Mass Spectrometry

Question 1

Define mass spectrometry:

Answer 1

Devices that weigh and count molecules (or atoms)
A mass spectrometer is an ion optical device that produces a beam of gas phase ions from samples, sorts these ions according to their mass-to-charge ratios and determines the intensity (abundance) or each ionic species
Molecules MUST have a charge

Question 2

Describe the basic mass spectrometry plot:

Answer 2

A mass spectrometer measured mass/charge ration (m/z)
A simplified mass spectometer produces an abundance/intensity scale
Base peak = largest peak in spectrum (100%)
Intensity distributions of m/z values

Question 3

Describe why multiple peaks can occur in mass spectra

Answer 3

Multiple parent molecules in the mixture
Fragmentation of parent
Isotopes (unavoidable)
Not because of impurity or degradation etc.

Question 4

Describe the molecular mass that is measured by MS:

Answer 4

Molecular weight is weighted average of the atomic masses of each isotope in the molecule
MS sees exact mass (monoisotropic mass - the sum of the most abundant isotopes of elements in a compound (need to ionise, can be single protonation)

Question 5

Describe the advantages of mass spectrometry:

Answer 5

Sensitivity
- Detector currets as low as 1000 ions/sec of a specific m/z
Specificity:
- m/z ratio of parent molecule is characteristic
- Pattern of fragmentation gives diagnostic fingerprint
Structural elucidation
- Fragmentation gives information about chemical groups in molecule - especially tandem MS
- Capable of de novo sequencing of peptides
Universal detection
- All molecules have mass, and most can ionise (useful for detector of separation methods - HPLC, GC/LC, capillary electrophoresis etc.)

Question 6

Describe inductively-coupled mass spectrometry (ICP-MS):

Answer 6

Ionised atomic plasma at 10,000 K (high temperatures)
Form singly-charged atomic ions
Lose structural information
Large differences in sensitivity between elements
Limit of detection can be different
Most useful for trace metal analysis

Question 7

Describe accelerator mass spectrometry (AMS)

Answer 7

14C/13C ratios for carbon dating or tracer analysis
Extreme sensitivity (more sensitive than decay counting)

Question 8

Describe molecular mass spectrometry:

Answer 8

Intact or partially fragmented molecules
- Non-polymers (small molecules) such as metabolites
- Polymers - especially proteins (doesn’t say much about sequence)
- MW of complete proteins
- Identification of known proteins in complex mixtures
- Post-translational modification
- De novo sequencing
Can be used for identification, structural elucidation and quantitation (e.g. detecting cocaine in urine)

Question 9

Describe the general mass spectrometer components:

Answer 9

Machines can be very complex or simple
Vacuum system controls frequency of collision of ions with neutral gas molecules
Mean free path is inversely proportional to pressure
Large pressure differences with compartments separated by apertures of low conductance
Expensive pumps (mechanical, diffusion, turbo, molecular cyrogenic)

Question 10

Describe the pressure and mean free paths in MS machines:

Answer 10

Vacuum system - 1 torr
Analyser - 10^-7 torr - mean free path 500m
Ion source - 10^-3 - mean free path 5cm
Higher pressure = shorter flight path

Question 11

Describe sample introduction for the three different states and their respective techniques:

Answer 11

Gases
- GC/MS - gas liquid chromatography interfaced with MS
Liquids
- FIA (flow injection analysis)
- HPLC (high pressure liquid chromatography - interfaced with MS = LC/MS)
- CE (capillary electrophoresis)
Solids
- Matrix-assisted laser desorption ionisation (MALDI) - co-crystalise analyte with a matrix containing a chromophore that absorbs light

Question 12

List the types of ion sources for use in MS:

Answer 12

Electron ionisation
Atmospheric pressure chemical ionisation
Electrospray ionisation
Matrix-assisted laser desorption ionisation

Question 13

Describe electron ionisation (EI):

Answer 13

Ionise molecules by collision with high energy electrons (70eV)
Volatile and thermally stable compondes only
Usual mode for GC/MS
Reproducible mass spectra, well-understood fragmentation patterns
Large libraries for fingerprinting
Often no molecular ion

Question 14

Describe atmospheric pressure chemical ionisation (APCI):

Answer 14

Compounds of low/moderate polarity

Question 15

Compare the quantitative performance of APCI vs. ESI:

