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Mass Spec Flashcards

1
Q

what is mass spectrometer

A

instrument used to define the covalent structures of substances (biomolecules & proteins) by ionizing, separating, and detecting molecular and fragment ions according to their mass-charge ratios (m/z)

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2
Q

what make mass spec a powerful analytical tool

A
  • very sensitive: able to produce structurally informative data from tiny amounts of starting material (eg. femtomoles or less)
  • Able to obtain structurally informative data from complex mixtures of samples – don’t need highly purified, homogenous samples (eg. blood plasma samples, urine extracts, perfumes, protein digests etc)
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3
Q

what are the 3 key components of a mass spectrometer

A

ion source: convert biological samples into gas phase ions

mass analyser: Separate gas phase ions based on m/z ratio

detector: detect and quantitate ions (see which species are high or low in abundance)

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4
Q

what is the most abundant peak interpreted as

A
  • Most abundant peak is given a value of 100% intensity
  • other components are expressed as a relative percentage of the 100% peak
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5
Q

describe hard ionization technique + eg

A
  • Generates enough energy to convert the biological samples into a gas phase ion but will usually have an excess of E from the ionization process, leading to fragmentation of the biological sample

eg. E Impact (EI) - can only ionize small molecules, 1-1000 Da

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6
Q

describe soft ionization technique + eg

A
  • Enough E is provided to ionize the biological sample but no huge excess E which leads to fragmentation of the sample
  • can ionize much bigger biological molecules ( larger protein complexes, not just individual proteins, and larger DNA fragments, polysaccharides, and biological polymers)

eg. Electrospray Ionization (ES or ESI)
– peptides, oligosaccharides, proteins, greater than 500,000 Da
eg. Matrix Assisted Laser Desorption Ionization (MALDI) – peptides, proteins, DNA, up to 500,000 Da

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7
Q

describe e impact ionization (EI)

A
  • Performs ionization from gas phase - samples must be in the gas phase before EI
  • Sample is introduced into the source by heating it from a probe tip until it evaporates or from an on-line gas chromatography
  • Difficult to convert large biological molecules into the gas phase without causing decomposition (esp. if large hydrophilic proteins). Therefore, can only ionize relatively small molecules (esp. hydrophobic molecules = easy to convert)
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8
Q

explain the process of EI

A
  1. Gas phase sample is bombarded by a beam of high E electrons coming from heated rhenium or tungsten filament (energy = 70 eV)
    * Can use magnets to focus the e- beam and maximize chances of inducing ionization
  2. The e- beam then comes close enough to an outer valence e- of the sample to repel it (due to same – charge), excising the e- from the outer valency orbital of the molecule
    * So Ionization occurs by loss of an e- to give M+. (a radical cation)
    * Can only produce a single + charge on the sample
  3. 70 eV in the e- beam will produce a great excess of E since an average bond E is only 5 eV, leading to bond breaking and fragmentation of the biological molecule (Most of the molecular ions will decompose into fragments via uni-molecular reactions)
  4. Now that the ions have got a charge, can start to control the movements of gas phase ions in the mass spec by introducing plates with a + charge for repulsion or - charge for attraction
    * eg. + charge on ion repeller (back plate) to repel the M+ into the mass analyzer
  • Not an efficient process – only a minority of molecules in sample will actually undergo EI
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9
Q

describe MALDI

A

-E comes from firing pulses of laser light at the biological sample
* Ionization from solid phase

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10
Q

explain the MALDI process

A
  1. Sample is embedded in a low molecular weight UV-absorbing “crystalline” matrix. The matrix has absorption maximum near the wavelength of the pulsed laser used to ionize the sample
    * Sample is dissolved in a solvent and mixed with the matrix. The mixture is placed on a metal target where they will dry and co-crystallize, producing a 3D solid crystal lattice made up of the sample and the matrix
  2. Introduce the metal target into a mass spec under vacuum & fire pulses of laser at target
  3. The matrix will absorb the laser pulse and enough E will be transferred to the sample to ionize it (& generate gas phase ions)
  4. In every MALDI experiment, will generate both + and – charged ions
  5. Once have charged gas phase ions, can now control their movement
    * But can only analyze 1 charge state at a time
    * SO depending on which charge ion we’re looking at, can put a charged back plate to repel the ions into the mass spec
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11
Q

describe MALDI lasers

A

-Most MALDI uses UV lasers: λ = 320-360 nm
Older version = nitrogen gas UV laser
More recent = solid-state UV laser
- Adv: able to fire more pulses of laser E at a quicker repetition = generate more ions = able analyze more samples in a fixed time frame

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12
Q

describe MALDI matrices

A
  • Low molecular weight organic molecules
  • Have double and triple bonds and conjugated ring system – act as chromophores for absorbing E from laser and allow E transfer process for ionization

alpha-cyano-4-hydroxycinnamin acid (aCHCA) - peptides and proteins

2,5-dihydroxybenzoic acid (2,5-DHB)- carbohydrates

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13
Q

what were early issues with MALDI

A
  • Ions of the same m/z ratio coming from the target have diff. speeds when entering MS. This is due to uneven energy distribution by the laser pulse, depending on where in the crystal the ion is formed
  • Some molecules will be nearer to the surface & will obtain higher E transfer from the laser while some = buried further in the crystal lattice, receiving much less laser E
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14
Q

what were some improvements for MALDI

A

Delayed Extraction
* Molecules nearer to the surface with higher E will move further from target plate after ionization
* Buried molecules with less E will move a shorter distance

  1. directly after ionization, ensure that no back potential is placed on the target to repel the ions into the analyzer
  2. Wait a few milliseconds
  3. SO that when the back potential is applied, slower ions (nearer to the potential) will receive more E and will be accelerated more to catch up with the faster ions (receive less repulsion from potential because they are further away)
    * This compensates for the diff in ionization efficiency caused by ionization from a 3D crystal lattice
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15
Q

what is the advantage of electrospray ionisation (ES)

