Mass Spectrometry 1-2 Flashcards
(102 cards)
0
Q
What is a mass spectrometer?
A
An instrument used to define the covalent structures of substances by ionizing, separating and detecting molecular and fragment ions according to their mass-to-charge ratio (m/z)
1
Q
Who invented Mass spectroscopy? What for and when?
A
Sir J.J Thomson
Demonstrated the mass to charge ratio of an electron
Showed the existence of isotropic elements
Nobel prize 1906
2
Q
Good MS instruments can measure up to femtomoles or less. What unit is a femtomole?
A
10^-15
3
Q
What two Advantages of MS put it above NMR and X-Ray Crystallography in terms of importance?
A
- It’s exquisite sensitivity- can detect substances down to even the atomic level, e.g can analyse transcription factors
- Can analyse complex mixtures of different molecules, unlike NMR and X-ray which require homologous mixtures. No purification or preparatory steps
4
Q
What doesn’t an Mass Spectrometer do?
A
It DOESN’T measure MASS. It measures the mass:charge ratio of a charged ion.
5
Q
What are the three main components of a Mass Spectrometer?
A
- Ion Source —> 2. Mass analyser —> 3. Detector
6
Q
What is the Job of the ioniser and the ion source? And what difficulties does it’s requirements cause?
A
The ioniser - generates ions of the target sample and converts to the gas phase
The ion source - is your sample which will be separated and later detected, it can be ionised in several ways e.g. EI, FAB, ES and MALDI
Most biological molecules are in the solid or liquid phase, so must be transformed first.
7
Q
What does the mass analyser do?
A
Separates on basis of the mass:charge ratio
8
Q
What does the detector do?
A
Provides the quantitative data, tells you the abundance and components of your sample.
9
Q
What is the first thing that must be done in a MS experiment
A
Your solid/ liquid phase sample must be converted into the gas phase
10
Q
What is the mass spectrum?
A
It is a record of the ions that are detected; abundance is plotted on the y-axis and the m/z ratio on the x-axis
11
Q
What units are on the intensity (y) axis?
A
NONE! (Usually) Just an arbitrary % unit that implies relative abundance. This is the quantitative info we receive from experiment.
12
Q
What units are on the M/Z ratio (x-axis)
A
NONE! They have no units, arbitrary value corresponding to its ratio.
13
Q
What are the four main ionisation methods? What do they measure, at what weight ranges and how intense are the methods on the samples?
A
- Electron impact (EI) - hard. small molecules exclusively - 1-1000 Da
Hard technique as high energy beam can cause covalent bond breakage and fragmentation - Fast Atom Bombardment (FAB) - Soft/hard. Measures peptides and oligosaccharides. Max = 10,000 Da (big peptides/small proteins). Some fragmentation due to excess energy, less than EI
- Electrospray Ionisation (ES/ ESI) - soft. Measures oligosaccharides and proteins up to and above 500,000 Da. Energy is sufficient for experiment, no fragmentation.
- Matrix Assisted Lasers Desorption Ionisation (MALDI) - soft. Peptides, proteins AND DNA!
14
Q
Why is the method Electron impact ionisation a misnomer?
A
It doesn’t/ rarely involves collision of the electron into the samples atoms. Instead the close proximity of the electron around the valence electrons of the samples atoms causes repulsion and expulsion of an electron, giving charge.
15
Q
Why does EI ionisation have a 1000 Da/ 1kDA size limit?
A
Because it requires the sample to be in the gas phase prior to ionisation.Higher molecular weighted species are harder to convert to the gas phase.
16
Q
Steps of Electron impact ionisation.
A
- Sample introduced into source by heating it from a probe tip or plate
until it evaporates OR comes from an on-line gas chromatograph system - Gas phase sample is bombarded with electrons from Rhenium or tungsten (energy = 70 eV. Very high energy)
- Ionisation occurs by loss of electron (through electron repulsions) gives a singularly charged (+ve or -ve) radical ion e.g. M+. is a radical cation
17
Q
Why does EI ionisation cause fragmentation?
A
Because the energy of the electron beam 70eV is much greater than that of a covalent bond ~5 eV. This causes bond breakage and fragmentation.
