W6 Mass Spectroscopy

Question 1

what is mass spectrometry

Answer 1

an analytical technique used to measure the mass to charge ratio of ions

used to identify unknown compounds via molecular weight, quantify known compounds and determine structure

Question 2

components of MS

Answer 2

inlet (sample introduction) > ions source (gas phase ion generation) > mass analyser (ion manipulation) > ion detector (convert number of ions into signal) > data system > mass spectrum

Question 3

function of ion source

Answer 3

to convert molecules to gas-phase ions via either MALDI or ESI

MS measures ion > unit is mass to charge ratio (m/z)

Question 4

what is the process of electrospray ionisation (ESI)

Answer 4

1. liquid sample is sprayed from the tip under an electrical potential between 0.7-5 kilo volts (kV)
2. nozzle generates a fine spray of charged droplets (at high flow rate, dry gas/heat used to remove solvent from droplet)
3. as size of charged droplet decrease > charge density on its surface increases
4. droplet explodes into individual charged ions

Question 5

pros of ESI

Answer 5

mass range up to 70kDa

good sensitivity

soft ionisation method (no fragmentation

no disruption of non covalent bond

compatible with liquid chromatography (to analyse highly complex mixtures)

analyte can carry multiple charges (high MW to lower m/z)

Question 6

cons of ESI

Answer 6

low tolerance for salt, ion pairing reagents, detergents and complex mixtures

hard to ionise non polar compounds

difficult to clean

multiple charging increase complexity in mixture analysis

Question 7

what is the procedure of matrix assisted laser desorption ionisation (MALDI)

Answer 7

1. compound is dissolved in solvent containing matrix that helps with ionisation process
2. matrix applied on metal plate > dried to form crystalline structure
3. laser directed at sample > matrix absorbs energy > matrix vaporise and cause sample to be carried into gas phase together
4. sample ionised (single positive charge) and released from matrix into gas phase
5. ionised molecules directed to MS > measure m/z ratio

Question 8

purpose of matrix in MALDI

Answer 8

help transfer the charge to sample molecules without causing them to break apart

Question 9

resolutions for the 4 mass analysers

Answer 9

quadrupole: low with scan speed of 1s

linear ion trap: low with scan speed of 1s

orbit rap: high to ultrahigh with scan speed of a few seconds

time of flight: high with scan speed of milliseconds

Question 10

how to do protein identification using proteomics

Answer 10

1. proteolytic digestion of proteins before analysis by MS
2. LC to separate peptide mixture for better detection
3. MS1 to obtain precursor ions’ m/z (peptide MW)
4. MS2 to identify peptide sequence
5. identified peptide matched back to possible proteins

Question 11

how to do protein quantification using proteomics

Answer 11

1. ions are selected to pass first quadrupole
2. ions are fragmented
3. ions are selected to pass the third quadrupole