A
  • Adv. = ionization from liquid phase (most bio. molecules = prefer to be in liquid phase)
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16
Q

explain the ES process

A
  1. Dissolve sample in a suitable solvent in a narrow glass needle that is coated with gold/ pallidum/ platinum at the tip. Depending on what aa is present, pH/pKa of solvent will give proteins in solution a charge
  2. Introduce the needle into the electrode
  3. A high voltage (3-4 kV) is applied to the tip (generate a strong electrical potential)
  4. A back pressure is generated so the sample will emerge from the tip as an aerosol of highly charged tiny droplets (containing peptides, proteins)
    * Droplets = charged due to the pH, pKa of solvent, but also the high electrical potential
  5. Drying gas (nitrogen) that flows around the outside of the needle is then applied to the droplets -this evaporates the solvent from the droplets
  6. Because solvent is evaporating, the droplets will get smaller and smaller, causing the charged ions to move to the surface
  7. At a critical point (Rayleigh limit) the same charged ions are forced too close together so the small droplets explode into ever smaller droplets
  8. Process continues until all the solvent is rid of, and left with gas phase ions
  9. Once have gas phase ions, can control movement into mass spec analyzer
    * Can generate both + and – charged ions - only analyze 1 charge state at a time
    * Manipulate by separating out only the charge we’re interested in (increase sensitivity)
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17
Q

What is the flow rate ES sources operate at

A

Early ES sources operated at flow rates of a few microliters/min (too much is released & need to keep adding back samples to generate the data)
Newer sources = nanospray sources - operate at flow rates of 10-30 nl/min
* Can have starting volume of 1 microliter and leave it spraying for several minutes
* Allow for many mass spec. experiments on a single sample loaded
* NanoES is much more sensitive than ES
* Sample quantities are typically in the subnanogram range

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18
Q

what does ES allow to do in addition

A

introduce the sample using high resolution Liquid Chromatography separation step prior to MS - very powerful method for analyzing complex mixtures
* Can inject sample directly from liquid chromatography into MA to generate mass spec data (because ionize from liquid phase) SO ES = favored for analyzing proteins and peptides

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19
Q

what are mass analysers and what do they do

A

separate biological molecules by m/z ratio:
* Quadrupole
* Time-of-flight (TOF)
* Ion trap
* Orbitrap
Depend on charge of gas phase ion for controlling their movement in electromagnetic field

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20
Q

what are 3 key performance characteristics of mass analysers

A
  • Upper mass limit: the biggest molecule that can be successfully separated based on its m/z ratio
  • Ion transmission: out of all ions produced in the source, how many will pass through the mass analyzer and get detected
  • Resolution: how good is the mass analyzer at separating ions with very similar m/z ratio
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21
Q

describe a quadrupole mass filter

A
  • made of four parallel rods
  • Each diagonal pair of rods is connected electrically
  • The electrical field is obtained by the application of a voltage made up of a Direct Current component (to one pair) and a Radio Frequency potential (to the other pair)
    -Can change the voltages on the electrode to change the strength of the quadrupole field
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22
Q

describe the process of quadrupole mass filters

A
  1. Gas phase ions are directed out of the source into the quadrupole into the space between the 4 electrodes
  2. Depending on the m/z ratio, some ions will be in harmony with the quadrupole field and will travel straight through the quadrupole to the detector & a signal is obtained.
  3. Some ions with a diff m/z ratio will not be in harmony with the quadrupole, so they will get attracted to one of the electrodes and be annihilated, thus will not reach detector and will not get a signal in the mass spec
    * If use a calibrant with a known m/z ratio, can produce a calibration curve which links m/z ratio to a specific quadrupole field
    * SO for a quadrupole experiment, can choose a m/z range (ex. 200 – 2000) & quadrupole will allow ions to pass through to reach the detector, one m/z at a time as the entire mass range is scanned
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23
Q

what is the quadrupole performance criteria (lowest of all 4)

A

upper mass limit: able to analyze m/z ratio up to only 4,000 Da
* low sensitivity: due to scanning over a certain mass range & annihilation of ions, cannot detect every ion produced in the source (lose ions all the time)
* Relatively low resolution (unit resolution to about 3,000)

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24
Q

what are the pros of quadrupole

A
  • Low cost
  • Rapid scanning (robust)
  • Small
  • Ideal for Gas Chromatography-MS (EI) & widely used for electrospray-MS
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25
Q

describe ion trap analysers

A

operate on a similar principle to quadrupole analysers but do not operate as a filter
* also generate a quadrupole electromagnetic field but instead of having 4 electrodes on the same plane, they are in a sandwich arrangement
-composed of a ring electrode in the middle with cap electrodes (enter and exit electrode) on each end

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26
Q

describe the process in ion trap analysers

A
  1. Ions from the source will pass through the entrance electrode
  2. Ions are then trapped within the electrodes by the electromagnetic quadrupole field
  3. The trapped ions will start to circulate (spin around inside the ion trap)
  4. Next = alter the strength of the electromagnetic field
    * Depending on the m/z ratio, some ions will receive an E boost, their trajectories will increase, and they will exit through the exit cap, appearing as a signal on the detector
    -Mass spectrum is produced by scanning the RF voltages to eject all ions through the end cap
    -Must calibrate with a standard known m/z ratio (know which voltage is used to eject which m/z)
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27
Q

what is the performance criteria of ion trap

A
  • Has higher upper mass limit, better ion transmission, better resolution than quadrupole
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28
Q

what is the limitation of ion trap

A

relative small space
-if you produce a lot of ions, will not be able to capture and track them properly.
-some will collide into each other or w/the sides of the electrode, losing them in the analysis

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29
Q

how to overcome the ion trap limitation

A

high performance on trap = linear ion trap
-ions are trapped over a larger linear volume which helps overcome problems of ion interference and increases storage capacity
-get a bigger signal in MS: ions have more room to rotate around each other, don’t collide w/each other or sides = better sensitivity = better ion transmission

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30
Q

what is an orbitrap

A

-ions trapped by static electrostatic field
-electrode made up of an outer barrel electrode and an inner spindle electrode
-static electrostatic field is generated between the spindle and barrel electrodes

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31
Q

how does an orbitrap work

A
  1. ions are introduced into the orbital electrode
  2. because of the EM field, ions start to rotate around the spindle electrode, not just statically, but also horizontally (axial direction)
  3. ions w/different m/z ratio oscillate along the axial direction of the spindle electrode at a different rate
  4. FT converts time-domain signal to m/z
  5. must generate a calibration curve with a calibrant with a known m/z ration which relates a certain m/z ratio to a certain frequency of ion oscillation along the axial direction
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32
Q

what is the performance criteria of an orbitrap

A

-best performing of all 4
-highest mass accuracy (analyze up to v.high Mw molecules)
-highest resolution
-highest ion transmission (almost 100%)

