Stuart Haslam - Mass Spec + PTMs Flashcards
What is mass spectrometer?
Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions.
But
it is also used to define the covalent structures of substances by ionizing, separating and detecting molecular and fragment ions according to their mass-to-charge ratios (m/z)
Looks at the ratio of mass & charge of gas phase ions NOT just mass
Why is Mass spec useful for biological molecules?
Mass spectrometry can be carried out on very tiny amounts of material (e.g. femtomoles or less (10-15mole)) and can be used to study very complex mixtures eg urine extracts, perfumes, protein digests et.c
Basically….
- Exceptionally sensitive technique which means you can use very small starting amounts – more sensitive than NMR
- Do not have to have highly purified homogenous samples! - ability to handle complex mixtures
What three parts can a basic MS be divided into?
Wide variety of Mass spec instrumentation on the market but they can all be divided into three main components…
- Ion sources – we analyse gas phase ions but most biological molecules don’t exist in this state - hence, conversion of sample into gas phase ions occurs at the ion source
- Mass Analyser - Separate gas phase ions based on their mass to charge ratio
- Detector - Detect and obtain signal as well obtain quantitative measurement (abundance)
Outline what a basic mass spectra looks like?
Mass spectra - Expressed graphically
X - (m/z) –> Mass (atomic mass units) to charge ratio - Reported mass is dependant of the ionisation state of species
Y - (intensity) –> relative measure - highest ion count corresponds to the highest peak – given an arbitrary value of 100. Hence, the other peaks are expressed as a percentage of the max.
Intensity and m/z don’t have specific units associated with it
What are the three main ionization methods?
Purpose of Ionization Methods is to change biological molecules into gas phase ions
- Electron Impact (EI - Hard) - described as a hard ionization meaning it is a high energy ionization technique. This can lead to excess of energy resulting in fragmentation of the biological molecules – small molecules, 1-1000 Da
- Electrospray Ionization (ES or ESI - Soft) – peptides, oligosaccharides, proteins, greater than 500,000 Da
- Matrix Assisted Laser Desorption Ionisation (MALDI - Soft) – peptides, proteins, DNA, up to 500,000 Da
Note - Both ES and MALDI use counterions to give their species charge but molecule itself doesn’t become ionized
Soft Ionization - just enough energy to convert to gas phase ion is used but no excess energy leading to fragmentation
Outline the Electron impact ionisation-MS procedure.
Electron Impact Ionisation – EI-MS
Main prerequisite – the sample has to already be in the gas phase before ionization – limits size as converting large molecules into gas is challenging
How is gas formed? Sample introduced into source by heating it from a probe tip until it evaporates or from an on-line gas chromatograph set-up (use to separate molecules in gas phase)
Ionization? Bombardment - Gas phase sample is bombarded with high energy electrons coming from rhenium or tungsten filament (energy = 70 eV)
What happens during electron bombarment during EI-MS?
No actual collision!
- High energy electron beam comes into close proximity with biological molecule – negatively charged electron beam repels out an electron from the outer orbital of the molecule
- Ionization occurs by loss of an electron to give M+ (radical cation) – yields gas phase radical cations!
Fragmentation?
Most of the molecular ions decompose into fragments (70 eV beam >> 5 eV bonds) via uni-molecular reactions - Electron beam is at 70 eV whereas the average bond in a molecule is at 5 eV meaning that there is a great excess of energy which allows for fragmentation
Before running MALDI ionisation what do we need to do?
Matrix Assisted Laser Desorption Ionisation (MALDI) – ionization from solid phase
Sample is embedded in a low molecular weight UV absorbing “crystalline” matrix which is chosen to have an absorption maximum near the wavelength of the pulsed laser that is used to ionise the sample.
Basically we need to co-crystalize the sample into a matrix
Outline how MALDI is used to create gas phase ions?
Note - Ionizations occurs in a vacuum
- Input of energy comes from pulses of laser energy – targeted to crystals on metal target
- The matrix absorbs the laser pulse, and enough energy is transferred (energy transfer process) to the sample to produce gas phase ions.
