Flashcards in Biotech Final - Lecture 5: Literally any and everything you need to know about MS Deck (32)
What does the mass spec. instrument do, very generally?
The mass spectrometry instrument produce, separate, and detect the m/z ratio of ionized molecules present in the gas phase.
What is the mass accuracy of the mass spec. instrument?
High mass accuracy (less than 1 kDa)
What is the sensitivity of the mass spec.?
What are the different ionization methods available for mass spec.? (The ones we went over)
MALDI and ESI
What are the different mass analyses available for mass spec? (That we went over)
TOF and quadrupole
Mass spectrometry can be used for a variety of applications, these include?
1) Protein identification
2) Determination of multiprotein complexes
3) Determination of protein modifications
4) Structure dynamics of proteins
5) Expression proteomics
The identification of an unknown protein using MS involves what?
2) In-gel trypsin digestion
3) Sample spotting
4) MALDI-linear or MSMS
What is MALDI?
Matrix assisted laser desorption ionization.
Thus the sample is combined with a UV absorbing matrix and dried to form co-crystals. A laser then rapidly vaporizes the crystal, The matrix facilitates desorption into the gas phase and protonation of the anlyte. The desorbed ion is then accelerated out of the ion source, into the flight tube.
What are the two different modes of operation of TOF?
Linear and reflector.
Linear mode gives what type of readout?
How is a mass fingerprint analyzed?
The MALDI-TOF linear provides a mass fingerprint (set of peaks). These are analyzed by computer analysis. All known protein sequences are in a database and the program performs an in silico digest using trypsin. The calculated mass values, with input such as modification options, are compared with the observed mass spectra. Potential identification. ensues.
What are the characteristics of a TOF mass analyzer?
1) Ions are formed in pulses
2) Small ions reach the detector before large ones
3) Measures the time for ions to reach the detector
Why is a vacuum system necessary in MALDI-TOF?
High vacuum is required to avoid ion collisions.
What is the flight tube?
Field free region where ions drift at a velocity inversely proportional to the square root of their m/z ratio.
What is the reflector?
Ion mirror that subjects ions to a uniform repulsive electrical field to reflect them. This is tilted to focus ions toward the detector.
What is the collision cell?
Gas cell (inert Helium for example) for collision induced dissociation (CID) to enhance fragmentation in PSD analysis.
What is the timed ion selector?
A velocity selector that allows a single precursor ion of a selected mass and their fragment ions to pass to the detector.
What is applied to the sample plate?
An accelerating voltage.
What is PSD?
PSD refers to a method of detecting and measuring the masses of fragment ions that are formed from a selected precursor ion.
How can PSD fragmentation be increased?
Using a higher laser intensity or by using a collision cell.
PSD fragment ion velocities are the same as their precursors. Why is this?
Velocity is determined by initial acceleration, given at the ion source.
What is a timed ion selector?
A type of velocity selector that allows only selected precursor ions and their fragments to pass through to the reflector.
What are the characteristics of MALDI-TOF: linear mode? (in terms of what it measures, what is formed and what is the readout)
Measures the mass-dependent TOF.
Parent ions are produced
No sequence information
Get a mass fingerprint.
What are the basics of MS-MS fragmentation?
1) Parent or precursor ion is selected by virtue of TIS.
2) Ions begin to fragment at the peptide bond due to collision energy
3) Each individual peptide molecule will have enough energy to fragment at one peptide bond, position will vary (get different sizes)
4) The proton can reside on the N or C-terminus
What is a b-ion?
N-terminal protonated peptide
What is a y-ion?
C-terminal protonated peptide
Why is protein sequencing possible?
1) Fragmentation occurs in a predictable fashion (i.e. at peptide bonds)
2) The resulting fragment ions masses are consistent with the known molecular weights of dipeptides, tripeptides, etc...
3) Not necessary to have full ion complement for accurate sequencing
If one were to identify a protein, what steps would need to be done?
1) Detect the mass of the intact protein (SDS)
2) In gel trypsin digestion
3) MALDI-linear and in-silico computer analysis
4) MS-MS fragmentation
High throughput protein identification with LC/MS/MS entails what?
1) Start with cells and lyse
2) Obtain protein extract
3) Cleave using trypsin
4) Partial separation using HPLC
5) MALDI-TOF linear
6) MS/MS (select one peak using TIS and fragment using collision cell
7) Obtain peptide spectrum and partial sequence
8) Repeat for all peaks
9) Identify protein