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33
Q

What is a TOF analyser

A

an evacuated metal tube

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34
Q

explain the TOF analyser process

A
  1. ions from source will hit the evacuated tube where there is no electrical potential and travel the distance to the detector at another end
  2. Ions are separated by differences in velocities as they move in a straight path towards the detector
    * Ions with a small m/z ratio = higher velocity
    * bigger ions with larger m/z ratio = lower velocity
    * Calibrate with calibrant of known m/z which relates m/z ratio to the velocity (amount of time it takes for an ion with a certain m/z to travel to the detector)
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35
Q

performance characteristics of TOF

A

measuring ions travelling down a tube so:
* “unlimited” upper mass limit mass range
* Close to 100% ion transmission – help sensitivity BUT: early analysers had poor resolution and mass accuracy

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36
Q

TOF limitations

A

Assumed that ions with the same m/z ratio will have the exact same E when they enter the flight tube (similar issue to MALDI)
* Ions with same m/z but diff starting E will travel at diff speed and hit detector at diff time, affecting mass spec quality

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37
Q

how to solve TOF limitation

A

a reflectron = corrects for the effects of unequal kinetic energy distribution of ions and gives a longer flight path

  • an ion mirror that reverses the direction of travel of the ions
  • ions of greater KE (higher V) penetrate further into the TOF before being reflected so have a longer flight path
  • ions with lower starting E, initially slower = catch up by travelling a shorter flight path
    -able to do so because they have a charge we can manipulate; put ion mirror of the same charge at the end of the flight tube to repel it for a 180º turn
  • compensates for the E difference so now ions w/ same m/z ratio hit the detector at the same time, giving a better spectra quality
    -linear analysers have low resolution (<1000); reflectron gives much higher resolution (>3000)
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38
Q

what do ion detectors do

A

all detectors will amplify the signal to give higher sensitivity of detection

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39
Q

explain PM-photomultiplier

A

detects photons
1. ions strike a dynode, results in e emission
2. e then strike phosphorus screen, releases burst of photons
3. photons pass into multiplier where amplification occurs in a cascade fashion
4. number of photons is converted into an electrical signal, showing as a peak in the MS chart

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40
Q

explain EM-e multiplier

A

detects electrons
1. ions striking the dynode, will emit secondary electrons
2. amplification occurs when they continuously strike the next dynodes resulting in a cascade effect that produces more and more secondary electrons
3. e- are then converted into an electric signal and converted into a peak in the spectra

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41
Q

explain MCP (micro-channel plate array detectors)

A
  • for simultaneous detection of multiple m/z values
  • have hundreds of electron multipliers arrayed over a surface
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42
Q

explain the combinations of all components

A
  • EI sources most common w/quadrupole analysers = cheap and small but low resolution
  • Quadrupole = low performance but that is all that is required due to small generation of m/z ions in EI source
    -GC-MS is widely used (a gas chromatograph (GC) coupled to MS); good for analysing low Mw non-polar compounds, metabolites
  • MALDI sources + TOF analysers
    -MALDI-TOF = “mass fingerprinting” complex mixtures of polymers
  • ES sources most common w/ triple quadrupole (although ES can generate millions Da ions), ion trap, orbitrap or Q-TOF instruments (hybrid instrument)
    -ES is especially useful for multiply charged molecules ex. peptides and proteins
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43
Q

explain hybrid experiments

A
  • have 2 or more analysers in tandem ex. the Q-TOF
  • Other common arrangements are TOF-TOF (usually combined with a MALDI source) and linear ion trap-orbitrap.
  • Hybrid instruments are used for MS/MS experiments (2D MS analysis)
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44
Q

give examples of hybrid instruments

A

4800 MALDI TOF/TOF Analyzer - Femtomolar sensitivity

MALDI AXIMA Resonance QIT
* Quadrupole, Ion Trap, TOF Mass Analyzer system
* ~1000 resolving power

Orbitrap Ascend TribridTM Mass Spectrometer
* Quadrupole, Multipole, C trap, orbitrap, linear ion trap
* Sub fmol sensitivity
* High resolution capability, 1,000,000

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45
Q

what type of data does MS contain

A
  • Molecular ion signals
  • Fragment ion signals
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46
Q

what is a molecular ion

A
  • Made from intact biological molecule
  • major type of ions that is generated from soft Ionization techniques - MALDI, ES
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47
Q

explain real molecular ions

A
  • generated by Electron Impact (EI)
  • Because molecule lost a single electron, directly generating singly + charged radical ions (M+.)
    -Their m/z will be equal to mass of molecule (m/1- because mass of e- lost is small and charge = 1)
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48
Q

explain pseudo molecular ions

A

Generated by MALDI/ES
-Because charge is generated by adding a counter ion during the ionization process
-if observing + charged species, need to add + charged ions (ex. protons, metal
ions ex. Na+, K+, Li+ or if observing – charged species, can remove a proton)

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49
Q

explain ions generated by MALDI

A

-Can get singly charged cations and anions
-m/z = mass of peptide +/- mass of added/ removed ion
eg. if peptide = 100
[M+H]+ = (100 + 1)/1 = 101
[M+Na]+ = (100 + 23)/1 = 123
[M-H]- = (100 - 1)/1 = 99

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50
Q

explain ions generated by ES

A

-Can get multiply charged cations and anions i.e. [M+nH]n+, [M+nNa]n+, [M-nH]n-
- m/z = (mass of peptide +/- mass of added/removed ions) / charge
eg. if peptide = 100
[M+2H]2+ = (100 + 2)/2 = 51

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51
Q

explain electrospray spectrum

A

Because generates multiply charged ions, spectrum will be more complicated, producing multiple molecular ions peaks, even for a single molecule
Eg ES spectrum of multiply charged ions of myoglobin