- Process not well understood - believed to be similar to “flash evaporation” - From this process we get both +ive and -ive charged ions BUT we can only analyse one type of ion
- Depending on the charge we want to analyse we would place the complement (positive or negative) charge potential on target – leading to repulsion and movement towards detector
What lasers are used for MALDI?
What are some common MALDI matrices used?
How was delayed extraction used to improve the quality of MALDI spectra?
Problem –> Ions of the same mass coming from the target have different speed which is due to uneven energy distribution.
Molecules at different positions in the 3D crystal matrix experience different rates of ionization/energy transfer - e.g. deeply buried molecules would take longer.
Solution - Delayed extraction –> ensures ions have the same velocity when they enter the analyser
Outline the Electrospray ionisation procedure.
Electrospray Ionisation (ES or ESI) – Ionization occurs in the liquid phase which is favorable for biological molecules as this resemble their native state within cells.
Procedure
- Sample dissolved in suitable solvent
- Sample introduced through a narrow glass capillary coated with gold at the tip - A high voltage (3-4 kV) is applied to the tip
- Capillary tip funnels solution towards a covering electrode – There is a large electrical potential between needle tip and the electrode (due to coating and application of a high voltage of tip)
- Consequence of large electrical potential - Sample emerging from the tip is dispersed as an aerosol of highly charged droplets
- Charged? – depending on solvent used and pKa our protein can be charge + large electrical potential created by electrode & metal tip can also make our peptides charged
How do we get ionization from the highly charged droplets produced in ES-MS?
What is Nano-electrospray of Nanospray ionisation?
Early ES sources operated at flow rates of a few microlitre/min
Nanospray - Newer source sources operate at flow rates of 10-30 nl/min – nanoliter per minute
Consequence - NanoES is much more sensitive than ES - produces smaller droplets than conventional ESI resulting in more efficient ionization - increased signal from sample + a lot less starting material needed
- Sample quantities are typically in the subnanogram range
- NanoES is carried out with “needles” into which about 1 ml of a solution of the sample is added – allowing for many MS and MS/MS experiments on a single sample (MS exp requires very little)
- It is also possible to introduce the sample using on-line nanoLC - very powerful method for analysing complex mixtures - coupling with liquid chromatography allows for separation followed by analysis
What is the role of the analyzer and what are the different kinds?
ANALYSERS – separates our ions by their mass to charge ratio
- QUADRUPOLE
- TIME-OF-FLIGHT (TOF)
- ION TRAP
- ORBITRAP
What are the three main factors to consider when analyzing the effectivness of an MS-analyzer?
3 key factors to consider:
- The upper mass limit – largest m/z ratio that it can successfully separate
- Ion transmission – How many ions produced by source can be separated by the analyser - efficiency
- Resolution – ability to separate ions with a similar m/z ratio
What is a Quadrupole Mass filter? What are it’s components?
How does a quadrupole mass filter seperate ions based on their m/z ratio?
How does it work?
- Pass sample in between the quadrupole
a) Some ions (red) will be in harmony with the magnetic field -move towards the detector
b) Some ions (blue) will be out of harmony with the magnetic field - attracted to one of the poles and will be destroyed - resulting in no signal - Hence, by varying the strength of the Quadrupole Electromagnetic field we can select for ions of a specific m/z to pass through to the detector
How to decipher the m/z value from the field strength?
- By calibrating using molecules of known m/z ratio’s we can generate calibration curves for m/z value vs. quadrupole field - allowing us to calculate the m/z ratio of the ion in our sample
How does a quadrupole mass filter perform? Is it a high resolution analyzer?
Key performance characteristics of quadrupole mass filter:
- m/z ratios of up to about 4,000 can be observed – Lowest upper mass range
- Relatively low resolution (unit resolution to about 3,000) - inability to seperate peaks
- Low Ion trasmission - as we adjust the field strength many ions will not pass through the detector
- Lower sensitivity - When using the quadrupole mass filter we perform rapid scanning - scan quickly through quadrupole field meaning that we DON’T detect all ions produced from the source reducing the sensitivity
Overall Low performance compared to other analyzers but!
- Ideal for GC-MS (EI ionization - already in gas phase)
- Widely used for ES-MS
- Low cost
- Robust
Outline what a ion trap analyzer is?
How does a Ion trap analyzer work?
