Biotech Final - Lecture 5: Literally any and everything you need to know about MS

Question 1

What does the mass spec. instrument do, very generally?

Answer 1

The mass spectrometry instrument produce, separate, and detect the m/z ratio of ionized molecules present in the gas phase.

Question 2

What is the mass accuracy of the mass spec. instrument?

Answer 2

High mass accuracy (less than 1 kDa)

Question 3

What is the sensitivity of the mass spec.?

Answer 3

Subpicomole amounts.

Question 4

What are the different ionization methods available for mass spec.? (The ones we went over)

Answer 4

MALDI and ESI

Question 5

What are the different mass analyses available for mass spec? (That we went over)

Answer 5

TOF and quadrupole

Question 6

Mass spectrometry can be used for a variety of applications, these include?

Answer 6

1) Protein identification
2) Determination of multiprotein complexes
3) Determination of protein modifications
4) Structure dynamics of proteins
5) Expression proteomics

Question 7

The identification of an unknown protein using MS involves what?

Answer 7

1) SDS-PAGE
2) In-gel trypsin digestion
3) Sample spotting
4) MALDI-linear or MSMS

Question 8

What is MALDI?

Answer 8

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.

Question 9

What are the two different modes of operation of TOF?

Answer 9

Linear and reflector.

Question 10

Linear mode gives what type of readout?

Answer 10

Mass fingerprint.

Question 11

How is a mass fingerprint analyzed?

Answer 11

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.

Question 12

What are the characteristics of a TOF mass analyzer?

Answer 12

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

Question 13

Why is a vacuum system necessary in MALDI-TOF?

Answer 13

High vacuum is required to avoid ion collisions.

Question 14

What is the flight tube?

Answer 14

Field free region where ions drift at a velocity inversely proportional to the square root of their m/z ratio.

Question 15

What is the reflector?

Answer 15

Ion mirror that subjects ions to a uniform repulsive electrical field to reflect them. This is tilted to focus ions toward the detector.

Question 16

What is the collision cell?

Answer 16

Gas cell (inert Helium for example) for collision induced dissociation (CID) to enhance fragmentation in PSD analysis.

Question 17

What is the timed ion selector?

Answer 17

A velocity selector that allows a single precursor ion of a selected mass and their fragment ions to pass to the detector.

Question 18

What is applied to the sample plate?

Answer 18

An accelerating voltage.

Question 19

What is PSD?

Answer 19

PSD refers to a method of detecting and measuring the masses of fragment ions that are formed from a selected precursor ion.

Question 20

How can PSD fragmentation be increased?

Answer 20

Using a higher laser intensity or by using a collision cell.

Question 21

PSD fragment ion velocities are the same as their precursors. Why is this?

Answer 21

Velocity is determined by initial acceleration, given at the ion source.

Question 22

What is a timed ion selector?

Answer 22

A type of velocity selector that allows only selected precursor ions and their fragments to pass through to the reflector.

Question 23

What are the characteristics of MALDI-TOF: linear mode? (in terms of what it measures, what is formed and what is the readout)

Answer 23

Measures the mass-dependent TOF.
Parent ions are produced
No sequence information
Get a mass fingerprint.

Question 24

What are the basics of MS-MS fragmentation?

Answer 24

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

Question 25

What is a b-ion?

Answer 25

N-terminal protonated peptide

Question 26

What is a y-ion?

Answer 26

C-terminal protonated peptide

Question 27

Why is protein sequencing possible?

Answer 27

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

Question 28

If one were to identify a protein, what steps would need to be done?

Answer 28

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
5) Identification

Question 29

High throughput protein identification with LC/MS/MS entails what?

Answer 29

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

Question 30

How is a peptide identified using database searching?

Answer 30

Database tries to find peptide whose in silico MS/MS spectrum best matches the experimental one.

Question 31

What aids in unambiguous determination of the POI?

Answer 31

1) Number of fragment ions
2) Database parameters
3) Other aids such as mass fingerprint

Question 32

Peptide identification using a database search works best when?

Answer 32

The PUF is listed in the database.