Analytical Techniques In Biochemistry-Mass Spectrometry

Front 1

What does mass spectrometry detect? This allows it to measure what?

Back 1

Detects: charged ions in gas phase

Measures: mass to charge ration (m/z)

Front 2

The potential mass spectrometry methods can be:

EI, CI, FAB, ESMS or MALDI.

What does each acronym stand for?

Back 2

EI: Electron Impact

CI: Chemical Ionisation

FAB: Fast Atom Bombardment

ESMS: ElectroSpray Mass Spectrometry

MALDI: Matrix Assisted Desorption/Ionisation

Front 3

What 2 mass spectronomy methods are most common?

Back 3

ESMS and MALDI

Front 4

ESMS and MALDI allow you to detect proteins, nuceli acids and post translational modifications. State 4 other uses of mass spec? (What else can it detect?)

Back 4

Answer can include any 4 of the following:

Protein sequencing, Modifying nucleic acids, Enzyme mechanics, Protein folding, Proteomics and Non-covalent assemblies

Front 5

There are 4 main steps involved in the process of ionisation for mass spectrometry.

Step 1 forms a fine spray of droplets. What property does these droplets have? How is this achieved?

Back 5

High surface charge densities, due to high potential on capillary needle

Front 6

Step 2 involves the evaporation of carrier solvent. What does this do to the droplets?

Back 6

They shrink, charge density on surface increases

Front 7

Step 3 involves explosive fragmentation. This occurs as the droplets reach what? What change occurs at this point?

Back 7

Raleigh limit, droplet surface charge density exceeds surface tension

Front 8

In step 4, desolvation continues. What does this form?

Back 8

Gas phase ions, either +ve or -ve.

Front 9

Because ESMS and MALDI can produce multiple charged ions on a single protein, the m/z ratio will be higher. What range is the m/z raio typically in for MALDI and ESMS?

Back 9

500-2,000 m/z

Front 10

For ESMS, masses up to what can be observed?

Back 10

150,000 Da

Front 11

State 2 advantages of using ESMS?

Back 11

Accurate: 1 Da in 10,000 (0.01%)

Doesn't require a strong vacuum

Front 12

State one form of ESMS?

Back 12

Fourier Transform Ion Cyclotron Resonance

(FTICR)

Front 13

What does FTICR produce?

Back 13

Deconvolution allows a Guassian distribution (average isotropic mass)

Front 14

State 2 advantages of using FTICR?

Back 14

Very sensitive and a very high resolving power

Front 15

For larger proteins, what form of MS is used?

Back 15

Matrix-Assisted Laser Desorption Ionisation (MALDI)

Front 16

There are 5 main steps involved in detection using MALDI. State step 1.

Back 16

Sample mixed with crystalline matrix and irradiated with a laser

Front 17

State step 2 involved in detection using MALDI.

Back 17

Matrix absorbs energy from laser, sample is volatised through pulses

Front 18

State step 3 involved in detection using MALDI.

Back 18

Sample molecules pick up protons from the matrix

Front 19

In step 4, an electric field accelerates the ions. For step 5, a high m/z is produced, and so what is measured? Why is this measured?

Back 19

Time of flight (TOF), all ions have same KE (=1/2mv^2), heavier ions move more slowly

Front 20

What data would be seen from a MALDI detector?

Back 20

1 peak per component

Front 21

State 2 uses of MALDI.

Back 21

Rapid ientification of a protein of a known sequence

Proteomics

Front 22

For protein sequencing using MS, there are 3 steps involved. State step 1.

Back 22

Use precision enzymes or chemicals to produce mixture of peptides broken at known locations.

Front 23

Step 2 is analysis using MS. What 2 forms of analysis can be used?

Back 23

ESMS coupled to HPLC

Directly via MALDI: provide MW and 15 a.a. in on each fragment

Front 24

State step 3 involved when sequencing a protein using MS.

Back 24

Sequencing: Compare massses of fragments with those predicted from genome

Front 25

What enzyme cuts on C terminal of lysine or arginine?

Back 25

Trypsin

Front 26

What enzyme cuts on C terminal of Phenylalanine, Tryptophan or Tyrosine?

Back 26

Chymotrypsin

Front 27

What enzyme cuts on C terminal of glutamate?