Mass = always the same, but each peak represent a differently charged state (+20, +12) – higher charge, lower m/z ratio
* Can use algorithms and software in data analysis to convert m/z ratios to a single mass of the myoglobin
* Because ES can generate multiply charged species, but the quadrupole mass analyzer is not good at looking at ions with high m/z ratio, the ions with low m/z ratio can still be observed in the operating range of the quadrupole

52
Q

explain how fragment ions are generated + example

A
  • Generated because hard Ionization techniques produces excess of E, resulting in fragments of ions, not molecular ions
  • Eg. EI spectrum
  • EI can generate molecular ions in some cases eg Alkaloid EI spectra M+. peak at 340
  • Because alkaloid is a large organic molecule and its structure has integrated, conjugated ring systems which is much better at absorbing excess E associated with EI
53
Q

what information do fragment ions carry

A

Fragment ions = more information-rich than molecular ions
* Each fragment ion = piece of info that can be put together to work out overall structure of the biological molecule

54
Q

what is collisional activation decomposition MS or 2D MS (MS/MS)

A

-allows soft ionization techniques (MALDI & ES) to also generate fragment ions, allowing us to work out the overall structure of larger biomolecules
-Uses a process called collisional activation to introduce additional energy inside the MS to the molecular ions to induce them into fragments

55
Q

how does collisional activation work

A
  • 2 (or more) analysers connected in tandem (hybrid instruments)
    1. Ions produced in the source are selected by the first analyser
    2. Selected ions then pass through a collision chamber located between the two analysers where the ions are COLLISIONALLY ACTIVATED by exposure to a collision gas (eg. alcohol, helium)
    3. During collision, there is a transfer of kinetic energy which is enough to induce the fragmentation of molecular ions
    4. Fragment ions & any non-fragmented molecular ions are then separated by the second analyser and detected to give a MS/MS spectrum
56
Q

describe triple quadrupole

A

-first instrument that allowed MS/MS spectrum

  1. Use a quadrupole electromagnetic field of a certain strength to allow only ions with a certain m/z ratio to pass (Molecular ions are selected by the first quad)
  2. Selected Molecular ions = fragmented in the second quadrupole in the collision chamber with collision gas
  3. fragment ions m/z ratio are analyzed in the third quadrupole, generating fragment ion spectrum
57
Q

what are the pros and cons of a triple quadrupole

A
  • very popular for quantitative proteomics - robust, reliable and relatively inexpensive.
  • BUT because quadrupoles = low resolution, using 3 quadrupoles together can cause errors to build up. Early triple quadrupoles = produce low quality fragment spectrum which is difficult to interpret and derive molecular structure
58
Q

describe Q-TOF

A
  • Combine quadrupole with TOF mass analyser
  • Very powerful MS/MS instrument in terms of sensitivity and resolution
  • had enormous impact on the growth of proteomics
59
Q

explain Q-TOF in MS

A
  • The quadrupole is set to allow all ions through and the TOF is the mass analyzer
  • Pulses of ions are accelerated into the TOF by the “pusher” and the MS spectrum is recorded
  • Collision cell does not have any gas in it
60
Q

explain Q-TOF in MS/MS mode

A
  • The quadrupole is set to allow selected ions through to the collision cell which contains the collision gas
  • TOF is the mass analyser
  • Pulses of fragment ions are accelerated into the TOF by the “pusher” and the MS/MS spectrum is recorded.
61
Q

explain the TOF-TOF instrument process

A
  1. Molecular ions produced by MALDI source passes through the first TOF which selects for molecular ions of a certain m/z ratio to go into the collision chamber.
  2. Selected molecular ions are kinetically fragmented and are accelerated out of the collision chamber into another TOF with a reflectron.
  3. The fragment ions then travel to the detector to produce MS/MS spectrum.
62
Q

Applications of MS/MS technology + eg

A
  • can identify protein & sequence from MS/MS spectrum

Eg. ES-MS/MS Peptide sequencing
1. Proteins are digested into peptides
* Trypsin = protease of choice because it cleaves on the C-terminal side of K and R residues which are basic aa, a good source of carrying a + charge.
* SO tryptic peptides have a minimum of 2 charges (N-terminus + C-terminal K or R)
2. Tryptic peptides are passed into ES
3. Multiply charged ions (2+ or 3+) produced from the peptides are selected & sent to the collisional chamber
* Adv: These ions require less collisional energy than singly charged ions
4. Result = multiply charged peptide fragment ions spectra

63
Q

recognizing multiply charged ions

A

to select for certain multiply charged ions to go into collisional chamber, multiply charged ions are readily recognised by the interval between the isotope peaks

Eg. a peptide with mass of 1000 Da
* If singly charged [M+H]+ : m/z = 1001 (13C peak is m/z = 1002): separation is 1 amu
* If doubly charge [M+2H]2+ : m/z = 1002/2 = 501 (13C peak is m/z = 1003/2 = 501.5) : separation is 0.5 amu
* If triply charge [M+3H]3+ : m/z = 1003/3 = 334.33 (13C peak is m/z = 1004/3 = 334.66) : separation is 0.33 amu

64
Q

explain C isotope clusters

A
  • Organic molecules are mostly built of carbon
  • Carbon has a naturally occurring heavy isotope – 13C
  • 1.1% of all C on the planet is 13C
  • Therefore, 1.1% of all C in the peptide = 13C
  • 13C has a diff mass than 12C — 1 Da heavier
  • So, 13C will give a diff m/z signal than 12C in the peptide mass spectrum
  • Therefore, biological molecules don’t just give a single molecular ion peak
  • It gives an isotope cluster (have isotope peaks from both 12C and 13C)
  • Every biological molecule gives a distinct isotope cluster in the mass spec
  • Differently charged molecular ions can be differentiated by the interval between isotope peaks
65
Q

what is the advantage of recognizing multiply charged ions

A

majority of impurity-derived signals = singly charged, ability to differentiate differently charged ions will allow separation of impurities from interested data during analysis

66
Q

interpreting MS/MS fragment ion spectrum + eg

A
  • Peptide sequencing in MS works because peptides = asymmetrical molecules
  • They have distinctive N-ter (with amino group) & C-ter (with carboxyl group)
  • The diff functional group ends have a different mass, allowing differentiation between fragment ions that come from N or C terminus

Eg. fragmentation of singly charged peptides (MALDI)
* Fragmentation occurs at the peptide bond that the extra proton is added to
* Fragmentation can occur from N-terminal side or from C-terminal side – each = follow different fragmentation pathway