18
Q
What is the general ionisation reaction?
A
M + e- —> M+* + 2e-
19
Q
Why aren’t divalent radicals generated?
A
The back board of the instrument carries a charge that is selected to repel the newly formed ions, therefore they are likely to be shot out of the ionisation chamber before subsequent ionisation. Also, ionisation isn’t efficient, so two is unlikely.
20
Q
What does FAB stand for?
A
Fast atom bombardment (ionisation)
21
Q
In FAB, the sample is dissolved in a small drop of a low volatility liquid matrix, such as?
A
Monothiol glycerol
22
Q
Where is the matrix placed in FAB
A
On a small metal target at the end of a probe which is then inserted into the FAB source and bombarded with accelerated atoms or ions
23
Q
In FAB, what atoms or ions are used to bombard the target sample?
A
the inert gas Xenon or an ion like Caesium (Cs+)
24
What happens in FAB ionisation?
A vacuum is created prior to bombardment in atoms/ions hit the top layer/surface of the matrix, their is an energy transfer event sufficient enough to induce ionisation of those surface particles. Ions of which you can now control the movement of.
25
What phase does FAB start with?
The liquid phase
26
Why is the matrix in FAB important?
Because without the matrix the sample will be ionised all at once, which will cause difficulties downstream separation. Through ionising only the surface we get a slow, controlled ionising cycle.
27
Why is a vacuum created in FAB?
To remove any contaminating gases e.g. oxygen or nitrogen
28
What are the drawbacks of FAB?
Ionisations occurs to the matrix also, which contaminates the very sensitive detection system. If detergents (easily ionised) were used in preparation, and even 0.01% remains there will be significant contamination.
29
What happens when the surface monolayer is destroyed in FAB?
The target is at high voltage relative to source extraction plates and the ions desorb from the matrix and are accelerated into the analyser
30
What does MALDI stand for?
Matrix Assisted Laser desorption Ionisation
31
What phase does MALDI start with?
The solid phase, bitch. In crystalline form.
32
Draw the FAB instrument setup.
No seriously, do it.
33
What are the key steps in MALDI?
1. Put under vacuum
2. Sample is embedded into a low molecular weight UV absorbing 'crystalline matrix' matrix, chosen to have an absorption maximum near that of the wavelength of the pulsed laser used to ionise the sample. Usually UV.
3. Matrix absorbs the laser pulse and enough energy is transferred to the sample to ionise it
34
How is the crystalline matrix formed in MALDI?
The sample is dissolved in liquid matrix which is subsequently dried to form crystalline matrix.
35
What is unusual about the process of MALDI? ☝️
The process isn't well understood, thought to be familiar to flash evaporation.
36
Draw the MALDI setup
You done it yet?
37
What are the two main UV lasers used in MALDI? What are their specs (wavelengths and repetition rate )?
1. Nitrogen gas UV laser: wavelength = 337nm and repetition rate = 1-20Hz
2. Solid-state UV laser: wavelength = 355nm and repetition rate = 0.1-1.5 kHz
38
How long has MALDI been around for?
20-30 years. Bruh.
39
What are the two most common MALDI matrice compounds? And what are they specialised in encapsulating?
1. Alpha-Cyano-4-hydroxycinnamin acid (alphaCHCA) - peptides and proteins
2. 2,5-dihydroxybenzoic acid (2,5-DHB) - carbohydrates
40
Why are alphaCHCA and 2,5-DHB used in MALDI matrices?
They are unsaturated (repeating double and single bonds), allows for efficient absorption and transfer of ionisation energy. They still ionise, but are easily recognised on spectra.
41
What is a major drawback of MALDI, that in early designs caused poor resolution?
Due to unordered crystal Matrix, ions of the same mass coming from the target have different speeds. This is due to an uneven energy distribution (energy spread) when the ions are formed by the laser pulse, depending on where in the crystal the ions are formed. This difference in velocity leads to different reading of the deflection path in the analyser leading to poor resolution.
42
What is the definition of resolution in mass spectroscopy?
the ability to distinguish two peaks of slightly different mass-to-charge ratios ΔM, in a mass spectrum.