- Ions produced by the source – enter the ion trap
- Generate a 3D quadrupole electromagnetic field trapping ions in ring electrode where they circulate
- We can manipulate the strength of the electromagnetic field – influencing the energy of the rotating ions and thus the radius of rotation within the ion trap - eventually ions with specific m/z ratio’s are ejected from the ion trap
- Hence, a mass spectrum produced by scanning the RF voltages to eject ions through the end cap
Performance characterisitcs of Ion trap analyzers?
Performance characteristics:
- Mass range - Ions within a selected m/z range are trapped within the electrodes – higher than Quadrupole
- Higher resolution than quadrupole
- Similar, if not better, ion transmission relative to quadrupole – remember not perfect when scanning we lose ions in the trapping process – not 100% efficient collide with the edges of the trap and not get emitted
How has the ion trap analyzer improved in recent years?
Outline what an Orbitrap is and how it works?
Data collection explanation
Ions with a different m/z ratio will travel along the spindle at a different rate (frequency) around the axial direction (Z)
The rate of oscillation is captured in the time-domain but has to be converted into Frequency domain - facilitating comparison.
The Frequency domain of our ion of unknown mass can then be compared to a calibration curve setup with molecules of known mass.
Performance characterisitcs of an Orbitrap?
Performance characteristics:
- High resolution – separation of similar m/z
- High mass accuracy
- Good upper mass range
- Good ion transmission
- Overall – Highest performance but this comes at a price – Cost!
What is a TOF analyzer? How does it work?
Performance characterisitcs of TOF?
Performance Characteristics:
- Ion transmission - Very good – high sensitivity
- Extremely high mass range - theoretically unlimited – as long as you can generate an ion you can produce a mass spectrum
- Intially, low resolution but the development of reflectrons has allowed for higher resolution
Outline how the reflectron was used to increase TOF resolution?
PRINCIPLE OF THE REFLECTRON – Ion mirrors
Sometimes, as seen with delayed extraction, we can get molecules with the same m/z but with kinetic different energy – reflectrons correct for the effects of kinetic energy distribution
The reflectron is an ion mirror that reverses the direction of travel of the ions - ions of greater kinetic energy penetrate further into the ion mirror and therefore have a longer flight path (visa-versa) - in turn equalizing total flight path between species with the same m/z
- Linear analyzers have low resolution (<1000); reflectrons give much higher resolution (>3,000)
Has the reflectron resulted in increased TOF resolution?
YEAAAHHHHH BUDDYYYYY
What is the role of Ion-detectors?
Ion Detectors - Allows us to detect separated ions and obtain quantitative information (relative quantities)
Detailed understanding of the physics not required but you should have a general idea of how they work
Outline how a PM-photomultiplier is used as a MS detector?
Outline how an EM-electron multiplier is used as a MS detector?
Outline how a micro-channel plate (MCP) array detector is used in MS?
Why does EI combine well with quadrupole analysers?
Why does MALDI combine well with TOF analyzer?
What type of analysers work well with ES ionization?
Why would we want to combine multiple analzers?
Hybrid Instruments – More & more common – used for 2D MS or MS/MS analysis
There is an ever-increasing range of “hybrid instruments” which have two or more analysers in tandem e.g. Q-TOF, TOF-TOF (MALDI source) and linear ion trap-orbitrap.
Combining mass analyzers we can escape some of the problems/disadvantages with specific mass analyzers
Note - it is also possible to combine two of the same analyzers
The main instrument manufacturers have their favoured instrument geometries, and they compete on performance (resolution, sensitivity etc), price, robustness etc
What are the two main pieces of information you can obtain from a MS spectra?
What should you consider when looking at Electron Impact (EI) molecular ions?
ELECTRON IMPACT - Hard ionization
RADICAL CATIONS M+ formation
Outer orbital electron is ejected to release a single positively charged radical cation
Mass of electron is negligible, so m/z of a molecular ion is equal to the mass of the molecule
What should you consider when looking at ES or MALDI molecular ions?
Soft ionization techniques – produce pseudomolecular ions – meaning we need a counterion associated with the ion
Counter ion needs to be taken into consideration
What type of molecule increases the liklihood of getting a molecular ion signal when using EI?
