Lab 5 - MALDI-TOF Mass Spectrometry

Question 1

What are the tasks of this lab?

Answer 1

• Preparation of reagents for digestion
• In-gel digestion
• Extraction of peptides
• MALDI-TOF MS spotting

Question 2

What is a mass spectrometer used to obtain?

Answer 2

Information of the mass of a molecule

Question 3

In biological application, what is mess spectrometry most commonly used for?

Answer 3

To rapidly identify novel multiprotein complexes and map protein modifications that regulate enzymatic activity

Question 4

What other field if centered on mass spectrometry platforms?

Answer 4

• Proteomics (the systematic analysis of all the proteins that are expressed in a given cell which revolutionized protein characterization)
• Disease research
• Drug discovery

Question 5

How is mass spectrometry carried out?

Answer 5

1) Proteins are first proteolytically cleaved with proteases at the peptide bond
2) Masses of the individual peptides are determined

Question 6

What will we be digesting in this lab?

Answer 6

The affinity-purified protein from last week with a protease called trypsin to unambiguously prove that you purified PTEN

Question 7

What does trypsin do?

Answer 7

Specifically cleaves the peptide bond following lysine and arginine residues

Question 8

What will the masses of the individual tryptic peptides produce?

Answer 8

A “mass fingerprint” for your protein of interest

Question 9

How can the tryptic peptide masses generated by mass spectrometry be used if the protein is unknown?

Answer 9

To search a protein sequence database to identify your unknown protein (powerful tool for elucidating protein-protein interactions and novel functional biological complexes)

Question 10

What is a mass spectrometer composed of?

Answer 10

• Sample inlet
• Ion source
• Mass analyzer
• Detector
• Vacuum system
• Instrument control system
• Data system

Question 11

What is the main process underlying mass spectrometry?

Answer 11

The measurement of the mass-to-charge ration (m/z) of gas-phase ions which are derived from a substance of interest (a protein in our case)

Question 12

How is the mass-to-charge ratio of gas-phase ions characterized?

Answer 12

By the movement of these ions within an electric field

Question 13

In this lab, what will peptides derived from proteolytically cleaved PTEN be dissolved in?

Answer 13

A solution of UV-absorbing compound referred to as the matric and placed on a target plate for the mass spectrometer

Question 14

What happens as the solution dries?

Answer 14

The matrix crystallizes and the peptide molecules become embedded within the matrix crystals

Question 15

What do pulses of UV laser light do?

Answer 15

Vaporize small amounts of the matrix and therefore the peptide ions, and are carried into the gas phase in the process

Question 16

How does ionization of the peptides occur?

Answer 16

By the protonation in the acidic environments which are produced by the acidity of the matric compound and by the addition of a dilute acid to the sample

Question 17

How are ions then detected>

Answer 17

Utilizing a time-of-flight mass analyzer (TOF)

Question 18

How does a TOF analyzer work?

Answer 18

By giving an ion a fixed amount of kinetic energy using high voltage power supply which allows the ions to accelerate into a generated electric field

Question 19

What happens to the ions following the acceleration?

Answer 19

Enters a field free region where it travels at a velocity that is inversely proportional to its m/z

Question 20

How does an ion with a high m/z ratio travel compared to an ion with a low m/z ratio?

Answer 20

More slowly

Question 21

What are some characteristics of the TOF mass analyzers?

Answer 21

• Have essentially an unlimited mass range
• Can acquire data rapidly
• Are extremely sensitive

Question 22

What will we be doing in the weeks ahead?

Answer 22

• Analyzing PTEN from your SDS-PAGE gel that was kept from the affinity chromatography molecule
• The band of interest will be isolated and digested at specific peptide bonds with a protease
• Analyzed using MALDI-TOF MS/MS

Question 23

Describe the protocol for preparing samples for analysis by MALDI-TOF mass spectrometry.

Answer 23

1) Calculate the amount of ammonium bicarbonate you would need to weight
2) Verify the pH
3) Prepare destaining solution
4) Excise the band of interest from your gel and place it in a 1.5 mL siliconized microcentrifuge tube
5) Add destain solution and place in bench top shaking incubator at 37 degrees C for 15 mins
6) Spin down and discard supernatant
7) Dehydrate gel by adding acetonitrile
8) Spin down tube and remove acetonitrile
9) Spin down and remove acetonitrile
10) Place gel in speed vacuum for 20 mins

Question 24

Describe the protocol for in-gel digestion.

Answer 24

1) Allow resuspended trypsin to that and place immediately on ice to minimize heat exposure
2) Prepare trypsin digestion bugger
3) Re-hydrate your dried gel piece by adding trypsin
4) Place tube with gel piece suspended in trypsin digestion buffer on ice and incubate for 30 mins to allow trypsin to enter the matrix and interact with PTEN
5) Add ammonium bicarbonate and place back on ice
6) Remove the siliconized tube containing gel piece and place a strip of parafilm around the lid of the microcentrifuge tube to minimize evaporation
7) Plac gel sample in bench top shaking incubator for 2 hrs at 37 degrees C
8) Every 30 mins, check the amount of buffer remaining in the microcentrifuge tube because the gel needs to be covered *keep track of time and amount of AB

Question 25

Describe the protocol for the extraction of peptides.

Answer 25

1) Remove gel sample from the shaking bench top incubator and vortex the sample briefly and spin down
2) Add 5% formic acid solution to gel piece
3) Collect the supernatant and transfer to a fresh microcentrifuge tube
4) Place eppendorf tube containing your peptides onto ice bucket

Question 26

Describe the protocol for TOF-MS spotting on target plate.

Answer 26

1) Pipette 1 uL of ample directly on the MALDI plate
2) Add 1 uL of the UV absorbing MALDI matrix