Mass spectrometry Flashcards
What is the cycle for measuring the m/z ratio in mass spec? (after calibrating)
How is molecular mass defined?
How are peaks made in a spectrum? What is the x and y?
Which isotopes are the most abundant?
What happens in shift of the spectrum & 0x13C peak when mass increases?
Why are MS instruments held under vacuum?
Introduce sample
Generate ions to manipulate
Separate ions on/z ratio in mass analyser
Enters collision cell where further fragmented so 2nd mass analyser measures these resulting fragments
Detect ions with ion detecter & mass spec is recorded on a computer
1/12th of a mass of carbon-12
Electrical current generated when ion strikes the detector. X is m/z, Y is relative ion abundance
Monoisotopic (e.g 0 x 13C)
Shifts to the left, abundance of 0 x 13C decreases as abundance of heavier isotopes increase.
Increases average distance of ion before a collision
What type of technique is MALDI and SLD?
Why is MALDI a better technique?
How is an ion formed with MALDI?
How is this done in a lab?
What are the problems with MALDI? Specifically with large and small masses
Soft ionisation techniques
Analyse is mixed with a matrix which absorbs the laser wavelength to prevent damage & fragmentation of the ions
When matrix absorbs energy from laser- transfers proton between matrix & analyte- so protonates the analyte to form ions
Mix analyte & matrix in wells- let it crystallise & put in MS.
Large analytes like proteins will be out of the detectable m/z range in MS. Also low mass analytes interfere & matrix ions can form.
Why is electrospray spectra better to measure m/z with proteins than MALDI?
How are ions formed in electrospray?
What are the advantages & disadvantages of ESI spectra?
What are the differences between MALDI & ESI?
Can measure m/z of large molecules with analysers with a low m/z
Build up of charge produces mist of droplets out of needle tip with different net charges- drying gas removes solvent
Many charged peaks useful to find accurate mass & can be used to measure large proteins with analysers of low m/z range.
Presence lots of peaks complicate spectra when +1 species (lead to overlap)
MALDI= matrix, rapid, single charges of +1, sensitive, can use salts, can be used for complex mixtures
ESI = many charges, can’t use salts, can’t be used for complex mixtures, multiple peaks, huge mass range, link to liquid chromatography
What is the mass range of a mass spectrometer?
What is the mass accuracy of a MS? How does it vary?
What is the difference between mass resolution & resolving power? What is the definition?
What affects mass resolution?
What could you do to measure the mass if a molecule has a high m/z but in low resolution?
m/z range that mass analyser operates
How accurately it can provide m/z info (to the true value). With analyser type
Resolution = the data of the mass spec
Resolving power = function of the mass spec
Relevant to the separation of ions of 2 different m/z values- higher resolving power means separate ions closer together
The type of mass analyser, initial kinetic energy of ions
Measure average mass of ion
What are the 2 broad classes of mass analysers?
How does a time of flight TOF mass analyser measure the m/z?
Initial kinetic energy affects resolution- how can these be solved? What peak is made when ions hit the detector at different times?
How does a quadrupole analyser work in MS/MS mode in Q-TOF?
How is quadrupole different in MS mode?
What does the collision cell do?
Scanning: transmit ions along a timeline
Simultaneous transmission: all ions analysed at the same time
Measures time taken for ion to reach the detector with a known distance- as m/z of particle depends on time (larger mass takes more time)
With a reflectron- so both ions reach detector at same time for a sharper beak & more accurate m/z (diff Ek reach detector diff time = broader peak)
Calibrate to filter out ions which become attracted to rods & separate out- so ion of particular m/z (chosen) has stable trajectory to collision cell
Operated in RF only mode so doesn’t separate ions- ions are separated in TOF analyser
Fragments selected ion for RTOF to analyse it
What are the differences between denatured and native MS spectra?
What can native MS be used to detect?
What is the main drawback of the subunits?
What else can native MS probe with proteins?
Denatured = 2 overlapping spectra of 2 subunits, has wide distribution due to a low m/z range from protein unfolding
Native = Protein is still folded so only have 1 distribution spectra of entire protein/complex, more narrow & high m/z ratio, but don’t see the components in spectra
Mass change on protein: oligomerisation, ligand binding, subunit exchange
Subunits must have different masses in order to measure mass change
Protein composition- topology- put in collision energy & subunits fall off- so the remaining subunit is at a higher m/z and fallen subunit at lower m/z
What is ion mobility MS (IM-MS) used to measure? What can be calculated from this?
What is it dependent on?
What happens with a compact and extended ion in IM-MS?
How does their structure relate to charge and its cross section?
Arrival times & convert to collision cross-section & calculate theoretical cross-section
Flexibility/folding of the proteins
Compact: experiences less collision with buffer gas so arrives quicker at detector
Extended: more collisions, arrives later (further on spectrum to right)
More charge states a molecule has = more unfolded/extended/larger- larger cross section
What is cross linking MS used for? (XL-MS)
What are the steps of carrying this out?
What type of proteins is this most useful for?
Shows which peptides are cross linked together with distance restraints
- Add cross linker to target complex (spacer between reactive groups)
- Digest with trypsin
- Separate pieces with LC-MS
- Identify peptides-
Dynamic proteins (flexible)- as dynamic structures can’t be in a high res structure
What is the precursor being tandem MS/MS?
What is collision induced dissociation? CID
What is CID used for?
What is selected in MS1 of tandem MS?
What is MS2 for?
What do low energy collisions fragment mainly?
MS1 isolates ion of interest, then it enters collision cell for further fragmentation, & MS2 separates ions again
Ion fragments by colliding with neutral atom as translation energy converted to internal energy
Fragment peptides to obtain their sequence
The peak of the peptide- which then goes on to do MS2
Deduce sequence of peptide in terms of amino acids
Backbone & some side chains- but mainly NH-CH, CH-CO, CO-NH