Due to excess energy, the molecular ions may sometimes not appear in the spectra as they become fragmented
But Alkaloid system can absorb the energy so sometimes resulting in the formation of a molecular ion
Is it possible to have ions of the same species with the different charges on an ES spectra?
Yeah BUDDDYYY
Deconvolution - take out the charged component of myoglobin – gives single Molecular ion signal
How are the pseudo molecular ions formed and when are they picking up the charged ions?
Debate around the molecular mechanism that are occurring during MALDI ionization and ES fundamentally we need to get charges on our ions by using counterions – needs to be taken into consideration when performing m/z calculation
MALDI - something is happening during the energy transfer in the matrix which yields molecule picking up counterions
ES - Dissolving in solvent can be a source of counterions (normally use weak acid to dissolve peptide/protein)
During ES, what factors influence charge of the species?
- Solvent pH can influence the charge,
- pKa of the species
- Droplets are passed through a large electrical potential (Tip and electrodes) which can influence the charge of the species
What does the Rayleigh limit refer to?
Rayleigh limit is the point where the volume of the drop is too small to contain all the charge ions in a single drop (energetically unfavourable), since they repel.
The drop then explodes and repeats itself until naked ions are obtained
In nanospray how does the sampling cone attract charged ions?
Depending on the charge of the ion we can manipulate it’s movement via repulsion (same charge potential) or attraction (opposite charge potential)
Cone is just one region where we can apply a charge potential to attract desired ions and repel oppositely charged ions that are not desired
What allows ES sources to become associated with such a variety of adapters on instruments?
- Able to ionize large species
- Generates multiple charge ions (decreases m/z ratio) which makes it easier to analyse on the different analyzers
- It ionizes from the liquid phase which is biological friendly
- We can link this to liquid chromatography allowing us to separate species in our sample
What do MCP detectors offer spatial resolution?
Made of lots of electron multiplier detectors – each one amplifies our sample
Array plate with lots of electron multiplier detectors increase sensitivity as our chances of obtaining a signal
Spatial resolution - MALDI imaging – MALDI matrix forms a 3D structure with x, y, and z coordinates, allowing us to correlate where the signal is detected to the sample tissue slice
Explain again why quadrupole mass analysers are scanning analyzers?
By applying different quadrupole fields strengths, we allow different molecules with different m/z ratio’s to be in harmony with the electric field, allowing for interaction with the detector – we basically scan using different electric field strengths meaning that only a select few ions will interact with the detector at one time
In harmony - enters detector
Out of harmony - does not enter detector
How does crystallization of MALDI matrix occur?
Liquid matrix in one tube and sample in another - mix and apply to the MALDI plate
When on the plate, it the matrix will dry to crystallize
You can manipulate the environment to favour the crystallization process – work in vacuum to encourage drying, apply warm stream of heat, etc.
When quoting a value for resolution (e.g. 3000), what does the value mean since there is no unit?
Simply a numerical value to inform you about how well a mass analyser can resolve (separate/distinguish two ions with similar m/z ratio)
Higher the value – greater the resolving power
What does bottom-up and top-down proteomics refer to?
Bottom-up proteomics - small peptides which are pieced together to form overall protein
Top-down proteomics - examining intact protein - full length proteins - direct detection of protein Mw
Why is fragmentation useful?
It allows us to probe the protein’s structure with finer detail (more information about structure - e.g. A.A. sequence) –> helping us bridge the gap between the structure-function relationship
How can we obtain fragment data when using ES or MALDI?
What does a triple quadrupole set-up look like?
Outline the MS-mode of a Q-Tof set-up?
Outline the MS/MS-mode of a Q-Tof set-up?
Outline the set-up of a 4800 ABI MALDI TOF-TOF
Why is a ES-MS/MS setup useful for peptide sequencing?
Useful to use ES ionization as it produces multiply charged ions (2+ or 3+). Why is this useful…
- Multiply charged peptide ions require less collisional energy than singly charged ions to fragment – Why?
- In addition, the majority of impurity-derived signals are singly charged whereas peptides give multiply charged ions - helps us to separate analyte (peptide) and chemical background noise.
