Stuart Haslam - Mass Spec + PTMs Flashcards

Question

Outline what an Orbitrap is and how it works?

Answer 1

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.

Answer 2

Performance characteristics: 1. High resolution – separation of similar m/z 2. High mass accuracy 3. Good upper mass range 4. Good ion transmission - Overall – Highest performance but this comes at a price – Cost!

Answer 3

Performance Characteristics: 1. Ion transmission - Very good – high sensitivity 2. Extremely high mass range - theoretically unlimited – as long as you can generate an ion you can produce a mass spectrum 3. Intially, low resolution but the development of reflectrons has allowed for higher resolution

Answer 4

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)

Answer 5

YEAAAHHHHH BUDDYYYYY

Answer 6

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

Answer 7

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

Answer 8

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

Answer 9

Soft ionization techniques – produce pseudomolecular ions – meaning we need a counterion associated with the ion Counter ion needs to be taken into consideration

Answer 10

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

Answer 11

Yeah BUDDDYYY Deconvolution - take out the charged component of myoglobin – gives single Molecular ion signal

Answer 12

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)

Answer 13

1. Solvent pH can influence the charge, 2. pKa of the species 3. Droplets are passed through a large electrical potential (Tip and electrodes) which can influence the charge of the species

Answer 14

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

Answer 15

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

Answer 16

1. Able to ionize large species 2. Generates multiple charge ions (decreases m/z ratio) which makes it easier to analyse on the different analyzers 3. It ionizes from the liquid phase which is biological friendly 4. We can link this to liquid chromatography allowing us to separate species in our sample

Answer 17

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

Answer 18

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

Answer 19

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.

Answer 20

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

Answer 21

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

Answer 22

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

Answer 23

Useful to use ES ionization as it produces multiply charged ions (2+ or 3+). Why is this useful... 1. Multiply charged peptide ions require less collisional energy than singly charged ions to fragment – Why? 2. 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 3. Multiply charged ions are readily recognised by the interval between the isotope peaks

Answer 24

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

Answer 25

Important to identify as... 1. The multiply charged species more likely represent peptides from a tryptic digest 2. Less collisin energy required to fragment 3. Higher probability that singly charged species are contaminants (noise), hence the multiply chagred species represent the signal

Answer 26

Note - Experimental fragmentation observations best explained by assuming that a proton is transferred to the peptide bond where cleavage takes place 1. Clear fragmentation patterns/pathways - allows us to interpret the peptide MS/MS spectrum 2. 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)

Answer 27

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

Answer 28

Trypsin cleaves at the C-terminus of R and K!

Answer 29

1. What proteins are expressed and when? e.g. in specific cell types during specific periods in the cell cycle 2. What amounts? 3. How are they modified -phosphorylation, glycosylation, prenylation etc? 4. How do they interact with each other? 5. How do levels/types change during differentiation/disease etc?

Answer 30

1. Quick 2. Relatively Inexpensive 3. Good resolving technique for complex mixtures 4. Works with crude samples 5. Moderately tolerant to salts and detergents 6. Good visual representation of entire sample 7. Easy comparison 8. Relatively reproducible

Answer 31

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

Answer 32

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

Answer 33

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

Answer 34

Protein glycosylation Example - All cells have a sugar “coat” called the glycocalyx

Answer 35

Glycan - Glycosylated protein Lectin - Protein that recognizes glycan

Answer 36

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: 1. **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). 2. **Deoxy sugars** - lack a hydroxyl group --\> For example fucose – has a methyl group on carbon 6 3. **Pentose** (5 carbon sugar) - Xylose 4. **N-Acetyl family --\>** N-Acetyl group added to position C2 in the ring - e.g. glucosamine & galactosamine 5. **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

Answer 37

Due the complexity of drawing the sugars, they are simply represented by coloured shapes. BUT --\> When answering questions always include a Key 1. Hexose – Coloured Circles – coloured denotes the different stereo-orientation 2. N-Acetyl – Squares – using the same colour as stereo-orientation combination as hexoses sugars 3. Fucose – Triangle 4. Xylose – Star 5. Xylic acid - Diamonds