43
What was the solution to poor resolution in MALDI?
Delayed Extraction.
44
What does Delayed Extraction involve? Use diagrams in your answer.
The pulse of ions is kept in the source for a short time after the laser pulse in order to allow time for ions formed deep in the matrix to emerge and 'catch up' with those formed near the surface. The repelling electric field (backboard) isn't applied until nanoseconds later, thus ions with greater velocity have travelled further away from this field and are repelled less so. However ions of lower velocity are closers to the applied field and are repelled much more, allowing for these delayed ions to catch up.
45
Who won the Nobel prize for his/her work in the development of delayed extraction in MALDI?
Koichi Tanaka.
46
What does ES/ESI stand for?
Electrospray ionisation. ✨
47
At what condition is ES ionisation carried under?
Atmospheric pressure
48
At what phase does ESI start with?
Liquid phase
49
Why is it good that ESI starts in the liquid phase?
Because this is the state at which most biomolecules are found.
50
Describe the steps of Electrospray ionisation. Doooooo it bitch, do it.
1. Dissolve sample into buffer solution
2. Load into glass capillary
3. Tip of capillary coated in gold or other heavy metal
4. A high voltage (3-4 Kv) is applied to tip, large potential difference between tip and surrounding electrode
5. Sample emerging from tip is dispersed as an aerosol of highly charged droplets (millions of drops)
6. Drying gas (nitrogen) causes evaporation, protein molecules in closer proximity, become unstable due to high surface charge repulsion, causes droplet explosion at critical value (Rayleigh limit)
7. Millions more drops, cycle continues until you have naked gas phase ions
8. Ions drift into analyser according to voltage gradient
51
Draw ESI
For reals.
52
What voltage is applied to the capillary tip in ESI?
3-4 KV
53
What is the drying gas used in ESI?
Nitrogen
54
Charge depends on? And how does this affect ESI?
Buffer and pH. Which means some of your peptides have different charges.
55
At what flow rate range did old ESI operate at? What range is achieved by NanoESI? And which method is more sensitive (better)?
Micro litres per minute. 10-30 Nano litres per minute. NanoESI is much better.
56
What sample quantities are usually required in NanoES? And what's the added benefit of this?
Around 1 micro litre in the needle, or subnanogram protein content. This means that it is possible to carry out many MS experiments on one prepared sample.
57
What is NanoES usually linked up to, to produce a power method for analysis of complex mixtures.
NanoLiquidChromotography
58
What is the name of the Electro Spray that is used in the Q-TOF instrument?
Z-Spray Nanospray. Named so due to the pathway of its Electrospray.
59
What is the mass (kDA) of myoglobin? And in what ionisation technique are multiply charged ions created and analysed?
The mass is 16,953 kDA
Electro-spray ionisation (ES) is used to create multiply charged ions, whereas MALDI and FAD only exploit singly charged cations
60
What is a commonly exploited charge locii in proteins to create a positive charge? And why?
The amine group, because is it a good proton acceptor
61
What is another commonly exploited charge locii in proteins for creating negative charge, and why? Which is more typically used in ES analysis of proteins, positively or negatively charged species?
Carboxylic group, good proton donator. And positive charged species.
62
What are the two characteristics of protein mass spectra like that seen of myoglobin?
1. Normal distributions of intensities around the base peak
| 2. Increased spacing between the peaks and increased m/z as adjacent peaks are of one less positive charge
63
What are the two vital equations in calculating the mass of a given peak? What are the steps and how do you calculate the mass of the protein?
1. M= z (m/z - H)
2. Z2 = ((m1/z1)-H/ ((m2/z2) - (m1/z2)
Z2 is the charge of your selected peak, select an adjacent peak of lower m/z and calculate z2. Once the charge of your peak is calculated calculate mass using equation 1.
To calculate the actual mass of the protein, the mass of each peak must be calculated, an average of all the peaks is generated by summating their masses and dividing by the number of peaks.
64
What do each of the peak represent on a protein MS spectrum?
Each peak represents an intact protein molecule with some number of adducted protons.
65
What are the 6 main analysers used in MS?
