PTMs part 2 Flashcards
What are mucins and what amino acids are they rich in? What does this allow?
• Mucins (composes mucous) are a large family (20 genes in humans) of heavily glycosylated proteins
o Rich in serine, threonine and hydroxyproline enabling O-linked glycosylation (via hydroxyl group)
o Cysteine-rich regions (intra-molecular disulfide cross-links) found at N- and C-termini
What is the impact of glycosylation in mucins?
o Glycosylation makes mucins highly resistant to secreted bacterial proteases and proteolysis in general
o Able to contain water (moist environment)
Glycans are very hydrophilic
o Glycosylation and cross-links enable the formation of a sticky, moist gel layer (also enables aggregation)
How can epithelial cell cancers be predicted? Why?
• Epithelial cell cancers can be diagnostically predicted by the presence of altered mucin glycans in the plasma
o Loss of normal topology and polarization of peitohelial cells in cancer results in secretion of mucins in the bloodstream
o Glycans may be altered due to rapid cell division in cancers (timing is everything in glycans)
• Involvement in cancer-
o Because the cells divide rapidly, the proteins miss the action of enzymes that go down into these pathways
What are all cells covered in?
• All cells are covered in a complex array of glycans
Where are cell surface and secreted glycan proteins assembled?
• Cell surface and secreted (ECM) glycan-proteins are assembled in the ER-golgi
What is the most complex of all PTMs and why?
- Glycosylation is the most complex of all PTMs and encompasses a broad range of single and multi-sugar modifications (linear, branched)
- Glycans generate much more combinatorial diversity than amino acid sequence of other PTMs
Is glycosylation a biochemically efficient process?
• Many enzymes (glycosylatransferases/glycosidases) in a complex, inefficient biochemical process
What are glycosyltransferases?
o Glycosyltransferases- enzymes that add sugars
What are glycosidases?
o Glycosidases- enzymes that remove sugars
What are the structural units of glycans?
o Monosaccharides are the structural units of glycans
What are glycans?
Glycan- a generic term for a sugar or assembly of sugars, in free form or attached to another molecule, used interchangeably with (oligo)saccharide or carbohydrate
What is a monosaccharide? Where is its first carbon?
A monosaccharide is a carbohydrate that cannot be hydrolyzed into a simple form. It has a potential carabonyl group at the end of the carbon chain (an aldehyde group) or at an inner carbon (a ketone group). These two types of monosaccharides are therefore named aldoses and ketoses, respectively. Free monosaccharides can exist in open-chain or ring forms.
• Carbon 1- anomeric carbon: where it is attached to a hydroxyl group and directly to oxygen
Which structures are commonly found in N- and O-glycans?
o Monosaccharides commonly found in N- and O-glycans
What are the different types of monosaccharides?
Pentoses Hexoses Hexosamines Deoxyhexoses Sialic acids (Sia)
What are pentose monosaccharides?
Pentoses- five-carbon neutral sugars e.g. D-xylose (Xyl)
What are hexose monosaccharides?
Hexoses- six-carbon neutral sugars e.g. D-glucose (Glc), D-galactose (Gal) and D-mannose (Man)
What are hexosamine monosaccharides?
Hexosamines- hexoses with an amino groupa t the 2-position, which can either free or, more commonly, N-acetylated e.g. N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-galactosamine (GalNAc)
• Building blocks of glycosylation
What are deoxyhexose monosaccharides?
Deoxyhexoses- six-carbon neutral sugars without the hydroxyl group at the 6-position (e.g. L-fucose [Fuc])
What are sialic acid monosaccharides?
Sialic acids (Sia)- family of nine-carbon acidic sugars, of which the most common is N-acetylneuraminic acid (neu5Ac, also sometimes called NeuAc)
What modifications can the hydroxyl groups of different monosaccharides be subject to?
The hydroxyl groups of different monosaccharides can be subject to phosphorylation, sulfation, methylation, O-acetylation or fatty acylation
How is a glycosidic bond formed? How are these subsequently labelled?
• Glycosidic bond formation
o Formation of a disaccharide between glucose and fructose= sucrose
o Two monosaccharides are brought together such that two hydroxyl groups are close to each other
o In an enzyme-catalysed reaction (a glycosyltransferase), a water molecule is eliminated, leaving a bond between C1 of glucose and C4 of fructose (an alpha 1,4 bond)
Forms the glycosidic bond
o Glycosidic bonds are labelled alpha or beta depending on the anomeric configuration of the C1 involved in the glycosidic bond
Up is beta
Down is alpha
What is maltose?
o Maltose is 2 Glc in an alpha linkage
What is lactose?
o Lactose is 2 Glc in a beta configuration
Can humans digest sugars in an alpha or beta linkage better?
Humans are more able to digest sugars in an alpha linkage
What are common classes of animal glycans?
o Proteoglycans o Glycosylphospatidylinositol (GPI0-anchored glycoproteins o Glycoproteins o Glycosphingolippid o O-GlcNac glycoproteins
What are examples of proteoglycans?
Herparan sulfate
Chondoitin sulfate
Dermatan sulfate
What are N-glycans?
N-glycan
• N-llinked via amido-nitrogen of asparagine
• N-glycan (N-linked, N-(Asn)-linked oligosaccharide) is a sugar chain covalently linked to an asparagine residue of a polypeptide chain, at the consensus peptide sequence: Asn-X (except proline)-Ser/Thr
What are O-glycans?
O-glycan
• O-linked via hydroxyl group of serine and threonine
• O-glycan (O-linked oligosaccharides) is frequently linked to the polypeptide via N-acetylgalactosamine (GalNac) to a hydroxyl group of a serine or threonine residue and can be extended into a variety of different structural core classes
What classes can N-glycans be divided into?
o N-glycans share a common pentasaccharide core region and can be generally divided into three main classes: oligomannose (or high-mannose) type, complex type, and hybrid type. Sometimes glycans are considered paucimannose (few mannose)
Describe the why the biosynthesis of N-glycans is such a contradictory and evolutionary confusing pathway
o IMPORTANT- it is EXTREMELY biochemically inefficient because some parts of the products made are often wasted/reversed in the end product (don’t use everything that is made in a sense)- but if seemingly irrelevant enzymes that mediate this pathway are mutated, then the pathway no longer works and it is fatal for the embryo/can lead to developmental disorders
Describe how the core oligosaccharide in the N-glycan biosynthesis pathway is synthesised
o Begins on the cytoplasmic face of the ER with the transfer of GlcNAc-P from UPD-GLCNac to Dol-P to generate dolichol pyrophosphate N-acetylglucosamine (Dol-P-P-GlcNAc). The reaction is catalysed by GlcNAc-1-phosphotransferase
o A second GlcNAc and five Man are transferred in a stepwise manner from UDP-GlcNAc and GDP-Man, respectively, to generate Man5GlcNA2-P-P-Dol on the cytoplasmic side of the ER. Each of the sugar additions is catalysed by a specific glycosyltransferase
o Man5GlcNac2-P-P-Dol precursor translocates across the ER membrane bilayer so that the glycan becomes exposed to the lumen of the ER. This translocation is mediated by a flippase
o ManGlcNAc2-P-P-Dol is extended by the addition of 4 Man transferred from Dol-P-Man
o Assembly of the Dol-P-P-glycan precursor is completed with the addition of three glucose residues donated by Dol-P-Glc
o This creates the 14-mer Core oligosaccharide Glc3Man9GlcNAc2 (the core oligosaccharide)
o The core is added to N-X-S/t by an oligosaccharyltransferase (OST)
How are monosaccharides protected from degradation for use in the biosynthesis of N-glycans?
Way that monosaccharides used for this process are protected from being broken down in other pathways is to protect them with a nucleotide (UDP)-> this is how they’re stopped from going to metabolism
What is dolichol and what is it used for in the biosynthesis of N-glycans?
