the amino acid side chains or at the protein's C- or N- termini. They can extend the chemical repertoire of the 22 proteinogenic amino acids by modifying an existing functional group or introducing a new one such as phosphate.
• Other forms of post-translational modification consist of cleaving peptide bonds, as in processing a propeptide to a mature form or removing the initiator methionine residue.
• The formation of disulfide bonds from cysteine residues may also be referred to as a post-translational modification.
• Sites that often undergo post-translational modification are those that have a functional group that can serve as a nucleophile in the reaction.

Common PTMs and their residues

PTM site detection by LC/MSMS

Phosphorylation

• Importance of phosphorylation/De-phsophoryaltion:
• Yeast: 120 kinases (out of ~6000 total genes), so human may have 1000 kinases.
• Many well known pathways are controlled by phosphorylation.
• Diseases and drug are developed according to these enzymes.

Phosphorylation plays a key in many biological processes

Phosphoproteomics workflow

Immobilized metal affinity chromatography (IMAC)

• Using Fe related compound (positive charge) to interact with phosphopeptides (negative charge).
• Using magnet to capture and enrich the phosphopeptides.

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The challenge of the phosphoproteomics

• The phosphorylation sites have numerous possibilities.
• Mono-phosphorylation – one site in one peptide.
• Multi-phosphorylation – multiple sites in one peptide. Usually, only consider to tri-phosphorylation.
• The identical peptide can have different phosphorylation sites.

Phosphosites vs. Phosphopeptides

• Phosphosites are NOT Phosphopeptides!!!
• A phosphosite is a serine/threonine/tyrosine with a distinct location in the protein sequence, which is phosphorylated.
• Quantitative information for each site is derived from ALL detected peptides, which are phosphorylated at this position (=combined/condensed information)

Example

Example

Example

Phospho (STY) table

• This table is usually sufficient to make qualified assumptions about changes in the phosphorylation status of proteins between samples
• Key quantitative values are: – Occupancy (mainly applicable for SILAC data) – Intensity__1/2/3 OR Ratio H/L__1/2/3

What does __1, __2, __3 mean?

Example

• There is quantitative information derived from single, double or triple(&higher) phosphorylated peptides
• Using this intensities is MUCH better then looking just at the summed site intensities, because in a biological system the same protein may have distinct functions when it is differentially phosphorylated

Localization probability

A filter cutoff of at least 0.75 is used for well-localized sites

Occupancy

How to get occupancy

Example

Example

Example

Kinomics

• Kinomics can be generated based on array chip.
• Using florescent light for detecting the phosphorylation reaction.
• Using the intensity of the light for the quantification.

PhosphoSitePlus

• PhoshoSitePlus is a database for storing the information of kinase and phosphorylation site.
• https://www.phosphosite.org/homeAction.action

Motif detection

• Motif is a region (a subsequence) of protein or DNA sequence that has a specific structure or function.
• Motifs are candidates for functionally important sites.
• Presence of a motif may be used as a base of protein classification.
• It is also an important feature for phosphorylation site.

Acetylation

• Acetylation is a highly conserved and reversible post-translational modification. It mainly takes part in regulating gene expression through modifying nuclear histones, but can also regulate several metabolic enzymes and metabolism pathways.
• The acetylation of the ε-amino group of lysine, which is common, converts a charged side chain to a neutral one.
• Acetylation/deacetylation of histones also plays a role in gene expression and cancer. These modifications are effected by enzymes called histone acetyltransferases (HATs) and histone deacetylases (HDACs).
• Two general mechanisms are known for deacetylation. One mechanism involves zinc binding to the acetyl oxygen. Another family of deacetylases require NAD+, which transfers an ribosyl group to the acetyl oxygen.

Histone acetylation

• N-acetylation, or the transfer of an acetyl group to nitrogen, occurs in almost all eukaryotic proteins through both irreversible and reversible mechanisms. This type of acetylation is co-translational, in that N-terminus is acetylated on growing polypeptide chains that are still attached to the ribosome. While 80-90% of eukaryotic proteins are acetylated in this manner, the exact biological significance is still unclear.
• Histone acetylation is a critical epigenetic modification that changes chromatin architecture and regulates gene expression by opening or closing the chromatin structure. It plays an essential role in cell cycle progression and differentiation. It mostly happens on Lysine.

Histone acetylation

• Histone acetylation is regulated by two sets of enzymes HATs and HDACs which add or remove acetyl group respectively from both histone and non-histone proteins, hence regulating gene transcription.

Acetyl-proteomics workflow

Salmonella Typhimurium acetlyproteomics profile

GO

KEGG

Glycosylation

• Glycosylation is the reaction in which a carbohydrate (or 'glycan'), i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor) in order to form a glycoconjugate.
• Glycans serve a variety of structural and functional roles in membrane and secreted proteins.
• The majority of proteins synthesized in the rough ER undergo glycosylation.
• Glycosylation is also present in the cytoplasm and nucleus as the O-GlcNAc modification.

Glycosylation

• Five classes of glycans
• N-linked glycans attached to a nitrogen of asparagine or arginine side-chains. N-linked glycosylation requires participation of a special lipid called dolichol phosphate.
• O-linked glycans attached to the hydroxyl oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side-chains, or to oxygens on lipids such as ceramide
• Phosphoglycans linked through the phosphate of a phosphoserine.
• C-linked glycans, a rare form of glycosylation where a sugar is added to a carbon on a tryptophan side-chain. Aloin is one of the few naturally occurring substances.
• Glypiation, which is the addition of a GPI anchor that links proteins to lipids through glycan linkages.

Glycans in glycoproteomics

• GlcNAc
• A derivative of glucose and is a common component of many complex carbohydrates, including the N-linked and O-linked glycans that are attached to glycoproteins.
• NeuAc
• A derivative of neuraminic acid and is a key component of many glycolipids and glycoproteins. It is often found at the terminal position of N-linked and O-linked glycans and plays important roles in cell-cell interactions, immune system function, and disease processes.
• Both GlcNAc and NeuAc are both Sialic acid. They are important components of the glycan structures that are found on many proteins and lipids in the body.
• The specific types and arrangements of glycans can vary depending on the tissue, cell type, and developmental stage, and can have significant implications for protein function and disease.

Glycans in glycoproteomics

• Sialic acid
• This is a common monosaccharide that is often found at the terminal position of N-linked and O-linked glycans. Sialic acid can play important roles in protein function and regulation.
• Fucose
• This is another common monosaccharide that is often found on N-linked and O-linked glycans. Fucose can affect protein stability, localization, and interactions with other molecules.
• Mannose
• This is a monosaccharide that can be found on both N-linked and O-linked glycans. Mannose can play important roles in protein folding and quality control.

Types of glycan structures

Glycoproteomics

Glycoproteomics

Glycomics

• Glycomics is a broader field that encompasses the study of all glycans in a biological system, including those that are attached to proteins, lipids, and other biomolecules.
• Glycomics typically involves the isolation and analysis of glycans from biological samples, followed by the identification and characterization of their structures, functions, and interactions with other molecules
• Glycoproteomics is a focused subfield of glycomics that specifically studies the glycosylation of proteins, while glycomics is a broader field that encompasses the study of all glycans in a biological system.

Challenge of data analysis in glycoproteomics

GlycoBinder

• Use three different aspects to analyze the data
• Glycoforms
• Glycosite
• Glycan

Data analysis for glycoproteomics

Other (digestion and enrichment)

The benefit of top-down proteomics to post-translational modification