Therefore peptide-derived fragment ions will predominate in the MS/MS data even if singly charged contaminants are present at the same m/z value as the molecular ion of the peptide
- Multiply charged ions are readily recognised by the interval between the isotope peaks
How do we generate multiply charged peptide ions?
Digest our protein into multiple peptides
Enzyme of choice - Trypsin is the enzyme of choice for digesting proteins because tryptic peptides have a minimum of two charges (N-terminus plus C-terminal K or R) - tryptic digest gives us a positively charged residue at the N & C-terminal
How can we recognize the charge and presence of multiply charged ions in molecular ion spectra?
Important to identify as…
- The multiply charged species more likely represent peptides from a tryptic digest
- Less collisin energy required to fragment
- Higher probability that singly charged species are contaminants (noise), hence the multiply chagred species represent the signal
What is the charge of molecular ion from the peak below, why do we select it for further analysis?
Outline the key factors that allowing peptide sequencing by exanining MS/MS spectra?
Note - Experimental fragmentation observations best explained by assuming that a proton is transferred to the peptide bond where cleavage takes place
- Clear fragmentation patterns/pathways - allows us to interpret the peptide MS/MS spectrum
- Peptide sequencing works because peptides are not symmetrical – clearly defined N and C terminus (different functional groups) - result is that the end of the N and C terminus have different masses –> Allows us to work in ‘both directions’ (two ion series)
Outline the formation of b-ions
Important - remember any of the peptide bonds can broken to produce b-ions – changing the mass of the B-ion fragment –> moving along peptide from N- to C- terminus
B1 Ion - N-terminal amino acid + proton
B2 Ion - 2 N-terminal amino acids + proton
Outline the formation of a-ions
Outline the formation of y-ions
Outline how we put together b- and y-ions to determine the peptide sequence and what are some things to keep in the back of your mind?
Trypsin cleaves at the C-terminus of R and K!
When performing proteomic analysis, what type of questions are we thinking about?
- What proteins are expressed and when? e.g. in specific cell types during specific periods in the cell cycle
- What amounts?
- How are they modified -phosphorylation, glycosylation, prenylation etc?
- How do they interact with each other?
- How do levels/types change during differentiation/disease etc?
What is Two-dimensional gel electrophoresis and why is it used in proteomic analysis?
Advantages of using gels?
- Quick
- Relatively Inexpensive
- Good resolving technique for complex mixtures
- Works with crude samples
- Moderately tolerant to salts and detergents
- Good visual representation of entire sample
- Easy comparison
- Relatively reproducible
What protein stains are used in 2DGE?
Detection in 2DGE – Protein staining reagents
General methods - Coomassie blue, silver stain (more sensitive)
More specific - radiolabelling, fluorescent stains…
But - 2D gel doesn’t tell us what the protein is… this is where MS comes in
MASS SPECTROMETRY - allows for rigorous identification of protein
Outline the steps in the proteomics workflow known as “Mapping” or “Fingerprinting”
(Think starting with 2 different gel samples)
Outline an example of top-down proteomics performed
From genes to proteins, where does most of the functional diversity between proteins come from?
What are post-translational modifications and why are they important?
PTMs are the chemical modification of a protein after its translation – chemical modification (addition of new functional groups) of a protein after its translated
They have profound effects on protein function by altering their activity state, localization, turnover (half-life), and interactions with other proteins.
Over 200 different types of PTM, every amino acid can be modified.
The majority of proteins are modified – not an underestimate that the vast majority of proteins have one or more PTM
Which amino acids are more regularly modified?
When talking about PTMs, what are Mod-forms of a protein? Why are the important to consider?
Do all mod-forms of a protein normally exist within cells?
What is one example of a protein that is undergoes substantial PTMs? (hint - nucleus)
One of the best examples of proteins that are heavily modified are Histones
They undergo…
Methylation, phosphorylation, acetylation, etc.
- They contain Large of different sites –> Lys, Ser, Thr, etc on the histone tails
Huge implications in regulating gene expression
Apart from phosphorylation, what is the other most common type of PTM?
