Mass Spectrometry

Question 1

What are the main components of mass spectrometry instrumentation?

Answer 1

• Ionization method: MALDI, ESI
• Mass analyzers: Time of flight (TOF) and quadrupoles
• Detector
• Vacuum system

Question 2

What is ESI-MS?

Answer 2

Electrospray ionization

Question 3

What does ESI-MS produce?

Answer 3

Multiple charged ions directly from volatile liquid during desolvation

Question 4

What is ESI-MS usually coupled to?

Answer 4

Liquid chromatography (online LC-MS)

Question 5

What various techniques can mass analysis be accomplished by?

Answer 5

Biophysical (TOF, Quad, Cyclotron)

Question 6

What does MALDI-TOF mean?

Answer 6

Matrix assisted laser desorption ionization-time of flight

Question 7

What are the modes of operation for MALDI?

Answer 7

1) Linear mode
2) Reflector mode

Question 8

What is linear mode used for?

Answer 8

To perform peptide mass fingerprint (no sequence information)

Question 9

What is reflector mode used for?

Answer 9

To perform MALDI-MS/MS (sequence information

Question 10

What forms co-crystals?

Answer 10

The mixture of a UV absorbing matric and analyte when dried

Question 11

What happens when the laser rapidly vaporizes the crystal?

Answer 11

Matrix facilitates protonation of the analyte and the desorbed ion is accelerated out of the ion source

Question 12

What are some typical matrices used?

Answer 12

• 4-hydroxy-alpha-cyanocinnamic acid
• 2,5-dihydroxy benzoic acid (DHB)
• Sinapinic acid

Question 13

What happens in MALDI-TOF linear mode?

Answer 13

• Measures mass-dependent time of flight: KE = 1/2mv^2
• Singly protonated molecules produced
• Produces mass fingerprint of protein of interest
• No amino acid sequence information

Question 14

What is the mass equation used in MALDI-TOF linear mode?

Answer 14

m = (2KEt^2)/L^2

Question 15

What are the titles of the x- and y- axis for a MALDI-linear mass fingerprint?

Answer 15

x: mass (m/z)
y: % intensity

Question 16

How is a mass fingerprint analyzed?

Answer 16

All known protein sequences in database –> MS-Fit computer program in silicon –> calculated mass values –> mass spectrometry observed values –> list of potential proteins

Question 17

What happens in MS/MS fragmentation?

Answer 17

• Parent or precursor ion is selected (times ion selector)
• Ion begins to fragment at the peptide bond due to collision energy
• Each individual peptide molecule will have enough energy to fragment at one peptide bond (position will vary)
• The protein can reside on either the N-terminal or C-terminal end of the fragment ions
• Each individual fragmentation event forms one of two ion species

Question 18

What are the two ion species?

Answer 18

• y-ions (C-terminal ions)
• b-ions (N-terminal ions)

Question 19

How do fragment ions initially travel?

Answer 19

At the same velocity (based on mass of original peptide ion - due to travelling in a region of high vacuum)

Question 20

What does the reflector do?

Answer 20

Redistributes new K.E. to fragment ions leading to new velocities

Question 21

What sequence information is obtained from MALDI-MS/MS?

Answer 21

Partial

Question 22

From the position on the graph, where do y and b ions get fragmented on the amino acid sequence?

Answer 22

y –> from right to left
b –> from left to right

Question 23

What is the general overall flow for identifying an unknown protein?

Answer 23

1) SDS/PAGE
2) In-gel trypsin digestion
3) MALDI-linear - peptide mass fingerprint
4) Search database - tentative match
5) MS/MS - sequence data
6) Analyze data manually and by database searching
7) Protein identified