Answer 38

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 1. **N-glycosylation**, sugar linked to amide nitrogen in the side chain of asparagine 2. **O-glycosylation**, sugar linked to oxygen in the side chain of serine or threonine

Answer 39

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

Answer 40

Precursor --\> first step of N-linked glycosylation Core --\> Core sugars found on all N-glycans

Answer 41

**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)

Answer 42

- 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

Answer 43

**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

Answer 44

**Factors affecting glycosylation** 1. Protein sequence - underlying protein sequence will determine whether glycosylation occurs 2. Sugar metabolism - presence of sugar precursors for glycosylation, are they present? 3. Expression of glycosyltransferases – are they sufficiently expressed? 4. Competition between glycosyltransferases - they compete for the capping of the antenna structures 5. 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

Answer 45

Glycosylation is an energy dependent process so there must be valuable reason why it is maintained on an evolutionary timescale… 1. **Solubility** - Sugars are highly hydrophilic, hence when added to a protein they make a protein more water soluble 2. **Stability** - present itself in many forms – glycans can prevent proteolytic digestion, physically stabilize the protein 3. **Conformation** - glycans can affect the conformation & help stabilize it, orientate cell receptors away from the Plasma membrane, making it available to interact 4. **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) 5. **Cell-Cell and Cell-Matrix recognition** - involved in a large array of communication processes

Answer 46

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... 1. Non-immunoglobulin - not antibodies 2. Specific and reversible - binding/unbinding 3. Without altering the covalent structure – rules out enzymes (glycosyltransferases/hydrolayses)

Answer 47

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

Answer 48

Most are cell surface – hence have Transmembrane domains Lectin families 1. **Galectins** – small soluble lectins that bind to Beta-galactosidase glycans – range of functions – e.g. cross-linking proteins on the cell surface 2. **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) 3. **P-Type** - involved in the transport of lysosomal enzymes from the golgi to the lysosomes – bind specifically to Man-6-P 4. **I-type** - immunoglobulin like fold in their CRD - bind various sialylated glycoproteins and are commonly found on immune cells

Answer 49

Drug Design procedure... 1. 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. 2. From this data, structural based drug design was carried out to design drugs that bind and inhibit the neuraminidase active site

Answer 50

**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.

Answer 51

Permethylation - chemically modify the glycans to make them more hydrophobic – change -OH to O-Methyl groups Why? 1. Increases ionization 2. Increase sensitivity 3. Increased fragmentation Resulting in... 1. Increase signal to noise, more distinct peaks and higher m/z range

Answer 52

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)

Answer 53

YEAHHH BUDDYYY LIGHT WEIGHT

Answer 54

Note that fragmentation is favoured on the reducing end of N-glycans - HexNAc and sialic acid --\> facilitating antennae determination

Answer 55

**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

Answer 56

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)

Answer 57

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.

Answer 58

**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

Answer 59

Trypsin enzyme specificity – Lysine and Arginine Hence, the vast majority will have lysine or Arginine. But there can be occasions where we can get... 1. Mis-cleavages 2. Plus the actual C-terminus of the protein can be any amino acid.

Answer 60

Quality Control system in the ER 1. Chaperones/Lectins recognize the glycans 2. Lectin binding to glycan allows for checking of the protein fold 3. If not completely folded/incorrectly folded – the protein remains in the ER for further folding via chaperones or if not, they are recycled.

Answer 61

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

Answer 62

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

Answer 63

**Reducing end** - sugar molecule can undergo linearization **Non-reducing end** - sugar with the non-reducing end is locked into the cyclical form

Answer 64

The numbers refer to the different serotypes of HA and NA (different variants)

Answer 65

Specific CRD recognizes a specific carbohydrate sequence which is normally around 1-4 monosaccharides long

Answer 66

Idea - Energetically more favourable due to repulsion created by the high concentration of charge