Protein glycosylation
Example - All cells have a sugar “coat” called the glycocalyx
Outline the importance of glycan-lectin interactions
Glycan - Glycosylated protein
Lectin - Protein that recognizes glycan
What are the different sugar groups (monomers) found on glycoproteins?
Complex glycans are build-up of small monomeric units (monosaccharide – aldehyde or ketone with two or more -OH groups) –> Less monosaccharide in glycans than A.A. in our proteins
Groups:
-
3 common Hexose (6 carbon) monomers in mammalian glycans – Glucose, galactose and mannose
- All structural isomers – orientations of -OH groups (Glucose and Mannose are epimers of each other whereas galactose and glucose are also epimers). - Deoxy sugars - lack a hydroxyl group –> For example fucose – has a methyl group on carbon 6
- Pentose (5 carbon sugar) - Xylose
- N-Acetyl family –> N-Acetyl group added to position C2 in the ring - e.g. glucosamine & galactosamine
- Xylic acid family –> largest monoer - carboxylic acid added to C1 + different chemical additions to the sugar ring
Examples - N-glycolylneuraminic acid & N-acetylneuraminic acid –> differ by a single -OH
Note - humans cannot produce N-glycolylneuraminic as we lack the oxidizing enzyme
How are the different sugar groups represented?
Due the complexity of drawing the sugars, they are simply represented by coloured shapes.
BUT –> When answering questions always include a Key
- Hexose – Coloured Circles – coloured denotes the different stereo-orientation
- N-Acetyl – Squares – using the same colour as stereo-orientation combination as hexoses sugars
- Fucose – Triangle
- Xylose – Star
- Xylic acid - Diamonds
Outline the process of glycosidic bond formation + include the different outcomes (1-4, Beta, alpha, etc)
What are the two main types of protein glycosylation?
Glycoproteins - Best characterized of all the glycoconjugates
- As sugars are hydrophilic molecules, a high proportion of secreted and membrane bound proteins (orientated towards aq. Extracellular environment) are glycosylated
Two forms
- N-glycosylation, sugar linked to amide nitrogen in the side chain of asparagine
- O-glycosylation, sugar linked to oxygen in the side chain of serine or threonine
Provide an overiview of N-glycosylation - Where on the A.A. sequence does it occur? Where in the cell does it take place?
Protein N-Glycosylation
- Requires an consensus sequon …Asn-X-Ser/Thr…where X is any AA except Pro but note not every sequon will be glycosylated –> Hence, N-glycosylation cannot simply occur on any Asn
- Initiated in ER by en bloc transfer of a pre-formed lipid-anchored conserved glycan
- N-glycosylation is a co-translational modification – N-Glycan is added as the protein is exiting the ribosome (not fully folded) – but gets modified as it moves along the secretory pathway
What is the precusor N-glycan structure that gets transferred to the protein in the ER? What is the core N-glycan structure?
Precursor –> first step of N-linked glycosylation
Core –> Core sugars found on all N-glycans
Outline the process of N-glycan biosynthesis
Outline the different pathways that an N-glycan structure can take to produce high mannose N-glycans, Hybrid N-Glycans and complex N-glycans.
What are the the main antennae building pathways for an N-glycan structure?
What are capping structures? What role do they play?
What is one of the main reasons Glycan structures are useful as a recognition molecule?
Provide an overview of O-glycosylation - On which residues does it occur? Where in the cell does it take place?
O-Glycosylation
- Occurs on the Ser and Thr
- No consensus sequence but some “rules” e.g., nearby proline + tandem repeats of Ser/Thr
- This is a true Post-translation modification as it is initiated in Golgi by addition of a single sugar - usually GalNAc in mammals
- No pre-formed O-glycan structure - formed by the sequential addition of residues – usually starts with N-acetylglucosamine residue in mammals (GalNAc)
How many different O-glycan core structures are there? Which ones are the most common?
- At least 8 cores known in mammals
- Cores 1 & 2 very common in glycoproteins in general
a) Core 1 – GalNac-Gal via Beta 1-3 linkage
b) Core 2 – Core 1 + Beta 1-6 GlcNAc residue - Cores 1-4 are the most common on mucins
After the core is formed the antenna can be built
What proteins are most commonly O-glycosylated?