67
Q

Fragmentation from N-terminus (B-ion fragmentation pathway)

A
  • 2 e- movements break the peptide bond and form a triple bonded oxonium ion on the N-terminal residue (b-ion).
  • The rest of the peptide is neutral so not observed on spectra

Because singly charged, m/z value = mass of aa + 1 (N-terminal H)
* so, if n-terminal glycine, will observe m/z peak at 57+1 = 58
* able to identify N-terminal aa
* diff molecular ions will fragment at diff peptide bond
* so, able to deduce aa composition from N-terminal side

68
Q

explain a-ions

A

During collisional activation, excess E can cause further fragmentation from b-ion, resulting in neutral elimination of carbon monoxide, generating an a-ion
* lose 28 amu – get satellite signal 28 amu lower than b-ion signal

69
Q

Fragmentation from C-terminus (Y-ion fragmentation pathway)

A
  • 2 electron e- result in quaternary nitrogen with a + charge on the C-ter residue (Y-ion).

Because singly charged, m/z value = mass of aa + 19 (C-terminal OH & 2 added H – ionizing H & H from breaking peptide bond)
* so, if c-terminal serine, will observe m/z peak at 87+19 = 106
* able to identify C-terminal aa

  • diff molecular ions will fragment at diff peptide bond
  • able to deduce aa composition from C-terminal side
70
Q

points to remember during peptide sequencing

A
  • Take into account the ionizing proton when working out mass of peptide(+1)
  • Because trypsin = protease of choice - all tryptic peptides will have C-terminal K or R
  • Make sure b and y ion series match up
71
Q

explain proteomics

A

large-scale study of proteomes
* Ability to determine protein aa sequence from mass spec drives the development of proteomics
* Proteins = most important molecules in the cell - to understand cellular functions, must understand proteins

72
Q

what questions does proteomics answer

A

What proteins are expressed and when?
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?

73
Q

explain the process of a proteomics experiment

A
  • First step in a classical proteomics experiment = separation experiment
    1. extract proteins from cell line/ system of interest/ model organism
    2. Then, separate the proteins by:
  • Simplest: SDS-PAGE – separate protein by size
  • 2D gel electrophoresis: separate protein by to
    size & charge (each spot = an individual protein)
74
Q

why is a gel used in proteomics experiment

A
  • Quick
  • Relatively Inexpensive
  • Can obtain good resolution for complex mixtures (able to work with crude samples)
  • Moderately tolerant to salts and detergents (which would be required to extract proteins from cell)
  • Good visual representation of entire sample
  • Easy comparison
  • Relatively reproducible
75
Q

explain the detection in 2DGE

A

Protein dyes – allow visualization of the spots on the gel:
* Coomassie blue, silver stain, radiolabelling (radioisotopes), fluorescent stains, etc.

76
Q

what is an example of a proteomics experiment

A

observing protein expression in a prostate cancer cell line
1. Extract protein from control (healthy prostate tissue) & prostate cancer cells
2. Perform 2DGE, detect, and look for differentially stained spots – indicate differentially expressed proteins that are likely to be involved in the cancer process, so potential drug targets

77
Q

what is the limit with gel in proteomics experiment

A

can only tell what protein of a certain size and charge is differentially expressed (but can’t directly identify the protein)

Therefore, MS = used for the following identification step: Mass Fingerprinting

78
Q

explain the process of mass fingerprinting

A
  1. Excise the differentially expressed spots from 2DGE
  2. Perform in-gel tryptic digest to obtain a mixture of tryptic peptides
  3. Run the obtained tryptic peptides on MS (usually MALDI/ES) to get a series of peptide molecular ions
  4. Run database search (eg. against in-silico peptide of whole human genome)
    * Ex. if studying human system, can get an in-silico digestion on the whole human genome & the database will give info about all possible tryptic peptides that would be produced from every single protein in the human genome
    * Match unknown sample to the in-silico peptides & obtain aa sequence
    * Limitations eg. if the system of interest whole genome hasn’t been identified, etc.

Can also perform MS/MS to obtain aa sequence of each protein (even without database/ genome hasn’t been sequenced) & perform further bioinformatics analysis

79
Q

explain bottom-up proteomics

A

(most common)
1. Extract peptides
2. Fragment the peptides in MS
3. Build up peptide sequences to get full protein ID

80
Q

explain top-down proteomics

A

Start with intact protein and use MS/MS to fragment it

81
Q

explain biological complexity

A

not linearly related to the number of genes

  • Additional complexities & expansion of the functional diversity of the genomic content = due to different regulatory mechanism at each stage of the central dogma

biggest is the post-translational modification

82
Q

Introduce PTMs

A
  • the chemical modification of a protein after its translation.
  • There is a huge chemical variation in the # of PTMs
    -200+ different types of PTM, every amino acid can be modified.
  • have profound effects on protein function by altering their activity state, localization, turnover, and interactions with other proteins.
  • Vast majority of proteins in our cells have at least 1 form of PTM
83
Q

explain the distribution of aa PTM

A

Every aa can be PTM but some aa = more frequently PTM
* hydroxyl-related aa eg. serine, threonine, tyrosine or positively charge aa like lysine, arginine = modified more often
* smaller neutral aa = modified a lot less eg. Glycine, leucine, isoleucine, glutamine

84
Q

explain mod forms

A
  • Proteins exist as mod-forms
  • # of possible protein mod forms depends on # of possible modification sites it has
  • Calculated by 2 ^ number of modification sites
  • Each mod form can have different functionalities
  • Not every single mod form is present all the time
  • Each mod form = transient, dynamic, responsive modifications that allow fine- tuning of protein function
    eg. serine/arginine repetitive matrix protein - over 300 phosphorylation sites (2^300 mod forms) – not every mod form is present at one time
  • Some proteins = heavily PTM ex. histones – each mod form = important for epigenetic regulation of transcription/ translation
85
Q

what can PTMs do

A

PTM can be used to fine tune the activity of a protein – allow cells to be
more responsive/ adaptive to challenging environments
- get rapid response by PTM instead of having to produce more/ diff proteins by
slower gene expression
- also allow more dynamic range of protein functionality