Dolichol is a polyisoprenol lipid comprised of five-carbon isoprene units linked linearly in a head-to-tail fashion with phosphate at the end
• It is a lipid carrier -> sits in the membrane of the endoplasmic reticulum and carry the glycan from the cytoplasm into the interior of the ER
Are the number of isoprene units in dolichols constant? Give an example (yeast vs mammals)
The number of isoprene units varies-
• Yeast dolichol has 14 isoprene units, whereas dolichols from mammals may have up to 19 units
Is the glycosylation energy efficient? Why/why not? How many enzymes are involved?
o Lots of sugar and energy has gone into making this
This happens on every single protein that is on the membrane on a human cell- all of them have N-linked glycans associated with them
14 enzymes involved and 14 monosaccharides involved
In the N-glycan biosynthesis pathway, how is the core oligosaccharide processed? Where is the fate-determining step?
o Processing of core oligosaccharide
Following the attachment of the 14-mer core oligosaccharide to Asn-X-Ser/Thr, processing reactions begin to trim the N-glycan in the ER
Processing or trimming begins with the sequential removal of glucose residues by alpha glucosidases I and II in the lumen of the ER
• The core is broken down
o Glucosidase I and glucosidase II get rid of the glucoses
o ER alpha-mannosidase gets rid of a mannose
Before exiting the ER, many glycoproteins are acted on by ER alpha-mannosidase I, which specifically removes the terminal alpha1-2Man from the central arm of Man9GlcNAc2 to yield Man8GlcNAc2 isomer
• Determines the fate of the glycan
The majority of glycoproteins exiting the ER en route to the Golgi carry N-glycans with either 8 or 9 mannose residues, depending on whether they have been acted on by ER alpha-mannosidase I
Pathways in the Golgi lead to 3 types of final glycan structure: Oligomannose, complex or hybrid N-glycans
What final glycan structure will the core oligosaccharide be if alpha-mannosidase I did not act? What will it do?
• If alpha-mannosidase I did not act, then the oligosaccharide will be an oligomannose
o Will go to membrane and protein will sit in membrane
What final glycan structure will the core oligosaccharide be if alpha-mannosidase I did act? What will it do?
• If alpha-mannosidase I did act, then the oligosaccharide will be a complex or hybrid type-> will go to golgi apparatus for further trimming and processing
Describe the structure of the complex type of glycan structure, what its pathway involves and the most important reactions along its pathway
o Complex- only five monosaccharides remain and N-acetylneuraminic acids are added on the end
Complex pathway involves trimming mannoses and only have 5 of the original 14 monosaccharides remaining-> terribly inefficient
The most important capping or decorating reactions involve the addition of sialic acid, fucose, galactose, N-acetlgalactosamine and sulfate to the branches
• Diversity/heterogeneity of N-glycans is vast
Describe the structure of the hybrid type of glycan structure
o Hybrid- one arm that is high mannose, the other resembles an arm from the complex N-glycan
What is the fate determining step that decideswhether the core oligosaccharide from N- glycan biosynthesis will be a complex glycan structure or a hybrid glycan structure and why
Fate determining step for hybrid pathway- action of GlucNac transferase I and GlucNac tranferase III (this enzyme is really the step that differentiates between complex and hybrid pathway)
• GlucNac transferase III blocks complex pathway by adding a GlucNac to middle structure
Summarise the N-glycosylation of proteins
- Precurosr synthesis occurs in the cytosol
- Glycans are built on lipi-like precursor (dolichol phosphate; Dol-P) in the membrane of the endoplasmic reticulum (ER)
- Once the oligosaccharide has 7 monosaccharides (always the same in the same order), the glycan-Dol-P is flipped into the lumen of the ER by a flippase
- Further monosaccharide addition occurs until a core oligosaccharide comprised of 14 monosaccharides is built (this is also always the same)
- The core oligosaccharide is attached by en bloc tranfer to nascent popylpeptide chains in the ER by an oligosaccharyltransferase (OGT). This addition occurs at the consensus sequon Asn-X-Ser/Thr (where X does not equal Pro)
- Trimming begins by the removal of terminal Glc by glucosidases
- The nascent glycoprotein is transferred to the Golgi Apparatus where further trimming and processing occurs
- Addition of other monoasaccharides during this process results in N-glycan diversity- and N-glycans can be classified into 3 types:
a. Oligomannose
b. Complex
c. Hybrid
Where are sialic acids positioned?
- Always occur at the termini of the arms
- In most cases, Sia are located at the non-reducing terminal ends of carbohydrate chains as monomeric forms on glycoproteins and glycolipids and play important roles in ligand-receptor interaction and cell-cell communication •
What is the implication of sialic acids in cancers?
• Nine carbon sugars highly enriched in cancer
What are sialic acids?
• Sialic acids are acidic sugars and comprise a family of almost 40 naturally occurring derivatives of N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc) and deaminoneuraminic acid (KDN; 2-keto-3-deoxy-D-glycero-D-galactononurosonic acid) with modification by acetylation, sulfation, methylation etc.
What is the charge of sialic acids?
• Highly negative charge
What is the role of sialic acids?
• Play an important role in ligand-receptor interaction, viral interaction with cells, cell-cell interaction…
Describe the difference in Neu5Ac concentration between humans and apes, why this difference occurs and the consequence of this difference
• DNA sequences of humans and chimpanzees are 98% identical
o The first clear example of a major biochemical difference between chimpanzees and humans was the discovery that, unlike great apes, humans cannot synthesize the cell-surface SIALIC ACID N-glycolylneuraminic acid (Neu5Gc)
o In humans, the CMPNeu5Ac hydroxylase (that converts CMPNeu5Ac to Neu5Gc) gene has been inactivated by a 92-bp deletion that occurred in the human lineage after the divergence of humans and chimpanzees
o As a consequence, an excess of its precursor N-acetylneuraminic acid (neur5Ac) is found in humans
Therefore, neur5Ac is all over the surface of human neurons- most enriched sialic acid in human brains
What are the biological roles of glycans?
• Major functions of glycans
o Cell-cell adherence and communication
o Protection against proteolytic damage
o Stabilization of the extracellular matrix (ECM)
o Recognition of self vs non-self (immune response)
o Influence protein-protein interactions
o Signalling (O-GlcNAc predominantly)
What techniques do pathogens use to interact with host-cell proteins?
Some pathogens use molecular mimicry- allows them to interact with host cell proteins
Bacteria have also evolved structures to enable host cell binding via glycan-mediated interactions
When do cells interact/bind with each other
Cells interact/bind to each other when the glycan-binding proteins of one cell matches the glycan on the other cell
What is the role of glycans in influenza viruses?
• Influenza viruses- both neuraminidase and hemagglutinin are strongly associated with sugars
o The hemagglutinin binds to surface sialic acids and the neuraminidase acts as a sialidase (removes sialic acids) to allow the nascent virion to burst out of the cell after using the cell for replication
What is the role of glycans in HIV viruses?
• HIV binding to host cell is mediated by glycoprotein 41, glycoprotein 120 and leptin interactions
o Glycoprotein 120 interacts with lectin on cell surface-> glycoprotein 120 induces conformational change allowing glycoprotein 41 to initiate fusion to the cell
How can protein N-glycosylation protect against unwanted proteolytic degradation?
Protein N-glycosylation can protect proteins against unwanted proteolytic degradation by causing steric hindrance
• If glycan occurs close to trypsin digest site, the trypsin doesn’t cut because it can’t reach arginine/lysine to make that digestion
What is the implication of the fact that there are congenital disorders of glycosylation/what can this lead to?
• Aberrations in these processes lead to irregular development most likely through perturbed cell-cell interactions
• Congenital disorders of glycosylation (type I)
o Mutations in enzymes that mediate seemingly unimportant steps in the glycosylation pathway are extremely delibitating/lead to severe developmental disorders or fatal/nonviability
o Therefore, even if glycosylation is an inefficient process, cannot evolutionarily get rid of it because of terrible consequences of single mutations in enzymes mediating this pathway
How can glycoproteins be analysed using 2-D electrophoresis?
- 2-D electrophoresis
- 2-DE combined with stains (sugar-specific)
- 2-DE combined with electroblotting and anti-sugar antibodies (not as common)
- Affinity chromatography (lectin) and 2-DE
How can glycoproteins be recognised with a 2-D gel?
o Addition of sugars results in both mass and charge [acidic] shift
o Visible as pI ‘isoforms’ or variants across a 2-DE gel (x-axis)
o Multiple glycol-structures
o ProQemerald glycoprotein gel stain and then perform total protein fluorescent stain on same gel so that total protein and glycoproteins will be seen in differing colours in same gel
What is the problem with visualising glycoproteins on 2-DE gels?
o Problem (the tear drop shape)- when there are glycans present, the SDS can’t modify the peptide backbone so the proteins don’t take on a completely identical net-negative charges: form tear drop shape
What are lectins?
o Lectins- carbohydrate binding proteins that are found on cell surfaces that enable two cells to interact with each other via carbohydrate-carbohydrate protein interactions
o Have specificity for an individual sugar or glycan structure
What sugars is wheat germ agglutinin specific for?