Mucins – Most common form of O-glycosylated proteins
- Mucins are cell surface and excreted glycoproteins
- Mucins help protect mucus membranes by keeping them hydrated, acting as lubricants and prevent invasion by micro-organisms
- Heavy O-glycosylation occurs as a result of multiple patches of S/T tandem repeats
Why is O-GlcNAc an unusual form of protein glycosylation?
What are the factors that influence whether we get glycosylation of a protein or not?
Factors affecting glycosylation
- Protein sequence - underlying protein sequence will determine whether glycosylation occurs
- Sugar metabolism - presence of sugar precursors for glycosylation, are they present?
- Expression of glycosyltransferases – are they sufficiently expressed?
- Competition between glycosyltransferases - they compete for the capping of the antenna structures
- Physiological status - sensitive to changes in physiology – diet, stress responses (pH, oxygen tension), diseases states, etc.
All these factors combined give us…
Cell and tissue specific glycosylation
Why does the cell bother to glycosylate at all?
Glycosylation is an energy dependent process so there must be valuable reason why it is maintained on an evolutionary timescale…
- Solubility - Sugars are highly hydrophilic, hence when added to a protein they make a protein more water soluble
- Stability - present itself in many forms – glycans can prevent proteolytic digestion, physically stabilize the protein
- Conformation - glycans can affect the conformation & help stabilize it, orientate cell receptors away from the Plasma membrane, making it available to interact
- Organizational and barrier functions - mucins (heavily O-glycosylated) found at mucus membranes play an important role in hydration and protection (form a protective barrier between cells and pathogens)
- Cell-Cell and Cell-Matrix recognition - involved in a large array of communication processes
What is the definition of a lectin?
Recognition requires two partners – Glycan and a corresponding binding protein otherwise known as a lectin!
Definition - Lectins are proteins of non-immunoglobulin nature capable of specific recognition and reversible binding to carbohydrate moieties of complex carbohydrates without altering the covalent structure of any of the recognized glycosyl ligands - Kocourek and Horejsi, 1983, Clinical Biochem. 3, 3-6
Breakdown…
- Non-immunoglobulin - not antibodies
- Specific and reversible - binding/unbinding
- Without altering the covalent structure – rules out enzymes (glycosyltransferases/hydrolayses)
What are Carbohydrate Recognition Domains (CRDs) and how do they interact with glycans?
CRDs - Region on lectins that recognizes and binds to glycans
- Usually found in shallow indentations on surfaces of lectins
- Whole range of interactions between Lectin and Glycan
What are the most important lectin families in animals cells?
Most are cell surface – hence have Transmembrane domains
Lectin families
- Galectins – small soluble lectins that bind to Beta-galactosidase glycans – range of functions – e.g. cross-linking proteins on the cell surface
- C-type lectin - contain a characteristics Ca2+ ion in CRD – bind to a variety of different glycan structures – typically found immune cells (immune recognition/immune response)
- P-Type - involved in the transport of lysosomal enzymes from the golgi to the lysosomes – bind specifically to Man-6-P
- I-type - immunoglobulin like fold in their CRD - bind various sialylated glycoproteins and are commonly found on immune cells
What is a specific example of a pathogen that has hijacked the glycan-lectin interaction?
Explain how influenze has hijacked the sugar-lectin interaction allowing it to infect and release itself from the host cell
Outline Haemagglutinin specificity and how mutations in the receptor allow it to infect humans.
Outline the theory by which humans are thought to be infected by the influenze virus - note it is NOT direct!
What are Tamiflu and Relenza? What protein on the influenze virus do they target?
Drug Design procedure…
- Co-crystallization of NA with Sialic acid - determination of key binding interactions - a lot of Hydrogen bonding and ionic interactions as well important packing events.
- From this data, structural based drug design was carried out to design drugs that bind and inhibit the neuraminidase active site
How does Tamiflu (Oseltamivir) act as a neuraminidase inhibitor?
Outline how Tamiflu and Relenza in contexct of the influenze viral life cycle?
What does the field of glycomics involve?
Glycomics - studying what glycans are present within the system in order to further understand their functionality
- Determining the glycan repertoire in cell, tissues organs etc as a first step to defining functions.
- Prior knowledge of glycan biosynthetic pathways is essential.