1. Magnetic sector
2. Quadrupole
3. Time-of-flight (TOF)
4. ION trap
5. Ion cyclotron resonance (ICR)
6. Orbitrap
66
What are the three key factors when considering the appropriate analyser? And what do each mean?
1. The upper mass limit (range) - how large an ion can the mass analyser handle
2. The ion transmission - how many ions can you generate, separate and detect? For example if you can detect all your ions then this is high sensitivity, if only 70% then poorer sensitivity.
3. The resolution- how good is the analyser at separating ions with similiar m/z ratios. The better the resolution the more powerful the technique
67
What is the main objective of an analyser?
To control the flight of ions.
68
What is dubious about Magnetic sectors history?
They were used during WWII to separate an isotope of uranium for use in atomic bombs.
69
What is the m/z equation for magnetic sector instruments? What are each of the values and which are constant and which are variable?
m/z = B^2 R^2 / 2V
```
B= magnetic field - variable
R= radius of magnet - constant (flight path)
V = Acceleration voltage - constant (voltage you apply to accelerate ions)
```
We vary B using appropriate calibration. Can relate certain m/z ratios to certain B strengths. Values are proportional.
70
On Magnetic sector analysers, how good are their upper magnetic range, resolution and ion transmission values? What is their other major drawback?
Okay upper mass limit, good resolution but poor ion transmission for its only a scanning deflector.
Can only analyse certain ions, of certain flight paths at a given time. Only a fraction of the ions created are detected meaning low sensitivity.
The other major drawback is their size and expense.
71
What technique do most modern magnetic sectors use? what does this entail and to what benefit does this achieve?
They are usually 'double focussing' in that they have an additional electrostatic analyser for energy focussing, giving greater resolution.
72
What does focal planes integrated into most modern magnetic sector analysers do?
Allows ions of different m/z to be accumulated for better sensitivity.
73
When was the quadrupole invented and what are their basic features?
The mid 1950's
Uses a quadrupole ELECTRIC field, comprising of RF and DC components to separate ions
Consists of four parallel rods to which you apply an electric field.
74
Draw a basic Quadrupole setup
Yeah bitch. That's right.
75
Are each pair of the four rods connected electrically horizontally, vertically or diagonally? To which are each pair assigned?
Diagonally. A DC component to one, and an RF potential to the other.
76
How are ions of different m/z separated in the Quadrupole analyser?
Ion of specific m/z will be 'in harmony' with the Quadrupole electric field, and will thus pass through to the detector. However ions of different m/z ratio will not be in harmony and will be too greatly attracted toward the poles, resulting in annihilation, meaning they aren't detected.
77
What of the upper mass limit, resolution, ion transmission and cost of quadrupoles?
M/z ratios up to 4,000 can be detected (low)
Low resolution ( unit resolution to about 3,000)
Low cost, can be made portable
Termed rapid scanning, only ions of certain m/z ratios are detected at a time. Low sensitivity.
78
What are Quadrupoles usually used in combination with?
Gas chromatography prior to mass spectrometer analysis
| Widely used for ES-MS
79
What are ion trap analysers homologous to? Although in what aspect do they differ?
Homologous to principles of quadrupoles, although they do not operate as filters.
80
How are the electrodes arranged in ion trap analysers? And how are their electronic fields generated?
Arranged in a sandwich geometry, composed of a middle ring electrode with cap electrodes at either end. The fields are generated by RF and sometimes DC.
81
Why are ion traps analysers considered a scanning technique despite theoretically possessing the ability to detect every ion?
Because some ions annihilate on the sides of the chamber.
82
Outline the steps involved in Ion trap analysis.
1. Ions from source are ionised and sent to the ion trap chamber.
2. Electric field generated, ions are trapped and begin to rotate
3. Change the frequency of the E field progressively to sequentially eject ions of a desired m/z ratio.
4. Ejected ions directed toward detector
83
What is the modern redesign of the ion trap and why does it deliver better performance than it's predecessor?
It is the linear ion trap analyser. Ions are trapped over a larger linear volume, overcomes problems of ion interference and increases storage capacity of ions increasing sensitivity.
84
What does the acronym TOF stand for? And what is their key principle?