GlcNAc, Neu5Ac
What sugars is concanavalin A (ConA) specific for?
Mannose
What sugars is ricinus communis agglutinin specific for?
Ricinus communis agglutinin (RCA; Ricin) : GalNAc/galactose
• Ricin blocks all the sugars sitting on surface of respiratory cells -> quick death
How can glycans be identified on gels/MS after purification occurs? How does this work?
• Once purification has occurred, can treat glycans with glycan-specific enzyme and use these to see if can identify proteins on gels or MS
• Specific enzymes can break the carbohydrate-peptide bond
o Can selectively remove glycan moieties for analysis
• Remove the sugar and both the peptide and the sugar can be analysed by MS
What are 3 proteins that can be used to treat glycans for subsequent identification of proteins with gels/MS?
o PNGase F
o Sialidase
o O-GlcNacase
What makes glycoproteins hard to study with mass spectrometry and subsequent identification using shotgun proteomics?
• Comprised of multiple sugar structures in different combinations
• Unlike other modifications, glycosylation is not fixed in mass difference
o Heterogeneity of glycosylated structures means that signal is extremely diffuse- dilutes the signal from the glycoforms and from the glycopeptides
• Glycopeptide analysis is a challenge for MS and shotgun proteomics
o Glycosylated amino acid may have a wide variety of different glycan structures attached, leading to a pronounced heterogeneity (micro heterogeneity)-> pronounced suppression effects
o Different sites may be only partially glycosylated (macro heterogeneity)-> pronounced suppression effects and ambiguous site assignment
o Metastable fragmentation -> decrease in signal intensity
The glycan bond between sugars/bond between glycan and asparagine or serine or threonine is very weak-> glycan just falls off as energy was added (when you don’t mean to fragment something but something fragments
Without enrichment, glycopeptides ionize poorly in the presence of abundant analytes (e.g in MALDI-TOF MS)
In tandem-MS (MS/MS) using collision-induced dissociation (most common configuration), to dissociate glycan-peptides the very labile sugar linkages are broken and little/no signal is generated from the peptide backbone
o In source decay-> decrease in signal intensity+ unnatural glycopeptides
Unnatural glycopeptides- a couple of the monosaccharides have fallen off which leads to misidentification of the peptide structure
o Extremely hydrophilic-> poor retention of glycopeptides when using reversed-phase chromatography- peptide size dependent. Need to try HILIC
What is glycan microheterogeneity?
Microheterogeneity- what glycan is present at the site
What is glycan macrohetergeneity?
Macroheterogeneity- whether the site is occupied or not
What is needed to sequence glycans and their peptide structures?
Tandem-Mass spectrometry
What is extracted ion chromatography and how can we identify:
- Upregulated protein
- Downregulated protein
- Protein with different glycan structure
• Extracted ion chromatogram- how much of the analyte is there before it goes in the mass spectrometer
o Need to normalise data back to the abundance of the protein
o If protein is upregulated, peak will rise in height
o If protein is downregulated, peak will fall in height
o If protein has a different glycan structure, peak will shift from left/right
How can glycopeptides be characterised by MS? List techniques that could be used
• Characterization of glycopeptides by MS
o Isolation and identification (PMF or Tandem-MS)
o Comparative MALDI-MS (not typically useful due to size of sugar structures)
o Selective enrichment of glycopeptides
Affinity chromatography (lectin affinity)
o Hydrophilic interaction liquid chromatography (HILIC)
o Hydrazide chemistry/ titanium oxide
o Tandem-MS (of glycopeptides and sugar structures)
How are simple glycopeptide mixtures selectively enriched and with what?
• Selective enrichment of glycopeptides (simple mixtures)
o Cells broken open to release proteins which are digested with trypsin to create peptides (some of which have sugar attached)
o Peptides are passed through lectin affinity column and glycosylated peptides bind
Lectin affinity column allows for higher visualisation of glycans-> extremely enriched
Lectin affinity specific for glycosylated peptides
o Elute and get spectra
How can shotgun proteomics be used to analyse the glycomics and proteomics separately and how does it do so? What is its limitation?
• Old technique-The first step in shotgun proteomics is to remove the glycan- created 2 fields- the glycomics (people who analyse the glycan) and the proteomics (people who analyze the glycosylated peptides)
o Allows analysis of the peptide (Asn -Asp diagnostic deamidation catalysed by PNGase F)
Know where peptide was glycosylated due to tag
o Allows analysis of the released glycan structures
o Therefore in large-scale experiments it is impossible to know which glycan came from which protein
How is glycoproteomics performed using shotgun proteomics?
• Shotgun proteomics- current technique
o Mix of non-glycosylated peptide, oligosaccharide, glycopeptide
o Put mix in sodium periodate (NaIO4) which oxidises cis-thiols within the terminal sugar into aldehydes
o Aldehydes can be exploited as they can interact with hydrazide resin-
Hydrazide resin has nitrogen functionality on it
Immobilise all glycopeptides on columns
o Anything that’s not glycosylated passes through
o PNGAseF cleaves the bond between the peptide backbone and the oligosaccharide, leaving the glycan completely immobilised on the hydrazide resin
o The peptide can then be sequenced
How does hydrazide enrichment work?
Hydrazide enrichment- terminal sugars are treated with sodium periodate to oxidise cis-diols to aldehydes that react with immobilised hydrazide resin. PNGAse F then cleaves the peptide leaving the N-glycan covalently attached to the resin and the former glycopeptide is deamidated and analysed by tandem mass spectrometry
What are glycopeptide enrichment strategies?
- Hydrazide enrichment
- Titanium dioxide
- ZIC-HILIC (Zwitterionic hydrophilic interaction liquid chromatography)
How can the sialiome be explored?
o Exploring the sialiome using titanium dioxide chromatography and mass spectrometry
Bind to titanium dioxide-> cleave with PNGAseF and analyse the deamidated form of glycan-peptides without reference to the glycans
Describe how titanium dioxide enrichment for glycopeptides works
o Titanium dioxide-enables the formation of a multi-dentate complex with hydroxyl and carboxyl groups on sialic acids. PNGase F releases the peptide and leaves formerly sialidated glycans attached to the resin
More of a charge based interaction but referred to as multi-dentate because a lot of way in which they can interact
How are glycoproteomics performed with ZIC-HILIC? Outline procedure
o Cell with glycoproteins-> whole cell/membrane preparation-> Digest with trypsin-> use hydrophilicity of the glycan to enrich (hydrophilic peptide enrichment with ZIC-HILIC microcolumns)-> remove non-glycosylated peptides-> identification of glycopeptides by MS/MS fragmentation
How does ZIC-HILIC work and what are its advantages?
o HILIC binds based on the hydrophilic nature of the attached N-glycans
o This is not covalent and works as for any chromatography technique (hence no requirement for PNGase F)
Non-glycosylated peptides can also be analysed
Can analyse intact glycopeptides
With what spectrometer can intact glycopeptides be analysed?
o Intact glycopeptides can be analysed with orbitrap mass spectrometer
What is orbitrap mass spectrometry and its advantages? How can it be used to study glycopeptides?
o Intact glycopeptides can be analysed with orbitrap mass spectrometer
Normal ion traps have high throughput but low mass accuracy and resolution
Also don’t see low mass fragment ions
Orbitrap provides increased resolution and ultra-high mass accuracy
• Allows for focus of ions-> can distinguish the difference between isoforms of amino acids
HCD-Use of C-trap (normally used to store ions from the ion trap prior to orbitrap analysis) as a collision chamber (pseudo triple quad)
• Now additional HCD collision cell (octopole)
Orbitrap enables for the use of ultra-high fragmentation energies (called HCD)- higher energy collisional dissociation
• The higher energy completely shatters glycosidic bonds- but do see peptide sequence
What is important in identifying monosaccharide composition and structures/topology?
• Abundant B- and Y-ions from CID-MS/MS important to identify monosaccharide composition and substructures/topology
What provides the local hydrophobicity for efficient HILIC SPE enrichment?
N-glycans
What can abundant Y1-ion following CID MS/MS be used for?
• Abundant Y1-ion following CID MS/MS can be used for CID-based MS2-peptide identification
What is important for glycopeptide identification?