Outline the steps in the glycomics screening workflow
Why is it important to permethylate our glycans before MS analysis?
Permethylation - chemically modify the glycans to make them more hydrophobic – change -OH to O-Methyl groups
Why?
- Increases ionization
- Increase sensitivity
- Increased fragmentation
Resulting in…
- Increase signal to noise, more distinct peaks and higher m/z range
What should you keep in mind when calculating glycan mass?
Outline the different components taking into consideration in the calculation of the glycan mass?
Do organ specific glycosylation patterns occur?
What is one of the biggest obstacle when deciphering a glycan spectra? What information can be used to overcome this?
One of the struggles with Glycan MS is the fact that the building blocks can be structural isomers - all have the same mass – how can we differentiate them?
Simply by examining Molecular ions, we won’t be able to differentiate them…
But… we can bring in knowledge of the biosynthetic pathway to provide a higher-level structural characterization (e.g. we know what the core residues are, the different antenna pathways and capping sugars)
Can glycomics be used as a functional tool?
YEAHHH BUDDYYY LIGHT WEIGHT
What other technique can be used if we still can not figure out the structure of structural isomers?
Note that fragmentation is favoured on the reducing end of N-glycans - HexNAc and sialic acid –> facilitating antennae determination
What is glycoproteomics?
Glycoproteomics - Combination of Glycomics and proteomics
Glycoproteomics - Defining the glycosylation status of individual proteins and individual sites of glycosylation –> Basically, which glycans are present on which proteins and on which specific glycosylation sites
More challenging and time consuming than glycomics – dealing with more variables makes it more challenging
What does a glycoproteomics setup look like?
What are the key pieces of information you can use to breakdown and analysis a spectrum like this?
Why do proteins have to be proteolytically digested into smaller peptides prior to peptide sequencing using ES-MS/MS?
Bottom-up proteomics - break down the protein into peptides in order to deduce the bigger overall structure
Why?
Smaller, easier to handle, more informative fragmentation (easier to deduce/interpret)
When will the product of a tryptic digest not have a C-terminal Lysine or Arginine?
The enzyme specificity – Lysine and Arginine Hence, the vast majority will have lysine or Arginine.
But there can be occasions we can get mis-cleavages plus the C-terminus of the protein can be any amino acid.
Which form of MS ionization is able to form multiply charged ions?
ES ionization is the only ionization type that yields multiply charged species
With Trypsin we get terminal Lysine and Arginine residues which are able to pick up a +ive charge - contributes to charge
When will the product of a tryptic digest not have a C-terminal Lysine or Arginine?
Trypsin enzyme specificity – Lysine and Arginine
Hence, the vast majority will have lysine or Arginine.
But there can be occasions where we can get…
- Mis-cleavages
- Plus the actual C-terminus of the protein can be any amino acid.
How do glycans in ER helps to quality control protein folding?
Quality Control system in the ER
- Chaperones/Lectins recognize the glycans
- Lectin binding to glycan allows for checking of the protein fold
- If not completely folded/incorrectly folded – the protein remains in the ER for further folding via chaperones or if not, they are recycled.
Why is it more difficult to ionise hydrophilic molecules?
In MS we measure m/z of gas phase ions - hydrophilic molecules with large numbers of H-bonding are more difficult to enter in the gas phase – e.g. Methanol vs. methane
What is one factors that influence the liklihood of obtaining a particulr b- or y-ion fragments?
Different amino acids have different side chains, these side chains can influence the likelihood of b and y ion fragmentation – difference in abundance is shown by the intensity of the peak
What is the difference between the reducing end and the non-reducing end of a sugar?
Reducing end - sugar molecule can undergo linearization
Non-reducing end - sugar with the non-reducing end is locked into the cyclical form
What is the significance of the nomenclature of the different influenza mutants (H1N1…)?
The numbers refer to the different serotypes of HA and NA (different variants)
How many sugars do CRB (carbohydrate binding domain) normally bind to?
Specific CRD recognizes a specific carbohydrate sequence which is normally around 1-4 monosaccharides long
Why are ions of a higher more likely to fragment?
Idea - Energetically more favourable due to repulsion created by the high concentration of charge