86
Q

what is the most abundant PTM

A

Glycosylation
* Sugar molecules can function as recognition molecules
* All cells have a sugar “coat” = glycocalyx – contain glycoproteins in surface
* Cell surface = cell’s primary interface with the external environment
* many fundamental cellular processes = driven by the specific recognition of surface sugar molecules on one cell by sugar binding proteins (lectin) on another cell

87
Q

explain glycan-lectin recognition + examples

A

key to cell-cell communication to induce cellular processes

Eg
* trafficking of human neutrophil to the site of infection
* targeting of T lymphocyte to a lymph node
* the interaction between a human sperm & egg
* Pathogenic organisms can also hijack the Glycan-lectin recognition to infect specific human cells (viruses, bacteria)

88
Q

what are some common monosaccharide building blocks found in human & mammalian glycoproteins

A
  • Hexose family
    -Lectin recognizes the orientation of OH groups
    -Change in OH group orientation = changes the affinity of lectin with the sugars
    -Glucose, galactose, mannose
  • Acetyl hexose family
    -C2 group changes from OH to acetyl
    -Acetylglucosamine (GlcNAc), acetyl galactosamine (GalNAc)
  • Pentose family (5C sugars) – Xylose
  • Deoxysugars - Fucose (methyl on C6 rather than OH group)
  • Sialic acids (Large 9C acidic sugars – has COOH group on C1)
    Most mammals have 2 forms:
    -Acetylneuraminic acid (NeuAc)
    -Glycolylneuraminic acid (NeuGc)
    -Humans only have NeuAc due to partial gene deletion, resulting in inactive hydroxylase which can’t oxidize NeuAc to NeuGc
89
Q

what is the key for monosaccharide building blocks

A

yellow circle: galactose
blue circle: glucose
green circle: mannose
blue square: acetylglucosamine
yellow square: acetylgalactosamine
red triangle: fucose
orange star: xylose
purple diamond: acetylneuraminic acid
light blue diamond: glycolylneuraminic acid

90
Q

explain glycosidic bond formation (joining monosaccharides)

A

(joining monosaccharides)
* Dehydration reaction (eliminates water)
* Energetically unfavorable reaction (equilibrium favors indiv. monosaccharides rather than bonded polysaccharides)
* require enzymes or E from hydrolysis of high E phosphate bond when joining monosaccharides to sugar nucleotides
* generate specific ends – non-reducing and reducing ends with diff. chem groups on glycans

91
Q

describe glycans difference from other proteins

A

-have much more structural diversity than protein, DNA, RNA sequence due to:
* multiple potential binding sites – so glycans = branched molecules (vs linear DNA, RNA, proteins)

  • glycosidic bond formation can result in ⍺ or β glycosidic bonds depending on the orientation of sugar rings about each other (vs always same amide bond in polypeptide chain)
92
Q

what are glycoproteins

A

(addition of glycans onto proteins)
* most abundant, diverse form of PTM
* High proportion of secreted and membrane bound proteins are glycosylated

93
Q

what are the 2 forms of glycosylation

A
  • N-glycosylation: sugar linked to amide nitrogen in the N on side chain of asparagine
  • O-glycosylation: sugar linked to oxygen in the OH of side chain of serine or threonine
94
Q

describe protein N-glycosylation

A
  • Glycan is attached to Asn in the specific aa sequence of …Asn-X-Ser/Thr…where X is any AA except Pro
  • Initiated in ER
  • A co-translational modification: occurs as the nascent polypeptide chain is coming off the ribosome (while the rest of the polypeptide is being synthesized, before protein folding into final form)
95
Q

explain the process of protein n-glycosylation

A
  1. en bloc transfer of a pre-formed precursor (a pre-formed lipid- anchored conserved glycan) - contains 3 glucose, 9 mannose, 2 glcNac residues linked to a lipid carrier dolichol
    -Because all = same starting process/ same precursor, all protein N-linked glycans will share a conserved trimannosyl core structure (2 glcNac, 3 mannose), but have varying number of antennas, structural variation on the antennas
  • Then, followed by a series of processing steps – to modify the pre-formed precursor:
  1. Removal of the first 3 glucose and 1 mannose – gives man8glcnac2 structure – which then enters the secretory vesicle & is transported from ER to Golgi
  2. As man8glcnac2 passes through the cis & trans Golgi, it goes through more modifications exg. trimming & modification of branch structure, addition of terminal sugar residues
  3. Mature glycoprotein exits the trans Golgi in a secretory vesicle to the PM
    * If have membrane anchor, glycoproteins will stay on PM as a membrane protein
    * If no membrane anchor, glycoproteins will be secreted
96
Q

explain N-glycan biosynthesis

A
  1. Start: Glc3Man9GlcNAc2 (common precursor)
  2. Removal of glucose by glucosidases – gives Man9GlcNAc2
  3. Removal of mannose by mannosidases- gives Man5GlcNAc2 – first family of mature N-glycans: High Mannose N-glycans
  4. Greater processing = build up from the High Mannose N-glycans (add monosaccharides back on) by glycosyltransferases - to build up antennas, add capping groups – result in 2nd family: Complex N-glycans (glycan family that underwent most processing, have most diversity in structure)
  • 3rd family: Hybrid N-glycans – N-glycans that have both characteristics of High Mannose & complex N-glycans
97
Q

building up complex N-glycans

A

Building up antennas:
* utilizes lacNAc units (contain galactose & GlcNAc) – connected via β-glycosidic linkages– tend to form long straight structures
* have capping groups at the end formed from fucose and sialic acid in ⍺-glycosidic linkage - tend to form kinks

98
Q

what does an n-glycan structure contain

A
  • long antennas of β-linked lacNAc units projecting away from cell surface
  • ⍺-linked fucose and sialic acid capping groups – involved in recognition by lectin - diff combinations of ⍺-linked capping groups form the recognition group, driving the glycoprotein functionality

eg. Human blood groups = driven by the diff. glycan structures (diff. ⍺-linked capping groups) on red blood cells and epithelial membranes