- c/z-ions from ETD/ECD MS/MS important for peptide identification
- b/y-ions from HCD (or Q-TOF) MS/MS important for peptide identification but N-and O-glycans are detached
How can ETD/ECD MS/MS data be used for site determination of O-glycan
• Crucial c/z ions from ETD/ECD MS/MS for site determination of O-glycan
Which parts of glycopeptides provide local hydrophobicity for efficient RP-(C1B) LC retention
• Peptide provides local hydrophobicity for efficient RP-(C1B) LC retention
What part of glycopeptides ensures efficient positive ion mode ionization and why?
• Multiple basic groups ensures efficient positive ion mode ionization. Multiple charges brings ions into favourable m/z range and is advantageous for ETD
Why might O-glycan proteolytical sites be uncleaved?
• Steric hindrance and electrostatic repulsion from O-glycan may leave proximal proteolytical sites uncleaved
How can sialic acid containing glycopeptides be enriched?
• Sialic acid containing glycopeptides can be enriched using TiO2 SPE or hydrazide (reverse glycoblotting)
What are oxonium ions and why are they useful in identifying glycopeptides?
• Oxonium ions (e.g. m/z 204.087) from HCD MS/MS valuable to identify glycopeptide spectrum and trigger precursor ETD/CID
o Oxonium ions- single monosaccharide marker ion
Can tell the spectrometer to go back and analyse the sugar structure of a peptide if the oxonium ion is found
What is the glycoproteome?
• Glycoproteome- intact glycopeptide analysis
What is glycomics and how is it performed?
Glycan analysis (glycomics) • Sugar released from glycopeptide • Carbohydrate structure subjected to Tandem-MS • Sugar structure can be defined • For glycomics, can treat each structure like a small molecule with reproducible LC retention time and quantitation based on relative intensity o If structure is known, can predict it o A lot of quantitation is done at the LC level for the glycans
How is glycoproteomics performed?
• Glycoproteomics- Complex glycoprotein mixture-> digest proteome-> mix of glycopeptides and non-glycopeptides-> enrichment using hydrazine, titanium dioxide or HILIC just to capture glycopeptides-> use PNGase F which makes asparagine deamidated-> can do LC-MS/MS on formally glycosylated peptides
How is glycomics performed?
• Glycomics- release glycome during PNGase F digest-> look at abundance of different glycans using LC/MALDI-(MS) MS/MS and do quantitation by chromatography
How is site-specific glycoprofiling performed?
• Site-specific glycoprofiling (have glycan in place with peptide or protein- purify proteins-> use HCD and LC/MALD (MS) MS/MS
What kinds of PTMs do reactive oxygen species produce?
• Reactive oxygen species (ROS) induce oxidative stress and redox PTM
What are reactive oxygen species and what are their impacts on the cell?
o Free radicals (free electron)
Highly unstable, cytotoxic
React with cellular elements (e.g. protein, lipids (lipid peroxidation), DNA [highly mutagenic])
• Cause injury when present in excess or anti-oxidant defence (due to protein damage) is compromised
How are reactive oxygen species produced? Describe the process responsible for it
• ROS produced continuously as a result of biochemical processes
o Mitochondrial TCA cycle and oxidative phosphorylation
• ROS must be produced as by-products of aerobic metabolism, that is O2 and H2O2 are formed by accident when molecular oxygen oxidises redox enzymes designed to transfer electrons to other substrates
• Such enzymes are predominantly respiratory chain associated (dehydrogenases- NAD/NADP; FAD etc.). These use flavin cofactors to accept hydride anions [H-] from organic substrates- reduced flavins then transfer electrons onto secondary redox molecules (e.g. iron-sulfur clusters)
• Free electrons usually pass via ubiquinone (Q)- Q is naturally ‘leaky’ and facilitates partial reduction of targets
o Single electron transfers result in free radicals (ROS; O2-)
• If oxygen reacts with the reduced flavin [FADH2] before it has passed on the electrons, an electron can transfer to oxygen to create superoxide
What are the major oxidants in reactive oxygen species and where are they form?
- Major oxidants include OH-, H2O2, O2-
* Formation of O2-/H2O2 in the mitochondria occurs at 11 sites
What are the defence proteins to reactive oxygen species?
• Defence proteins include superoxide dismutase, catalase
What is the name of a biomolecule modified by ROS?
• When a biomolecule is modified by ROS, it is said to be oxidised
What is the name of a biomolecule repaired by ROS?
• When a biomolecule is repaired from ROS, it is said to be reduced
Describe the electron transport chain in the mitochondria
• Process-
o Mitochondrial ATP and O2- are linked by electron transfer from nutrients to O2-.
o Nutrients (glucose, fatty acids, amino acids) are enzymatically converted to common intermediates (acetyl-CoA, oxaloacetate, pyruvate), which enter the Krebs cycle to undergo further oxidation.
o Oxidant production is coupled to NADH and succinate. Electron flow through respiratory complexes via ubiquinone (Q) and cytochrome c (C ) and the reduction of O2 to H2O is couped to the formation of a transmembrane potential of proteins across the mitochondrial inner membrane (MIM) which is then utilised to drive ATP synthesis by complex V.
o ATP is then transported out of the mitochondria in exchange for ADP by ATP; ADP exchanger (ANT)
o Electron transfer flavoprotein oxidoreductase (ETFQO), dihydroorotate dehydrogenase (Dhodh), proline dehydrogenase (Prodh), succinate: quinone reductase (SQR), sn-glycerol-3-phosphate dehydrogenase (G3PDH) can also feed electrons into the Q pool following oxidation of their cognate substrates
Describe how the process of FAD accepting two electrons from succinate can produce reactive oxygen species.
• FAD accepts two electrons from succinate
o If oxygen reacts with the reduced flavin [FADH2] before it has passed on the electrons, an electron can transfer to oxygen to create superoxide. Two possible pathways-
1. If O2- (superoxide) escapes, flavosemiquinone can react with a second oxygen to create another superoxide. This pathway is more typical of SDH and FDH since these use Fe-S clusters to take electrons
2. NADH dehydrogenase pathway- Spin inversion and peroxy adduct formation leading to formation of H2O2. This pathway is more common in NADH DHase
How are reactive oxygen species produced when electrons are passed via iron-sulfur centers to ubiquinone in the mitochondrial transfer pathway?
• Electrons are passed, one at a time, via iron-sulfur centers to ubiquinone, which becomes reduced QH2
What are iron-sulfur centres of iron-sulfur cluster proteins? What are they composed of?
o The Fe-S centres of iron-sulfur cluster proteins may be as simple as:
With a single Fe ion surrounded by the S atoms of four Cys residues. Other centres include both inorganic and Cys S atoms, as in 2Fe-2S or 4Fe-4S centers
• Thiol from cysteines hold iron
How can intracellular free iron be generated by oxidative damage to [Fe-S] cluster proteins? What does this lead to?
cluster proteins
• Many important enzymes contain iron-sulfur clusters
o Aconitase, fumarate, hydratase, serine dehydratase etc.
• In the presence of oxidant (O2-), O2- can occupy this site instead of H2O
o The Fe-S cluster here consists of 4Fe-4S (where 3 of the 4 Fe are coordinated by 4S [including from a Cys amino acid] and the final Fe has 3S and one water
• When this occurs, the catalytic Fe is lost, the enzyme is inactivated, can unfold and is targeted for proteolytic degradation
• In this process, both free iron Fe2+ and H2O2 are produced
• This drives the Fenton reaction and leads to hydroxyl radical damage to DNA
What are the two roles of succinate dehydrogenase?
• Succinate dehydrogenase is a single enzyme with dual roles
o Converts succinate to fumarate in the citric acid cycle
o Capture and donate electrons in the electron transport chain
Why do we need iron in our body? What are the disadvantages of this?
• The iron conundrum
o Iron is an essential trace nutrient as it is a co-factor in many enzymes but excess free iron drives the Fenton reaction
o Iron is often tightly bound to iron storage proteins in the human host (e.g. ferritin, lactoferrin, transferrin, etc.) to limit oxidative damage and to keep iron away from bacteria
What is ferritin?
o Ferritin is a 24-mer subunit protein consisting of multimers of light (L) and heavy (H) subunits= 474 kDa in total.
What is the role of ferritin?