Time of flight analyser. Ions are separated by differences in velocities as they move in a straight path toward the detector.
85
What is the upper mass limit of TOF's?
Unlimited 🙌
86
What does a TOF analyser consist of and what are the steps involved in TOF analysis?
Simply a metal evacuated tube.
1. Introduce ions into tube using an accelerating potential charge
2. Speed of ions and thus time taken to transverse the tube is related to energy; the larger the ions the longer it takes and the smaller vice versa. m/z ratio is related to time of flight.
87
What are the advantages and disadvantages of TOF's?
Ads:
1. Cheap
2. Can be linked to other analytical techniques
3. Has no upper mass limit
Dis's:
1. Poor resolving power
2. Due to ions possessing different kinetic energies from MALDI ionisation, ions of the same m/z ratio will travel at different times
88
What was the resolution to the effects of unequal kinetic energy distribution on TOF analysers? And how does it work?
The reflectron (invented 1974)
Principle is to give a longer flight path to ions of slightly higher kinetic energy.
The reflectron is an ion mirror that reverses the direction of travel of ions. Therefore ions of higher kinetic energy penetrate further and consequently have a longer flight path, allowing for the lower energy ions to 'catch up'
89
What resolution do reflectrons give linear analysers like TOF in comparison to those without?
They increase the resolution from less than 1,000 to above 3,000
90
Why do some ions possess greater or less kinetic energy than equivalent ions?
Because the ionisation process isn't 100% uniform, especially in MALDI. Ions exposed to slightly different conditions, small difference in Kinetic energy can cause a huge loss in resolution.
91
Draw a reflectron.
I'm fucking serious, punk.
92
Which achieves a higher resolving power, a linear TOF analyser or a reflecting TOS? And at what Resolutions would we expect (FWHM)?
A reflecting TOF-MS achieves a higher resolution at around 3400 (FWHM) and a linear is around 600 (FWHM)
Full width at half maximum
93
What is best performing mass spectrometer at the moment? What makes it the best and what are the general features of one? Any disadvantages?
The FOURIER TRANSFORM ION CYCLOTRON RESONANCE (FT-ICR)
Highest resolution, highest sensitivity, highest mass accuracy, simultaneous detections.
Features; Magnetic B field, transmitter plate and trap plate.
Disadvantages: expensive, bulky and temperamental
94
Outline the steps involved in Fourier Transform Ion CYLCLOTRON RESONANCE (FT-ICR)
1. Accelerated ions are passed into uniform high magnetic field (B), causing them to adopt spiral paths
2. Ions of particular m/z are excited by oscillating electric field (RF) generated by electrodes on the transmitter plates, absorption of energy increases velocity and radius of spiralling ions
3. Electric currents detected by receiver plate
4. Time-domain signal of the IMAGE currents converted into mass spectrum by Fourier Transform
95
At what Tesla strength are the super-conducting magnets in Fourier Transform Ion Cyclotron Resonance found at?
10-20
96
How does FT ICR detect ions of different m/z sequentially?
By scanning the RF (radiofrequency) of the applied electronic field
97
What is the purpose of the Fourier transform in FT ICR mass spectroscopy?
It relates the strength of the magnetic field to the increased velocity (current) of the different m/z ions at different RF frequencies
98
What beneficial feature of Fourier transform ion cyclotron resonance mass spectrometers allows for its high sensitivity?
The ions analysed aren't destroyed as they simply pass the detectors with their generated current induced by the nearby electrodes
99
What is the second highest performing mass spectrometer? What features make it so?
Orbitrap;
Has high resolution
High mass acuracy
Good upper mass-range and ion transmission
Cheaper than FT ICR
100
Outline basic method of the Orbitrap MS. Go on bitch
1. Ions trapped by static electrostatic fields
2. Ions orbit around Central spindle electrode and oscillate in axial direction
3. The FREQUENCY of this axial oscillation relates to the ions m/z ratio
4. Fourier transform algorithm coverts time-domain signal to m/z
101
What components are found in an Orbitrap MS?
Outer electrode.
Inner spindle electrode which ions rotate around
Static electrostatic field
Magnetic field