99
Q

explain how glycans occupy space

A

have large hydrodynamic volume - occupy larger space than expected with its Mw

  • The unmodified protein = highly folded, compact
  • Attached glycan contain large degree of freedom of movement – able to move around and occupy space, making themselves available for key interactions
100
Q

describe protein-O-glycosylation

A
  • Occurs on Ser and Thr
  • No specific consensus sequence required but tends to occur when have nearby proline, tandem repeats of Ser/Thr
  • Initiated in Golgi (after protein is fully folded and exited ER into Golgi)
  • Occurs in a sequential process by the addition of a single sugar at a time - usually first added sugar = GalNAc in mammals
101
Q

explain how o-glycans are classified by core structures

A
  • All start with a GalNAc but have variations in the core
  • At least 8 cores known - 1 & 2 very common in glycoproteins
  • Core 1: add galactose on to GalNAc
  • Core 2: add galactose & GlcNac on to GalNAc

Once have core, can build up the antenna and end with the ⍺-linked cap to allow recognition by lectin

102
Q

what are the most common form of O-glycosylated glycoproteins

A

Mucins
* Large macromolecular structure with multiple repeats of serine, threonine, proline residues – so heavily O-glycosylated
*so, tend to absorb a lot of water & become hydrated gel-like molecules
* Protect mucus membranes by keeping them hydrated, acting as lubricants and prevent invasion by micro-organisms

103
Q

describe o-glcnac

A

the addition of a single GlcNac residue
* a specialized form of glycosylation that occurs on intracellular proteins

O-GlcNAc proteins =
* NOT elongated to more complex structures
* Localized to the cytoplasm and nucleus.
* Present in all higher eukaryotes studied.

104
Q

how is o-glcnac different from classical glycosylation

A
  • similar and as abundant as phosphorylation
  • O-GlcNAc proteins are also Phosphoproteins - often reciprocal
  • Highly dynamic modification – has a regulatory role
105
Q

what factors contribute to cell & tissue-specific glycosylation

A
  • Protein sequence
  • Sugar metabolism (– impacts which sugar structure gets added on – must have precursors available)
  • Expression of diff. glycosyltransferases/ glycosidases – affects which glycans are present due to competition between glycosyltransferases – eg. more fucosyltransferase than sialyltransferase = more fucosyl structures
  • Physiological status of the cell
  • Glycosylation changes with aging, embryonic state, diseases state
106
Q

what are the functional consequences of glycosylation

A

add a large hydrophilic group onto the protein:
* Helps with solubility (ex. maintain solubility of most serum proteins)
* Affects stability
- protects degradation by proteases eg. protect epitopes on a bacterial glycoprotein from immune recognition
- controls half-life of glycoproteins eg. affecting when proteins are taken out of serum circulation in the liver
* Help orientate specific functional domains in the protein eg. glycosylation of the stalk region of a cell surface receptor can help orientate the glycoprotein above cell surface, making it available for interaction with ligand
* Barrier functions eg. O-glycosylated mucins protect mucus membranes eg. by preventing mechanical damage from passage of food down GI tract, form protective barrier stopping pathogenic bacteria, fungus form getting through the mucus layer to infect underlying cells
* Function as recognition molecules – allow for specific Cell-Cell and Cell-Matrix recognition

107
Q

what are glycoforms

A

glycoproteins with the same protein sequence but diff attached glycan
* 1 protein can have many possible types of glycosylation = diff Glycoforms
* Different glycoforms can potentially have different biological functions

108
Q

Formation of diff. glycoforms protein will depend on:

A
  • the cell type in which the protein is expressed
  • the physiological status of the cell
  • may be developmentally regulated
109
Q

give an example of the importance of glycoproteins and its different glycoforms

A

Sperm-egg recognition
* involves glycan recognition
* Involves a family of glycoproteins: Glycodelin
- 28 kDa protein
- Has 3 Asn residues but has N-linked glycosylation at 2 of the residues
- Has 2 glycoforms:

Glycodelin-A (GdA) = female reproductive tract
* Contain complex N-glycans at both Asn residues
Glycodelin-S (GdS) = male reproductive tract
* Contain complex N-glycan only at Asn 63 but exclusively high mannose glycans at Asn28

110
Q

why can different glycoforms have different functions

A

because diff. glycans on glycoproteins change the recognition and interacting molecules

111
Q

explain Glycodelin-A (GdA)

A
  • Expressed in the Endometrium, amniotic fluid, pregnancy serum
  • Upregulated upon a successful fertilization event & the embryo starts to embed in the decidual endometrium
  • Immunosuppressive - because all developing fetuses contain 50% of genetic info from father, so 50% foreign to mother.
  • Suppression of mother immune system = prominent during Pregnancy
  • Pregnancy complications eg. Miscarriage/ premature birth = caused by breakdown in immunological tolerance
  • Inhibits sperm-egg binding (contraceptive molecule) so tightly regulated during menstrual cycle – downregulated at the most fertile time in the cycle around ovulation period & levels rise after ovulation
112
Q

explain Glycodelin-S (GdS)

A
  • Expressed in Seminal vesicles, seminal plasma
  • Immunosuppressive – after ejaculation, male sperm (genetically diff from female) has to survive in immunologically hostile environment in the female body - infertility can be caused by overactive female immune response targeting the sperm
  • Enhances sperm-egg binding
113
Q

describe influenza virus

A
  • RNA virus
  • 4 subtypes: A, B,C,D – A,B cause human diseases: A = most virulent
  • Expresses 2 key viral surface proteins involved in sugar recognition:
    (sugar recognition = essential for influenza virus replication)
    Hemagglutinin (HA) = lectin
    -18 serotypes of HA
  • Recognizes terminal sialic acid residues on glycoproteins
    Neuraminidase (NA)
    -11 serotypes of NA
    -cleaves off the terminal sialic acid of the glycan it recognises
114
Q

explain the influenza infection process

A

Glycan recognition can be utilized to initiate infection processes

  1. Hemagglutinin of influenza virus binds to host cell surface sialic acid glycoprotein
  2. the interaction causes endocytosis of influenza virus
  3. replication of genetic info & production of new influenza viral particles inside the host cell
  4. new influenza viral particles bud away from the cell surface
  5. they express hemagglutinin, which recognises host cell surface sialic acid glycoproteins, causing the new viral particle to get stuck on infected cell surface
  6. Neuraminidase cleaves off the terminal sialic acid, releasing new viral particles to infect other cells, continuing the viral life cycle.
  7. Neuraminidase also helps viral particle penetrate protective mucus that line the respiratory tract (by cleaving off sialic acid in mucins that protect mucus membrane barrier)
115
Q