Iron storage and protection
Iron transport
Anti-infection (elevated ferritin during bacterial infection to limit free iron further)
How is iron released from ferritin when it is needed?
o Iron is released from ferritin by lysosomal degradation when needed
What biomolecules are targeted by ROS?
o Sulfur-containing and aromatic amino acids (protein level)
o Iron-sulfur [Fe-S] cluster proteins are damaged
Part of the production of oxidant as well as damage caused by oxidant
o Sulfur-containing amino acids are highly reactive with free radicals (oxidants)
o 10 minutes exposure to H2O2 in the presence of sufficient free iron induces mutations/DNA damage [iron chelators are protective]
o Iron is released when [Fe-S] cluster proteins are oxidatively damaged
o DNA damage, protein oxidation, lipid peroxidation is a large result of oxidative damage in humans [atherosclerosis etc.]
What is the fenton reaction and what does it do?
o Fenton reaction generates the hydroxyl radical in the presence of free iron
H2O2+ Fe2+ -> OH- + FeO2+ + H+ -> Fe3++OH-+HO
Why is it hard to study methionine oxidation?
Methionine oxidation is very difficult to study (methionine sulfoxide reductases)
• The sulfur of methionine pick up oxidants during 2D gel process, during preparation of trypsin digests…
o Hard to know if oxidised methionine is biological or part of the process
What is an example of ROS being a major component of immune defence against bacterial pathogens?
• ROS are a major component of immune defence against bacterial pathogens-
o The phagosome (which takes up bacterial pathogens) has several features that aid in killing bacteria
What features does the phagosome have that aid in killing bacteria?
Low pH
Proteases/peptides (antimicrobial cationic defensins)/lysozyme
Reactive oxygen species (ROS)- oxidative stress- produced by myeloperoxidase (peroxynitrite) and NADPH oxidase
ROS and CI can generate hypochlorous acid
Reactive nitrogen species (RNS)
Decrease ferritin/transferrin- limit iron availability
• Doesn’t contain much ferritin/transferrin-> doesn’t give bacteria chance to steal iron and hence keeps its iron outside the phagosome
Which enzymes usually remove ROS?
• SOD, Kat and GSH usually remove ROS
How do SOD and GSH work together to remove ROS?
• SOD, Kat and GSH usually remove ROS
o A superoxide is reduced by supraoxide dismutase (SOD) to hydrogen peroxide
o Peroxide is either converted to water by catalase or to water by GSH
o Glutathione gets oxidised by glutathione peroxidase to GSSG
o GSSG is reconverted by GSH by GSH reductase
o Glutathione (GSH) is a short tripeptide (ECG) that mops up free oxygen via oxidized cysteine
What is the relationship between GSSG and GSH?
o GSH to GSSG ratio should be 400:1 in a healthy cell
GSSG is a dimer of GSH
What is the role of the SOD enzyme and what are its possible cofactors?
o Superoxide dismutase (SOD)
Converts superoxide (O2-) to H2O2
Several co-factors in bacteria including Fe-SOD (sodB), Mn-SOD (sodM) and Ni-SOD, Cu-Zn-SOD also possible (redundancy+ cell location)
What is the role of Kat?
Converts H2O2 to water and O2
How many catalases do E.Coli have and why?
E.coli has 2 catalases- one cytoplasmic and one periplasmic; others have only 1 (KatA) that can localise to either compartment
What is the advantage of some pathogens being able to secrete catalase into the extracellular environment?
Some pathogens have the ability to secrete catalase into the extracellular environment (competitive advantage and colonization benefit)
What are the primary defenses against both endogenous and exogenous ROS?
o SOD and Kat are the primary defenses against both endogenous (produced by self-organism) and exogenous (produced by a foreign body) ROS
What is the role of glutathione peroxidase?
H2O2 can be reduced by glutathione peroxidase that catalyses the formation of a disulfide bond between 2x GSH to form GSSG dimer
What is the role of glutathione reductase?
Glutathione reductase reduces the disulfide back to 2x GSH, also converting NADPH to NADP+
What are the roles of glutathione (GSH) in production of PTMs?
Alternatively, GSH can post-translationally modify protein thiols- SSG OR reduce intra- or inter-molecular disulfide bonds
• Post-translational modification called glutathionylation
What are peroxiredoxins?
o Peroxiredoxins (Prxs) are a class of antioxidant proteins that reduce oxidants by homodimer formation (disulfide bond)
What is the Prx2, its role and its mechanism of action? What are the consequences of this when there is excess ROS?
Prx2 is a member of the large family of 2-Cys Prxs
Prx2 resolves oxidative stress via an active site cysteine (referred to as the catalytic or peroxidatic Cys) that is oxidized to a disulfide formed with a resolving Cys on a second Prx2 molecule (homodimer formation)
Prx2 is then salvaged (reduced) by the actions of thioredoxin (Trx) or glutaredoxin (Grx) (reductases)
• Reductases reduce disulfide bonds
Excess ROS may induce over-oxidation of the peroxidatic Cys to Cys-SO2H (sulfinic acid-sulfinilation) and Cys-SO3H (sulfonic acid-sulfonilation) and these may not be recoverable by Trx/Grx (cannot be reduced and form disulfide bond anymore)
• Sulfinic acid is very active
What are the possible redox post-translational modifications of cysteine?
o S-sulfhydrylated (-SSH) o Cys-sulfenic acid (-SOH) o Cys-sulfinic acid (-SO2H) o Cys-sulfonic acid (-SO3H) o S-sulfenylamide (-SN) o S-glutathionylation (-SSG) o S-nitrosylated (-SNO) o Free thiol (-SH)/thiolate(-S) o Disulfide bonds (-S-S-)
How are modifications S-sulfhydrylated (-SSH) made from other redox PTMS?
o S-sulfhydrylated (-SSH)
From -SOH + H2S: oxidation
How are modifications Cys-sulfenic acid (-SOH) made from other redox PTMS?
o Cys-sulfenic acid (-SOH) From -SSH + Trx: reduction From -SN + -SH: reduction From -SSG + Grx :reducton From -SH/S + H2O2: oxidation
How are modifications Cys-sulfinic acid (-SO2H)made from other redox PTMS?
o Cys-sulfinic acid (-SO2H)
From -SOH + H2O2: Oxidation
How are modifications Cys-sulfonic acid (-SO3H) made from other redox PTMS?
o Cys-sulfonic acid (-SO3H)
From -SO2H + H2O2: Oxidation
How are modifications S-sulfenylamide (-SN) made from other redox PTMS?
o S-sulfenylamide (-SN)
From -SOH: oxidation
How are modifications S-glutathionylation (-SSG) made from other redox PTMS?
o S-glutathionylation (-SSG) From -SOH + GSH: oxidation From -SN + GSH: reduction From SNO+GSH: oxidation From -S/-SH + GSSG or GSNOL oxidation
How are modifications S-nitrosylated (-SNO) made from other redox PTMS?
o S-nitrosylated (-SNO)
From -S + GSNO and NO: oxidation
How are modifications Free thiol (-SH)/thiolate(-S) made from other redox PTMS?
o Free thiol (-SH)/thiolate(-S)
From -SNO + Trx/GSH/De-NSO: reduction
From -S-S- + Trx/reductases: reduction
How are modifications Disulfide bonds (-S-S-) made from other redox PTMS?
Disulfide bonds (-S-S-) From -SH/-S + GSSG: oxidation
What is the role of enzymatically reversible redox PTMS?
• Enzymatically reversible redox PTMs are protective (all except Cys-sulfinic acid and cys-sulfonic acid, which are not reversible)
What is the problem of irreversible redox PTMs and give two Cys-examples and how these are produced
o Cys-sulfinic acid and cys-sulfonic acid occur when there is too much hydrogen peroxide
They are considered completely irreversible-> causes protein to unfold and degrade
• Cys-sulfinic/cys-sulfonic acid form is toxic to cells
What is the role of cys redox PTMs?
• Redox signaling
• Modifications protect cysteines against ‘over’ or irreversible oxidation
o E.g. S-nitrosylation (SNO), S-glutathionylation (SSG), S-sulfenylamide (in protein tyrosine phosphatases; PTPs)
Redox activity on phosphatases can make them non-functional
• Disulfide bonds and thiol-disulfide interchange/exchange
o Of major importance in signalling and used to purify these PTM modified proteins/peptides
o
• Enzyme catalysis (e.g. SNO/SSG- protein-specific)
o SNO and SGG have been shown to activate and deactivate proteins
• Crosstalk (e.g. in PTPs; but also site level)
• Irreversible oxidation (cys-sulfinic/cys-sulfonic forms)- loss of function/degradation
Where can PTMs occur in the protein? Why?
• PTMs occur in exposed regions of proteins (sites that are accessible)
o If the site is not accessible for the modifying enzyme or solvent, then not likely to see very much in modifications
On which amino acids do redox PTMs usually occur?