Answer 15

Sensitivity:
- Polar compounds ESI>APCI
- Non-polar compounds APCI>ESI
BUT ESI is susceptible to ion suppression (decreased detection because of other compouns in the mobile phase or sample). This effect can also be enhancing, and varies between individuals
Choice of ionisation method is application depedent

Question 16

Describe the basics of electrospray ionisation (ESI):

Answer 16

Polar molecules
Includes fragile biomolecules of high MW
Least fragmentation
Best method for finding molecular ion
Used most in biomedical science, such as for proteins
Increasing the voltage leads to increased fragmentation, which is not random and is dependent on bond strength
Inert gasses lead to atoms colliding and the weakest bonds breaking first
This effect is quite reproducible

Question 17

Describe how ESI occurs:

Answer 17

Liquid stream electrostatically nebulised by spraying at atmospheric pressure
Charged droplets exposed to heat and/or drying gas (N2) leading to ion evaporation
Applicable to compounds with a wide range of polarities and MW tends to give multiple charge ions with large molecules e.g. [M+nH]n+
Ideally suited to analysis of proteins
Also excellent for small molecules that can be charged in solution

Question 18

Describe MALDI:

Answer 18

Very important for non-volatile analytes, especially proteins
Multiple samples on single plate allows rapid analysis
Tends to give singly-charged species

Question 19

List the types of mass analysis:

Answer 19

Time-of-flight (TOF)
Transmission quadrupoles (quads)
Ion traps

Question 20

Describe time-of-flight mass analyses:

Answer 20

Pulses of ions accelerated out of course with same energy (from electric field) - differ in initial velocity
Separated by drifting at different speeds in long elevated tube (1m)
Smaller=increased kinetics=increased speed (hit detector first as they are lighter)
Can achieve very high mass accuracy and sensitivity.

Question 21

Describe transmission quadrupoles:

Answer 21

Four parallel cylindrical rods with dc and rf voltages, causing ions to oscillate (13-20cm rods)
Acts as a m/z filter allowing passage of ions in a narrow m/z window (others have too high an amplitude and are deflected out of the quad)
Vary rf amplitude and dc potential to scan through m/z range
Easy to make, cheap, small and very common

Question 22

Describe the limit and mode of quads:

Answer 22

Upper limit ca 4000 Da (but multiple charging can bring m/z of proteins into this range)
Scan mode:
- Rapidly scans through a range of m/z values (determines spectrum)
- Low sensitivity
- Can retrospectively extract signal from any ion (extracted ion mode)
SIM (single ion mode):
- Monitors a single m/z continuously
- High sensitivity
- Best mode as HPLC detector for known analyte
Can switch between m/z values rapidly (monitor multiple ions)
Can also use quads as non-selective ion guides (rf only operation)
Transmits ions of all m/z value

Question 23

Describe triple quadrupoles (tandem MS, MS/MS) mass analyses:

Answer 23

Q1 partially replaces need for chromatographic separation
MS/MS - selected reaction monitoring (SRM) - analogous to SIM in single quad
Q1 fixed - precursor ion set
Q2 - fragmentation (collision induced dissociation)
Q3 fixed - product ion set
Only a single m/z goes through, leading to sensitivity
Complex samples require a degree of separation
Leads to enhanced selectivity and sensitivity relative to single quad SIM

Question 24

Describe multiple reaction monitoring (MRM):

Answer 24

Can be used to quantify multiple compounds in a single sample e.g. LC/MS/MS analysis of parent drug and 5 metabolites in plasma sample
Quantified using tetra-deuterated stable isotope internal standards

Question 25

Describe ion traps mass analyses:

Answer 25

Also quadrupoles, but confine ions in 3D, rather than along linear trajectory Ions with stable trajectories are trapped Fill with a broad range of m/z, then 'elute' (scan out) unstable ions to the detectors Easy to use, small footprint, low cost Excellent for structural elucidation Sensitivity/quantitative performance not quire as good as triple quad

Question 26

Describe the advantage of hybrid Q-TOF and Qq-TOF analysers:

Answer 26

TOF has a high mass accuracy, while Qq doesn't, so combine

Question 27

List some proteomics applications of MS:

Answer 27

Determination of MW Protein identification - De novo sequencing and database matching Post-translational modification Changes in expression (relative quantification)

