Mass Spectrometry Flashcards
What are the main components of mass spectrometry instrumentation?
- Ionization method: MALDI, ESI
- Mass analyzers: Time of flight (TOF) and quadrupoles
- Detector
- Vacuum system
What is ESI-MS?
Electrospray ionization
What does ESI-MS produce?
Multiple charged ions directly from volatile liquid during desolvation
What is ESI-MS usually coupled to?
Liquid chromatography (online LC-MS)
What various techniques can mass analysis be accomplished by?
Biophysical (TOF, Quad, Cyclotron)
What does MALDI-TOF mean?
Matrix assisted laser desorption ionization-time of flight
What are the modes of operation for MALDI?
1) Linear mode
2) Reflector mode
What is linear mode used for?
To perform peptide mass fingerprint (no sequence information)
What is reflector mode used for?
To perform MALDI-MS/MS (sequence information
What forms co-crystals?
The mixture of a UV absorbing matric and analyte when dried
What happens when the laser rapidly vaporizes the crystal?
Matrix facilitates protonation of the analyte and the desorbed ion is accelerated out of the ion source
What are some typical matrices used?
- 4-hydroxy-alpha-cyanocinnamic acid
- 2,5-dihydroxy benzoic acid (DHB)
- Sinapinic acid
What happens in MALDI-TOF linear mode?
- 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
What is the mass equation used in MALDI-TOF linear mode?
m = (2KEt^2)/L^2
What are the titles of the x- and y- axis for a MALDI-linear mass fingerprint?
x: mass (m/z)
y: % intensity
How is a mass fingerprint analyzed?
All known protein sequences in database –> MS-Fit computer program in silicon –> calculated mass values –> mass spectrometry observed values –> list of potential proteins
What happens in MS/MS fragmentation?
- 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
What are the two ion species?
- y-ions (C-terminal ions)
- b-ions (N-terminal ions)
How do fragment ions initially travel?
At the same velocity (based on mass of original peptide ion - due to travelling in a region of high vacuum)
What does the reflector do?
Redistributes new K.E. to fragment ions leading to new velocities
What sequence information is obtained from MALDI-MS/MS?
Partial
From the position on the graph, where do y and b ions get fragmented on the amino acid sequence?
y –> from right to left
b –> from left to right
What is the general overall flow for identifying an unknown protein?
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