• Suite of PTMs that predominantly occur on cysteine thiols (cys-SH)
o Methionine can also be modified (Met-SO)
What factors of the Cys-redox PTMs can be exploited for enrichment/identification?
• Since these PTMs are largely specifically reduced by different means in vivo, this can be exploited for enrichment/identification
o Enzymatically: Cys-SSG (glutaredoxin)
o Chemically: Dithiothreitol (DTT) or other global reducing agents (requires protection of non-modified -SH groups first
What is the problem with enriching redox PTMs?
• Protein-level enrichments are complex and low resolution
o Low stoichiometry modifications- occur on a small number of copies of a protein
What mass-spectrometry based methods can be used for the analysis of redox PTMs?
• Mass spectrometry-based methods for analysis of redox PTMs
o Enrichment by thiol-disulfide exchange post-specific or global reduction of reversibly redox modified peptides
What is the process/method of identifying cys-redox PTMs? Which step/how does this step change depending on the redox PTM of interest?
- Development of TDE for analysis of proteins/peptides containing reversibly oxidised cysteines
- Block biologically free thiols with alkylating agent (NEM- N-Ethyl Malamide or Iodoacetamide)
a. So you know that when doing Mass-spectrometry analysis, anything that is modified with alkylating agent was a biological free thiol (nothing happens to the other modifications)
b. Important to firstly protect any free thiols by alkylation - Selective or global reduction
a. Reduction with glutaredoxin- if interested in S-glutathionylation of proteins (SSG will reduce to free thiol)
b. Reduction with ascorbate- if interested in S-nitrosylation (nitrogen modifications) (nitrosylation will reduce to free thiol)
c. Reduction with hydroxylamine-if interested in S-acylation (acylation will reduce to free thiol)
d. Reduction with DTT- if interested in all reversibly redox modified files
i. Problems- do not know what type of PTMs they were, just that they were redox modified - Thiol-disulfide exchange affinity chromatography
a. Thiol group is located on column
b. Free thiols interchange and form disulfide with the column - Wash-
a. High volume, multiple washes removes false positives and non-specific binding
b. Anything that doesn’t bind- not redox modified - Reduce to elute-
a. Elute anything that binds with DTT and then analyse on gel/mass spectrometry
What are the principles for the enrichment of reversible Cys-redox PTMs? Summarise the steps
- Block free thiols using alkylating agents (NEM; IAA)
- Reduce reversibly oxidised Cys redox PTM with broad spectrum (e.g. DTT) or modification-specific (e.g. ascorbate) reducing agents to make new free thiols
- Bind newly free thiols to a resin (e.g. thiol-disulfide exchange) or label with thiol-specific reagent (e.g. oxICAT, iodoTMT)
o Thiol-disulfide exchange is selective and 2DLC-MS/MS enables compatibility with large-scale analysis of peptides from complex mixtures
90% enrichment- very efficient - Elute/enrich
- LC-MS/MS
o Bound/eluted- should see cysteines
o Unbound/washed content- should see alkylated cysteins but no un-alkyated cyspeptides
What percentage of cys are in protein amino acid sequences and what percentage of these will exist with irreversible modifications?
• Cys is about 1.3% of all protein amino acid sequence and 1-2% of these will exist as SO2H (sulfinic) or SO3H (sulfonic acid) Cys (irreversible modifications)
Are reversible or irreversible redox PTMs more common in disease conditions?
o Under pathological conditions, irreversible modifications rise in abundance (irreversible modifications become more common in disease conditions)
Can you use thiol-disulfide exchange for irreversible modifications?
• Can’t use thiol-disulfide exchange on these because they can’t be converted back (are not reversible)
What does the enrichment of irreversible redox modifications depend on?
• Enrichment exploits the fact that these irreversible modifications are acidic (highly negatively charged)
o Don’t bind well to strong cation exchange
o Use HILIC because irreversible modifications adds regional hydrophilicity to peptides much stronger than normal cysteine/free thiol forms
Describe the enrichment of irreversible redox modifications and how it works in principle, as well as its efficiency
• Enrichment hence depends on:
o Reduction/alkylation of disulfide bonded Cys or free thiols
o SCX (strong cation exchange) negative selection
Irreversible modifications don’t bind well to strong cation exchange as they are negatively charged
o HILIC positive selection
Irreversible modifications come right at the beginning of liquid chromatography gradient
o Only gets about 20% enrichment- not ideal
Describe the steps for a multiplexed and quantitative assay for reversibly oxidised peptides in I/R
• Protein extraction + free thiol alkylation (NEM=A)
• Reduce PTM cys (DTT)
• Block new thiols (MMTS)
o To protect from chemistry during trypsin digest and iTRAQ labelling
Protect free thiols made from previous step
• In solution digest (trypsin)
• iTRAQ label (1st amines)
o Can’t label Cys-SO2/SO3 H very well as if add iTRAQ label to primary amines, can destroy any separation gotten by SCX and HILIC (removes specificity of this)
• Pool and reduce protecting disulfide (TBP)
• TDE enrichment results in two labelled fractions
Why aren’t label Cys-SO2/SO3H labelled with iTRAQ?
o Can’t label Cys-SO2/SO3 H very well as if add iTRAQ label to primary amines, can destroy any separation gotten by SCX and HILIC (removes specificity of this)
Why are proteases needed?
• Proteins (given their sequences ad folded structures) represent a highly diverse substrate
o Proteins have a life-span, once they reach the end of that life-span or become toxic to the cell due to modification (e.g. irreversible redox PTM) or unfolding they must be removed
o Other proteins have functions that need to be tightly regulated at post-translational level (e.g. blood pressure regulation) and therefore are synthesized in an inactive form that requires a protease for activation
o Proteases themselves are often synthesized in an inactive form so that their effects are regulated (this form is called a zymogen or proenzyme)
What is the difference between exoproteases and endoproteases and give examples
• Proteases that remove amino acids from N- or C-termini are broadly classed as exoproteases (e.g. aminopeptidases and carboxypeptidases), while those that attack internal peptide bonds are endoproteases (e.g. trypsin)
What are classes included in exoproteases and what is their purpose?
o Exoproteases include methionine aminopeptidase (removes start codon methionine) and signal peptidases (signals important for defining where the protein will end up in the cell-cleaved by signal peptidases)
Aminopeptidases- cleave amino acids at the N terminus
Carboxypeptidases- cleave amino acids at the C terminus
What proportion of the human genome do proteases make up?
• Proteases represent a significant proportion of the Human Genome (2% of genes)
o 590 proteases in the human genome
What are the functions of proteases?
• Protease functions-
o Some enzymes (including proteases themselves) are synthesized in an inactive form (a proenzyme or zymogen) that needs to be activated (often by protease activity)
o The active protease then cleaves its targets; however, the activity must be tightly regulated and endogenous inhibitors can stop activity, or alternatively the protease can be removed by proteasomal degradation
o Protease inhibitors are therefore a large class of drugs used to treat diseases
Process-
Transription-> translation-> activation of zymogen by protease action-> active form of protein acts on substrate and has downstream effect-> active protein will be inhibited or degraded when no longer needed
How are proteases important in the coagulation cascade and what is the process of this cascade?
o Coagulation cascade
Sequential proteolytic cleavages of serine protease zymogens activates them, allowing the cascade to progress
• The intrinsic pathway (external damage) is activated upon exposure of blood to negatively charged exposed surfaces and depends on the proteolytic activation of factors XII, XI and IX
• The extrinsic pathway (internal/vascular damage) is activated when vascular tissue damage leads to activation of factor VII
• The pathways converge at proteolytic activation of the endopeptidase factor X (also known as the stuart-prower factor) which is capable of converting prothrombin into thrombin. Thrombin also cleaves and activates fibrinogen, allowing activated factor XIII to form a cross-linked polyfibrin clot
Can protease defects cause disease? How many hereditary diseases of proteolysis are there?
- Defects in proteases are associated with many diseases including neurological, inflammatory, cardiovascular diseases and cancers
- About 200 hereditory diseases of proteases/proteolysis
What are the two main types of proteases?
- Endoproteases
* Exoproteases
What are the two main classes of exoproteases
o Amino-peptidases
o Carboxy-peptidases
What are methionine amino-peptidases and how do they work? What determines the lifespan of proteins with this peptidase?
o Amino-peptidases
Methionine aminopeptidase removes the N-terminal [START CODON] Met
• The amino acid at position 2 reflects the lifespan of the proteins- this is known as the N-End rule
What is the function of di- and tri-peptidases and how do they do so?