Question 28

Describe electrospray analysis of Rho protein:

Answer 28

Charge envelope protein - charge state 40-70 which determines biomolecule mass Leads to deconvolution (mathematical transformation of a spectrum of several multiple charged peaks into one peak corresponding to a single charged ion) m/z is known, z is known, thus calculate m

Question 29

Describe protein identification in proteomics:

Answer 29

2D gel --> in gel digest --> tandem MS (LC-MS/MS or ion trap) --> de novo sequencing or submit mass spec patterns for database searching

Question 30

Describe peptide sequencing by tandem MS:

Answer 30

2D gel --> trypsin digest spot --> LC MS/MS Q1 - select precursor peptide ion Q2 - collision-induced dissociation Q3 - analysis of N- and C-terminus fragments

Question 31

Describe the advantages of the 2D gel method:

Answer 31

Well established technique Visual display of protein patterns Excellent resolving power Inexpensive to get started with manual techniques

Question 32

Describe the disadvantages of the 2D gel method:

Answer 32

Abundant proteins mask other proteins Limited protein capacity Low abundance proteins not visualised or separated Poor ability to handle acidic, basic, and membrane proteins Time consuming and difficult to automate Quantification not always reliable

Question 33

Describe isotope coded affinity tags (ICAT) for proteomics research:

Answer 33

Have labelled cysteines in light and heavy samples, which are pooled, digested with ICAT to labelled proteins and purified with ICAT labelled peptides or IEX and avidin (further separated by reversed phase LC), identified and quantified by MS and MS/MS (used to identify data about the protein)

Question 34

Describe the the funnel approach to quantitative complex mixture analysis:

Answer 34

Leading from vast and complex to small and simple Preparative SCX-HPLC (fraction collect) Affinity isolation of cys peptides Capillary RP-HPLC Mass analysis and automated quantification Selective protein identification (10% or less showing significant differential expression levels)

Question 35

Describe the advantages of MDLC method with ICAT reagents:

Answer 35

Sample complexity may require multidimensional chromatography By collecting only ICAT labelled cysteine peptides the complexity is reduced ``` Quantitative Looks at low-abundance and membrane proteins MDLC of peptides Complexity of peptide mix reduced High throughput Easy to automate ```

Question 36

Describe isobaric tag for relative and absolute quantification (iTRAQ):

Answer 36

Introduced in 2004 Simultaneously identify and quantify proteins from up to 4 samples Expands protein and proteome coverage by labelling all peptides, including those with post-translational modifications (PTMs) Increases confidence in identification and quantification from MS/MS spectra by tagging multiple peptides per protein

Question 37

Describe work flow of an IMS (imaging mass spectrometry):

Answer 37

Can image fresh samples (such as a brain slice), which is matrix spotted and each spot analysed to produce a mass spectrum Can be used to image live animals A. A tissue section is collected on conductive MALDI plate B. Matrix is deposited in a uniform manner over the surface of the tissue C. Spectra are acquired from each location (pixel) over the surface of the tissue D. 2D ion density images are reconstructed from the spectra Hundreds of protein images can be created from a single 12 um thick section of tissue from a single acquisition

Question 38

Describe stable isotype labelling with amino acids in cell culture (SILAC):

Answer 38

Proteins isolated by SDS-PAGE/2D-gels stained in the gel Protein in the gel reduced to open up S-S bonds and SH groups protected by treatments Proteins protealysed by enzymatically or cut chemically Peptides resolved by micro-bore or capillary HPLC Most eluent directed into the MS by ESI Masses of each peptide measured in real time as they elute from the column During elution, many peptides are fragmented in the MS and masses of fragments are measured MS data used to identify the protein by comparing the peptides to simulation of MS of enzymatic digest and to MS/MS fragmentation of such peptides from the protein sequences in the data bank If the protein is a new protein, proceed to obtain amino acid sequence data by sequencing or computationally

Question 39

Describe how ICAT can provide accurate quantification:

Answer 39

A pari of ICAT reagent labelled peptides are chemically identical and easily visualised because they co-elute, and there in an 8 Da mass difference - these ICAT pairs serve as mutual internal standards for accurate quantification Ratios of original amounts of proteins from the two cell states are strictly maintained in the peptide fragments