Back 27

V8 protease

Front 28

What chemical cuts on C terimal of methionine?

Back 28

Cyanogen Bromide

Front 29

What chemical cuts on N terminal of cysteine?

Back 29

2-Nitro-5-Thio-Benzoic Acid

Front 30

Of the Post Translational Modifications, what mediates phosphorylation?

Where can the OH group come from?

Back 30

Kinases,

OH group comes from serine, threonine or tyrosine

Front 31

What mass shift is seen with phosphorylation?

Back 31

+80 Da

Front 32

What does lysine acetylation control?

Back 32

Histone binding to DNA

Front 33

What mass shift is seen with lysine acetylation?

Back 33

+42 Da

Front 34

N-linked glycosylation. Oligosaccharides are attached to asparagine through what?

Back 34

N-acetylglucosamine

Front 35

What mass shift is seen through N-linked glycosylation?

Back 35

+203 Da

Front 36

Cell surface O-linked glycosylation only occurs at 2 amino acids. Name both.

Back 36

Serine and Threonine

Front 37

What mass shift is seen with cell surface O-linked glycosylation.

Back 37

+203 Da

Front 38

What mass shift is seen with reversable O-GlcNAc-ylation?

Back 38

+203 Da

Front 39

On what amino acid can lipidation occur?

Back 39

Glycine

Front 40

Lipidation involves the process of what?

Back 40

Myristoylation

Front 41

What mass shift is seen from lipidation?

Back 41

+210 Da

Front 42

Where can farnestylation occur?

What is removed and produced?

Back 42

Cysteine, 4 amino acids from C terminal

Last 3 amino acids removed, forms a methyl ester

Front 43

What mass shift is seen with farnestylation?

Back 43

+235 Da - 3 amino acids

Front 44

If you encounter a brand new protein, there are 3 steps involved. State step 1 and 2.

Back 44

Step 1: Trasitional sequencing using fluorophores

Step 2: Plan digest enzymes

Front 45

Step 3 involves building a database. What information is included for this database?

Back 45

Mass of peptide frangments and the sequence of 15 amino acids in on each fragment

Front 46

State 2 limitations of using MS as a form of protein sequencing?

Back 46

Only see 15 amino acids in from terminus

Can't distinguish between leucine and isoleucine

Front 47

For protein-ligand binding, what type of binding is needed for it to be sequenced?

Back 47

Covalent

Front 48

If a non-covalent bond between protein and ligand, what can be detected in the ligand?

Back 48

Bound metals

Front 49

The MS of DNA can be used to observe?

Back 49

ssDNA, ds DNA and Quadruplex DNA

Front 50

State 2 Considerations when using MS to observe DNA?

Back 50

Answer can include any 2 of the following:

Hydrogen bonds don't survive ionisation

Every phosphate group on ladder picks up metal in buffer, e.g. NaCl

Ion peaks of ssDNA seen instead of dsDNA, carry out at -50 to -15 degrees (cold spray)

Front 51

For ESMS and MALDI, compare the:

Ionisation

Mass Range

Back 51

Ionisation

ESMS: continuous

MALDI: Pulsed

Mass Range

ESMS: 1,000,000+Da

MALDI: 1,000,000+Da (70,000+ MALDI is better)

Front 52

For ESMS and MALDI, compare the:

Detection limit

Accuracy

Sensitivity to contamination

Back 52

Detection limit

ESMS: 10^-15 to 10^-21

MALDI: 10^-15 to 10^-18

Accuracy

ESMS: High MALDI: Not as good

Sensitivity to contamination

ESMS: Reduces sensitivity, must be salt free

MALDI: Reasonably tolerant (mM of salt tolerated)

Front 53

For ESMS and MALDI, compare the:

Spectral simplicity

Coupling with online separation

Noncovalent interactions

Back 53

Spectral simplicity

ESMS: Gaussian distribution

MALDI: Single peak

Coupling with online separation

ESMS: HPLC MALDI: Not needed

Noncovalent interactions

ESMS: Yes MALDI: No

Front 54

Which of the 2, ESMS or MALDI is used for tandem sequencing of proteins?

Back 54

ESMS

Front 55

What is tandem MS?

Back 55

Involves multiple steps of MS slection, with some form of fragmentation occuring in between