Di- and tri-peptidases are involved in recycling of amino acids
• Break bonds between amino acids (di- removes 2, tri- removes three)
What are the two main types amino-peptidases?
Methionine aminopeptidase
Di- and tri-peptidases
What is the main function of carboxypeptidases?
o Carboxy-peptidases
Carboxypeptidases are crucial in processes from digestion through to recycling and hormone activation
How is insulin made into an active hormone?
• E.g. Insulin is produced as a preproinsulin that contains a 24-mer signal peptide then removed by signal peptidase; proinsulin is then acted upon by endopeptidases and carboxypeptidase E to make the 51 residue insulin active hormone
o Tightly controlled at the post-translational level
How are proteases classified?
• Proteases are classified based on conserved catalytic domains
How many proteases do humans have? Include breakdown of extracellular, membrane and intracellular proteases
o Human- 567 proteases
273 extracellular proteases
16 membrane proteases
277 intracellular proteases
What are the main protease catalytic domains in humans and how many per domain?
- Threonine (28)
- Aspartate (21)
- Cysteine (148)
- Serine (175)
- Metallo (194)
What protease is trypsin classified as?
o Trypsin is classified as a serine protease-
How do serine proteases act?
Asp-102, His-57 and Ser-185 represent a catalytic triad found in all serine proteases
Arg and lys are attracted to the catalytic site by the negatively charged Asp189 (substrate-binding)
Protease then folds and then the serine moves in the catalytic triad and cleaves the bond between lysine and the next amino acid
How many proteases do murine have? Include breakdown of extracellular, membrane and intracellular proteases
341 extracellular proteases
16 membrane proteases
287 intracellular proteases
What are the main protease catalytic domains in murine and how many per domain?
- Threonine (26)
- Aspartate (2&0
- Cysteine (162)
- Serine (224)
- Metallo (205)
How many variants do mice have in comparison to humans?
Human-
o Humans only have 2 variants of trypsin
Mice-
—-Mice have 8-9 variants of trypsin (mice are omnivores-widespread diet)
Describe how substrate and cleavage site positions are labelled in protease recognition studies
• Proteases are classified based on their catalytic site amino acid composition
o In protease recognition studies, the substrate is given the designation P4, P3, P2, P1 and P1’, P2’ etc. For example trypsin cleaves substrates with K or R at P1 and any amino acid but proline at P1’
In protease studies, upstream amino acids from cleavage site are given designation of P’, whilst those downstream from cleavage site are given designation of P
On the protease, upstream of the protease cleavage site positions are given the designation of S’, whilst those downstream from cleavage site are given designation of P
o The catalytic site (active site) of the protease has the designation (S4, S3, and S1’, S2’, etc.)
These are not necessarily in sequence order in the protease
What are capsases and how do they work? What happens if they are deficient?
o Caspases- cysteine-aspartases are Cys proteases that target the C-terminal side of Asp
Caspase deficiency is linked with uncontrolled cell division (tumourigenesis)
Caspases are intimately associated with cascade that leads to apoptosis
What are calpains and how do they work?
o Calpains- calcium-activated cys proteases
Calpains can increase apoptosis while also working in concert with HIF and other proteins to increase cell division, migration and invasion
What information can the cleavage site give?
• Cleavage site location might give information about
o Consensus sequence identity for a particular enzyme
o Modifications present on cleavage site that may attract protein
o Consensus sequence genome location uniqueness
What are techniques for the analysis of proteases?
o Comparative MALDI-MS (or ESI-MS)
o Enrichment of ‘neo’-N- and / or C-termini
o Exploit activity of proteases themselves
Recombinant proteases
Zymography
How is comparative MS of native and fragmented proteins performed for analysis of proteases?
Should see image [spectral] differences (and similarities) in peptide maps
Comparative MS of native and fragmented proteins
• Take two protein spots identified as products of the same gene, but large variation in mass (suing MASCOT)-> excise and subject to tryptic digest-> MALDI-TOF MS-> spectrum
o Spectrum of the native protein should contain several peaks more than the fragment: both spectra should share some peaks in common and hopefully, the fragment should contain one novel peak corresponding to the newly formed N- or C-terminus
What is the neo-N/C terminus?
Neo-N/C-terminus: the amino acid that is the N/C terminus of the lower mass variant (fragment)
How is in-gel zymography performed for identifying active proteases
• In-gel zymography for identifying active proteases
o The SDS-PAGE gel is polymerized in the presence of a substrate- e.g. a protein standard [gelatin or casein] or even a complex whole cell lysate
o Your protein mix is introduced (non-reducing, non-denaturing]; a standard (e.g. a recombinant protease of interest) can also be used
Sample in nonreducing loading buffer and standard protein(ase) mix
o SDS-PAGE is performed and the gel visualised
Electrophoresis washed in Triton X-100 solution
Incubated in a suitable assay buffer
Stained and visualised e.g. Coomassie blue staining)
o Negatively staining regions are those where a protease is present
What can zymography be used for and how can it be used for this?
• Zymography can be used to identify new proteases and recombinant proteases to determine specificity
o Negative staining (bright staining)= proteases
o Can look at how the protease digests things to find out what its substrates are and what residues it recognises
Increase protease concentration-> run substrates on gel-> cut bands out and see where protease sites are
Peptide mapping with ITRAQ labelling
How are degradomic approaches used to examine proteases? Describe
• Degradomics approaches using negative selection of N-terminal peptides
o Proteome samples under different conditions (e.g. control versus protease-overexpressing cells) contain uncleaved, intact proteins and cleavage fragments (neo-termini) resulting from proteolysis
o All primary amines are blocked- demethylation or-isobaric- reagents such as ITRAQ or TMT
o Trypsin digest is conducted
Creates a huge number of neo-N termini but neo-N termini created in the trypsin digest do not contain a blocked primary amine
o Enrichment-
TAILS
COFRADIC
ChaFRADIC
o Mass spectrometry identification and quantification
What enrichment techniques can be used for protease analysis using degradomics approaches using negative selection of N-terminal peptides
TAILS uses a resin to remove tryptic peptides that bind via their unblocked alpha-amino groups, allowing for negative enrichment of blocked N-terminal peptides for MS
COFRADIC relies on a series of chromatographic fractionations and depletion of internal (that is unlabeled) tryptic peptides after derivatization with a hydrophobic reagent (TNBS)
ChaFRADIC is a COFRADIC variant that relies on the charge shift of nonterminal, tryptic peptides upon chemical acetylation that allows removal by SCX chromatography and negative enrichment of N-terminal peptides
How is TAILS enrichment performed?
TAILS uses a resin to remove tryptic peptides that bind via their unblocked alpha-amino groups, allowing for negative enrichment of blocked N-terminal peptides for MS
• Deplete free alpha-amine containing peptides (that is internal tryptic peptides) with HPG-ALD polymer that binds primary amines-> remove polymer
o Blocked amines get passed through, free alpha-amines are stuck in column
How is N-terminal amine isotopic labelling of substrates (N-Degradomics) performed? What would the iTRAQ ratios suggest?
• N-terminal amine isotopic labelling of substrates (N-TAILS): N-Degradomics
o Step 1: Sample
o Step 2: Cell lysis, denaturation, reduction and alkylation
o Step 3: whole protein dimethylation
Proteins are demethylated to block any primary alpha-amines (from ‘true’ N-termini [Met, Met + 1, or following removal of predicted signal] or ‘neo’ N-termini)
o Step 4: protease treatment/digest with proteases such as trypsin
o Step 5: N-terminal enrichment
Primary alpha-amines generated by trypsin digest (peptide N-termini and lysines) are bound to hyperbranched polyglycerol resin (HPG-ALD) while demethylated peptides are not
o Step 6: LC-MSMS
Dimethylated peptides are subjected to LC-MS/MS (can also do quantitation using isotopic dimethyl)
• ITRAQ ratio ~1.0: original N-terminus or basal proteolysis
• ITRAQ ratio»_space;1.0 : generation of neo N-terminus due to proteolysis
• ITRAQ ratio <1.0: loss of original N-terminus by proteolysis
How is COFRADIC performed for proteases?
COFRADIC relies on a series of chromatographic fractionations and depletion of internal (that is unlabeled) tryptic peptides after derivatization with a hydrophobic reagent (TNBS)
• SCX fractionation-> RP fractionation-> Derivatize free alpha-amines with TNBS-> RP fractionation
How is ChaFRADIC performed for proteases?
