L2: Mass Spec

Question 1

Human genome size

Answer 1

20,000-25,000 genes

3.15x10^9 bases

Question 2

True or false:

Post-translational modifications are directly coded by genes

Answer 2

False.

Question 3

If a protein has 50 known modifications, how many possible proteoforms exist?

Answer 3

50! or 3.04e64

Question 4

Y axis of mass spec measures what?

Answer 4

Intensity

Question 5

X axis of mass spec measures what?

Answer 5

mass/charge (m/z)

Question 6

Mass spectrometers can routinely detect ____ moles of sample

Answer 6

10^-15

Question 7

MS is used to detect what?

Answer 7

mass, identity, sequence or composition (Tandem MS), and PTM of a peptide

Question 8

Mass spectrometers work by pushing a) _____ using b) _____ fields.

Answer 8

a) gas phase ions

b) electric or magnetic

Question 9

When working with proteins and peptides, which two techniques are by far the most common methods for generating gas phase peptide/protein ions?

Answer 9

Electrospray and MALDI

Question 10

Basic components of a mass spectrometer

Answer 10

1. Source (e.g. MALDI): ionises sample into ‘flight’
2. Analyser (e.g. quadrupoles or time-of-flight): separates sample based on m/z
3. Detector (e.g. electron multiplier): detects ions
4. Computer: deconvolutes data and plots as abundance vs. m/z

Question 11

Time of flight analyser

Answer 11

• Ions accelerate to a detector with the same energy (E)
• Smaller ions reach the detector 1st
• Require a good vacuum (<10-6 torr)
• Simple but expensive
• Mass range up to 500 kDa
• Mass determined according to equation: m = 2E/v^2

Question 12

Quadrupole analyser

Answer 12

• Four parallel rods with a DC voltage and a superimposed AC (RF) potential
• Scan DC and RF to allow ions of increasing m/z to successfully pass to a detector
• Can capture specific ions and eject specific ions

Question 13

Advantages and Disadvantages of Quadrupole vs. ToF

Answer 13

Quadrupole advantages:

• Tolerant of poor vacuums
• Low cost

Disadvantages:

• Low resolution
• Analyse ions m/z <2000

Question 14

Orbitrap Mass Analyser

Answer 14

• DC electric field causes trapping of ions
• Ions oscillate radially around centre electrode and axially along z axis
• Ions are non-destructively detected via their z oscillation frequencies
• High mass resolving power (450K) and mass accuracy (1ppm)

Question 15

Tandem MS (MS/MS)

Answer 15

• Tandem MS analysers have 2 or more analyser chambers
• Some analysers can perform MS^n by re-trapping ions
• Peptide bonds are broken usually by Collision Induced Dissociation (CID) in the collision cell (argon or helium)

Question 16

MALDI-TOF Mechanism

Answer 16

• Sample to be analysed is mixed with a matrix compound
• Solvents vaporise, leaving only the recrystallised matrix, but now with analyte molecules spread throughout the crystals. Matrix and analyte are said to be crystallised in a MALDI spot
• Matrix has a strong optical absorption in either UV or IR range so that they rapidly and efficiently absorb the laser irradiation.
• The Matrix is often acidic, acting as a proton source to encourage ionisation of the analyte.

Question 17

MALDI-ToF effective mass range

Answer 17

500Da to 1,000,000+ Da

Question 18

Electrospray ionisation (ESI)

Answer 18

• Most common source in HPLC, known as a soft ionisation technique (intact molecules are detected).
• Solvent must be removed to produce gas phase ions
• Must only use mobile phase additive which are volatile
• Protein in droplets is stranded in gas phase and sucked into the spectrometer

Question 19

Biologists often submit samples in buffer and other
salts for analysis. What happens if you spray salt
solutions?

Answer 19

Electrospray requires your sample to be in a volatile solvent and not contain salts and other non volatile components as salt will crash out inside the source of the MS, corroding the MS source

Question 20

Why is HPLC used in MS?

Answer 20

To separate out the salt, and separate the protein.
Sample goes through HPLC and is injected onto an rpHPLC column. The protein sticks to beads inside the column and is eluted with acetonitrile gradient

Question 21

The same molecule with 2 charges will reach the MS detector in a) HALF / DOUBLE the time as it experienced b) HALF / DOUBLE the force

Answer 21

a) Half

b) Double

Question 22

Difference between monoisotopic and average mass

Answer 22

Monoisotopic mass is used for small molecules

Average mass is used for big proteins

Question 23

Trypsin cuts at which amino acids?

Answer 23

Lysine (K) and arginine (R)

Question 24

Basis for peptide mass fingerprinting

Answer 24

1. List of peptide masses is generated
2. Values of peptide masses are inserted into an algorithm e.g. MS-Fit
3. A protein is matched from a genome database to correspond to the masses based on known protein masses.

Question 25

Advantages and disadvantages of peptide mass fingerprinting

Answer 25

Advantages:
• Fast and sensitive

Disadvantages:
• Must have sequence in database
• Need to know which enzyme was used to digest fragment
• Protein can’t be heavily modified

Question 26

In an MS/MS ions search, fragmentation of the backbone occurs at which bond?

Answer 26

Peptide bond

Question 27

Most common ions produced by CID are what?

Answer 27

b and y ions

Question 28

How to find first y ion in a tryptic digest?

Answer 28

Look for
lysine = 128 +19=147
arginine = 156+19=175

Question 29

Largest protein database

Answer 29

uniprot but lots of redundancies

Question 30

Database with no redundancies

Answer 30

MSDB