explain how influenza was not naturally a human virus

A
  • Natural reservoirs = infect GI tract of bird (aquatic birds – duck, geese, chicken)
  • BUT mutation of hemagglutinin, changing the recognition of terminal sialic acid glycosidic bond, allows infection of other species as well – including humans
  • In natural bird reservoir, hemagglutinin recognizes terminal sialic acids in a-2,3 glycosidic bond
  • In human influenza virus, hemagglutinin recognizes terminal sialic acids in a-2,6 glycosidic bond
116
Q

explain how highly transmissible influenza strains can cause influenza pandemic

A
  • Ex. Most influenza recent pandemic = 2009 Swine Flu H1N1

H1N1
* Not direct transmission from bird to humans
* Get indirect transmission from a 3rd species: pig
* Pig expresses both a-2,3 linked sialic acids & a-2,6 linked sialic acids – can get infected with both avian and human influenza virus
* pigs act as mixing vessel – they are simultaneously infected with avian & human influenza, allowing genetic sharing & genetic reassortment, resulting in emergence of a novel virus – H1N1

  • Highly Infectious - 20-27% of the population infected
  • But not virulent - death rate of <0.02%
117
Q

what is a virulent influenza virus

A

Avian H5N1 influenza A
* Rarely transmitted to human population but 53% fatality rate
* If continues to mutate & become more easily transmissible from birds to humans = very dangerous

118
Q

how to exploit glycan knowledge for drugs

A

glycans = key to viral infection cycle, so can exploit knowledge of glycan structure & glycan binding proteins to inhibit virus infection (glycans =drug targets for developing antivirals eg. Tamiflu & Relenza (neuraminidase inhibitors)

119
Q

explain structure-based drug design

A

Hemagglutinin – lectin that binds sialic acid to initiate infection process
* Have shallow binding sites with weak interactions between sugar & protein
* So not good drug target

However, for Neuraminidase
* Binding site = Deeper cleft into the surface of enzyme & has stronger interactions between active site aa and sugar
* better drug target for structure-based analogues of the sialic acid
* Discovered 8 conserved aa in active site of influenza A & B neuraminidase important in sugar recognition & hydrolysis
* Can synthesize variants on sialic acid structure that changes the interactions with enzyme

120
Q

explain tamiflu

A
  • Change glycerol hydroxylated sidechain of sialic acid residue for an aliphatic (hydrophobic) C chain
  • Therefore, change interaction with Glu 276 in active site of enzyme – no more H bonding
  • Hydrophobic group will shift orientation of Glu 276 to form H bond with Arg224 instead, inhibiting action of the enzyme
  • Inhibition of Neuraminidase = Inhibit ability of new viral particles to bud off & release, stopping influenza virus life cycle
  • But not inhibiting viral entry, so if take the drugs too late after an already well- established infection, can be ineffective
121
Q

what is glycomics

A

studies designed to define the complete repertoire of glycans that a cell, tissue, or organism produces under specified conditions of time, location, and environment.

Glycomics studies glycans removed from proteins, so lose info. about which glycans = present on which glycoprotein/ at which site on glycoprotein

122
Q

explain the glycomics Screening strategy

A
  1. Extract glycoproteins (or also glycolipids) from biological tissues
  2. Remove the glycans from the glycoproteins:
    - Remove N glycans= enzyme PNGase F
    - Remove O glycans = chemical process: reductive elimination
    - Results in family of N and O glycans for analysis
  3. Best analytical technique to characterize structure of glycans = mass spectrometry (due to high sensitivity & ability to handle complex mixtures)
  4. Need to perform chemical derivatization reaction to convert hydrophilic glycans (due to OH groups and N-acetyl groups) to hydrophobic molecules.
    - Involve: Permethylation reaction: replace OH groups with O-methyl groups, replace N-acetyl groups with methylated N-acetyl groups – will dramatically improve data quality
123
Q

how to interpret the mass spec data for glycans

A

need:
* Mass of each glycan residue
* Take into account ends of molecules:
- Reducing end – with O Ch3 (+31)
-Non-reducing end – with Ch3 (+15)
- Tend to work with MALDI, so look at sodiated (Na) molecular ions, so add Na
mass as well (+23)

eg. m/z 2605
2605 - 15 -31 -23 = 2536 (use this)

Problem: structural isomers: (galactose, glucose, mannose) and (GalNAc, GlcNAc)
* Use knowledge from biosynthetic pathway that core = mannose, antennas = galactose

124
Q

how to study glycan biomarkers of human disease

A
  • Can conduct glycomics experiment on diff. species or diff cell types eg. healthy vs diseased cell types
  • Patient with hepatocellular carcinoma (liver cancer) has changes in their glycan pattern
  • Therefore, looking at glycosylation changes can enhance cancer diagnostic ability
125
Q

describe MS/MS sequencing for glycans

A

select individual glycan molecular ions and induce fragmentation to interpret fragment ion spectra – to give more detailed structural analysis

  • Tell specific position of molecules
  • Increase degree of structural definition of glycans
  • Able to deduce biological functions of the glycans
126
Q

what is glycoproteomics

A

defining the glycosylation status of individual proteins and individual sites of glycosylation

127
Q

explain the glycoproteomics process

A
  1. purify glycoproteins
  2. Perform proteolytic digestion to obtain a series of peptides & glycopeptides
    - If hv enough material, can perform initial glycomics experiment – allow identification of all glycans the glycoproteins are carrying to narrow down searching range of glycans
    - If not enough material, can go straight to glycoproteomics
  3. Permethylation to improve data quality
  4. Use NanoLC (or any chromatography) to separate peptides from glycopeptides
  5. Perform 1st round of MS to select for certain molecular ions - individual glycopeptides for fragmentation
  6. Performs 2nd round of MS (MS/MS) & observe fragment ion spectra of a specific glycopeptide
    -Because of glycopeptides, will get a combination of sugar fragment ions & peptide fragment ions (b, y, a ions)
    -Then interpret spectra to work out glycans and peptide sequences – can observe which glycan at present at which site of glycopeptide

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