ChaFRADIC is a COFRADIC variant that relies on the charge shift of nonterminal, tryptic peptides upon chemical acetylation that allows removal by SCX chromatography and negative enrichment of N-terminal peptides
• SCX fractionation-> acetylate free alpha-amines-> SCX fractionation-> select fractions without retention charge shift
What is the use of degradomics or terminomics?
• Degradomics or terminomics enables identification of protease substrates (e.g. MMPs)
o Look for protease recognition motifs
o Look for targets of degradation during disease
o Mainly N-terminal studies but some work being done on C-tails as well
What is non-enzymatic deamidation and how does it work? How many mass units does it add and where is it most common?
Non-enzymatic deamidation: Glutamine (Q) to glutamate (E), or asparagine (N) to aspartate (D)
o +1 mass unit (128-129 or 114-115)
o N D about 10 times more common
o Most common where glycine is found C-terminal to N or Q, but protected by valine (branched, hydrophobic)
Uncommon when there is a valine
o Chemical modification
Where was non-enzymatic deamidation first discovered?
o First discovered in cytochrome C- deamidated forms have different physicochemical properties and is a signal for degradation
How is deamidation analysed?
Deamidation-analytical
o 2-DE gels (spot trains)
o Comparative MS-post-tryptic digest and Tandem-MS
o There are no specific stains, no specific affinity approaches and no specific enzymatic strategies available
Is enzymatic or non-enzymatic deamidation a marker of aging?
o Deamidation is considered a marker of protein aging
Deamidation occurs non-enzymatically as a function of protein aging, not necessarily associated with PNGase-F
What is the purpose of deamidation?
Implicated in pathology- deamidation is a modification of old proteins that need to be degraded
What is the role of deamidation in crystallins?
Example- eye lens proteins (crystallins)
o Slow turnover during lifetime
o Deamidation leads to aggregation
o Aggregation leads to cataracts
o Deamidation-mediated cleavage reduces function as chaperone
What is the role of deamidation in tau protein?
Example- Tau protein (Alzheimer’s)
o Deamidated in neurons
o Leads to aggregation
o Neurofibrillary tangles
What things happen between transcription and translation?
o Isoforms- produced by e.g. endogenous proteolysis and RNA splicing
o Coding SNPs or mutations in the gene may cause different amino acids to be translated
o Post-translational modifications
What are proteoforms?
• Proteoforms- encompasses all possible combinatorial variations of a protein whether the variation is generated at the genetic, proteomic or modificomics level
o Other terms include protein variants, protein forms, protein specides
How many possible proteoforms can be made with PTMs and how is this calculated?
o Possible proteoforms= 2n
Where n is the number of possible amino acid site modifications
If more than one modification is possible at any given site then the number of proteoforms is multipled by m+1 (non-modifified, modified 1, modified 2…)
• E.g. a protein with 6 modification sites of which 1 site can be modified in 4 ways would be 25x51
What are the limitations to achieving theoretical proteoform diversity in reality?
• Limitations achieving theoretical proteoform diversity in reality
o Copy number- the actual copy number per cell limits the proteoform diversity
Copy number- how many copies of the protein are translated in the cell
o Gene expression- whether the gene/protein is expressed at all in a given cell type
What is top down-proteomics?
• Top down-proteomics-
o Tandem mass spectrometry of proteoforms creates fragment ions
o Proteoform fragments map to primary sequence and localise amino acid modifications and variants
o A protein is redefined as a set of proteoforms
What is bottom-up proteomics and its disadvantage?
• Bottom-up proteomics-
o Protein digestion produces peptides that are identified by tandem mass spectrometry, allowing inference of proteins present
o It is not generally possible to identify the proteoform of proteoforms from whichhe peptides are derived
Can unique proteoforms serve as diagnostic markers?
• Unique proteoforms serve as diagnostic markers and may unveil therapeutic strategies
What are the limitations of shotgun of bottom-up proteomics?
• Limitations of shotgun or bottom-up proteomics
o Loss of total proteoform context in bottom-up proteomics
What is an advantage of top-down proteomics?
• Top down proteomics/whole protein analysis-
o Get the cell-> get the proteins out of cell-> do mass spectrometry of total protein
o Gives context of total proteoform
What are the challenges of top-down proteomics/its disadvantages?
• Challenge of top-down proteomics is primarily technical-
o Requires efficient separation of proteins on a chromatographic time-scale compatible with MS
To acquire the data for a whole protein is very slow- peptides go very fast in the MS, but whole proteins require much more time because they are bigger biomolecules
For large-scale approaches need suitable separation commensurate with MS speed
o Requires fragmentation approaches that generate sufficient sequence coverage of large biomolecules (proteins) while maintaining labile PTMs for site elucidation
If the labile PTMs are not maintained, lose the proteoform context
o Requires an informatics/data analysis pipeline that can account for poor MS/MS coverage and modifications
o Disadvantage is time- takes a lot more time than bottom up proteomics
o Causes an enormous increase in complexity of data
Data intensive and difficult interpretation of resulting spectra
o Best performed on purified proteins
o Relies on electron-based fragmentation techniques (ETD)
What protein separation needs to occur prior to top-down proteomics and how can this be done?
• Protein separation prior to Top-down proteomics-
o Gel-free electrophoretic separation is possible with fractions validated on SDS-PAGE gels
Isoelectrofocusing in solution
o Affinity purification (AP) for specific proteins/proteoforms or complexes
o Chromatography (LC) using C4 resin for protein separation
What protein separation cannot be used to separate prior to top-down proteomics?
o Cannot use 2-D gels
2-D gels are inherently bad with big biomolecules- can’t get ino the gel
Had to get intact protein from the gel- will get extremely low yield
• Will not diffuse out of gel easily
What types of fragmentation can be used to fragment peptides?
Collision-induced dissociation (CID) Higher collision dissociation (HCD) Electron transfer dissociation (ETD) Electron capture dissociation (ECD) Infrared multi-photon dissociation (IRMPD) Ultraviolet photodissociation (UVPD) • Very nice for sequencing lipids Electron ionisation dissociation (EID)
What is collision-induced dissociation, what does it create and what does it do?
Collision-induced dissociation (CID)
• Create y and b ions
• Vibrational form of separation
• Break the peptide bond/amide bond between carboxy terminus of 1 amino acid and the amino terminus of the next amino acid-> makes sequencing the peptides extremely simple
What is higher collision dissociation, what does it create and what does it do?
Higher collision dissociation (HCD)
• Create y and b ions
• Vibrational form of separation
• Break the peptide bond/amide bond between carboxy terminus of 1 amino acid and the amino terminus of the next amino acid-> makes sequencing the peptides extremely simple
What is electron transfer dissociation, what does it create and how does it do so?
Electron transfer dissociation (ETD)
• Creates c and z type fragment ions
• Breaks the N-carbon alpha bond
o In ETD, fragmentation of large, multiply charged precursor cations occurs by transferring electrons from a singly charged radical anion
Molecules with a high charge state (+2 or above) are trapped in the ion trap and a radical anion introduced (infused as a gas; typically fluoranthene)
Fragmentation produces c- and z- type peptide backbone ions (cleavage at the N-Calpha bond rather than the amide bond cleaved in CID)
Why are CID/HCD fragmentation approaches inappropriate for large biomolecules with PTMs?
o Collisional approaches (CID/HCD) generate fragment ions by collision of an ion (parent/precursor) with an inert gas (e.g. argon) that converts translational energy into vibrational energy which breaks the amide bond in peptides to produce b- and y- ions
o For large biomolecules, CID/HCD produce too few fragment ions to enable elucidation of the sequence
o Additionally, as we have seen previously, CID/HCD result in loss of labile PTMs and thus do not allow PTM site elucidation
What fragmentation approach is used to fragment larger biomolecules with PTMs and why?
o ETD allows fragmentation of larger molecules with high charge states at the expense of time (MS) and with increased tandem-MS complexity (data analysis)
o ETD does not cause loss of labile PTMs, which are stable in electron-based approaches and thus site identification is possibleo
What are issues with performing top-down proteomics at large scale?
• Top-down proteomics at large scale
o Issue of time-scale vs separation- ETD allows fragmentation of larger molecules with high charge states at the expense of time (MS)
o Separation requires native state- LC or isoelectric focusing
o ETD suitable up to 50-80kDa and for relatively hydrophilic proteins
Hydrophobic protein limitation- they come out of solution in separation state
