Question 1 – Electrospray ionization (10 marks total)Tick all those that apply.
o Electrospray ionization uses an electron-emitting filament to ionize molecules
o Electrospray ions are generated in vacuum
o The number of charges of an analyte relates to its molecular surface area
o The ionization state of a protein in electrospray depends on the isoelectric point
o Electrospray ionization can be used to study noncovalent interactions
o Ions can be made in either positive or negative ionization mode
o Electrospray spectra of intact protein complexes typically show isotopes resolved
o Non-aqueous solutions can also be used in electrospray ionization
o Electrospray ions are always multiply charged
o Protein sequence mutations can usually be detected in electrospray ionization
Question 2 – Erythromycin (5 marks total)
Erythromycin is an antibiotic. Its spectrum and structure are shown here:
a. Looking at the structure which ionisation mode, positive or negative, would you use
to produce the mass spectrum and why? (2)
b. What charge state do the ions have in the spectrum shown here? (1)
c. What is the monoisotopic mass of the erythromycin molecule (show your working)?
(2)
Question 3 – Cytochrome C digest (5 marks total)
The spectrum below is from a mixture of peptides generated from a digest of cytochrome c.
a. Determine the charge states of the signals labelled 1 to 3. (3)
b. The group of signals A are slightly unusual, by looking at the spacing between the
signals, what can you say about them? (2)
Question 4 – tandem MS (10 marks total)
Tick all those that apply.
o One of the mass analyzers in tandem MS (or MS/MS, MS ) must be a quadrupole
o The peptide bond is preferentially broken in collision-induced dissociation
o ‘b’ type ions contain the N terminus of the peptide sequence
o Amino acid sequencing requires that proteins are digested first into peptides
o Collisional activation (CID) of analyte molecules can be compared to heating them
o All 20 natural amino acids are uniquely identifiable by their mass
o The type of post-translational modification (PTM) but not its position within a peptide
2
can be determined by tandem MS
o Tandem MS can also be applied to sequencing of oligonucleotides and carbohydrates
o In electron transfer dissociation (ETD), the sum of the fragment ion charges is lower than
the precursor ion charge
o It is not possible to distinguish phosphorylation and sulfation of an amino acid by MS
Question 5 – peptide fragmentation (10 marks total)
This is a tandem MS of a tryptic peptide. You saw the MS spectrum of this peptide in the
lectures. The mass of the intact peptide is 1569.692 Da.
a. There are two immonium ions labelled with their mass-to-charge ratio in the
spectrum. Label the signals with their single-letter amino acid abbreviation. (2)
b. The other peaks that are labelled with their m/z are y-ions. These fragments all
contain the C-terminus of the peptide. Using these m/z values and the residue
masses in the table provided, determine as much of the sequence as you can. Write
out the amino acid sequence from the N- to the C-terminus. (5)
c. (i) Calculate the difference in mass of the intact peptide ([M+H] + = m/z 1570.68) and
the y-ion with the highest m/z. (1)
(ii) What are the final two amino acids in this sequence? (2)
Question 6 – proteins
(10 marks total)
(i) The mass spectra to
the right show two
proteins. One is the
wild type version of a
protein and the other
is a mutant where
threonine 209 has
been replaced by
another amino acid.
By determining the
mass of each protein
and using the amino
acid residue mass
table, which amino
acid has the threonine
been replaced by? (6)
(ii) Are the masses
that you have
calculated the monoisotopic or the average
mass? Explain your
answer. (4)
Question 7 – proteins (10 marks total)
Fructose bisphosphate (FBP) aldolase is an enzyme that catalyses the formation of FBP from
glyceraldehyde-3-bisphosphate and dihydroxyacetone phosphate (DHAP).
The Class I FBP aldolase found in E. coli amongst other bacteria uses an active site lysine to
form a Schiff’s base with DHAP as part of the reaction mechanism. MS has been used to
confirm the amino acid sequence of the FBP aldolase and to identify the position of the
active site lysine.
a. The calculated mass of the FBP aldolase is 37,964.3 Da. The mass measured by mass
spectrometry was found to be 37,979.3 Da. DNA sequencing of the cloned gene
revealed 2 changes to the reported sequence: Val191 was replaced by Leu and
Ile308 was replaced by Asn. Do the MS data confirm these changes? Explain your
answer. (3)
Class I FBP aldolases are characterised by the
formation of a Schiff’s base intermediate with
dihydroxyacetone phosphate (DHAP). This
involves the reaction of an amine with a
carbonyl to give a hydroxylamine which
condenses to the imine, or Schiff’s base.
To trap the Schiff’s base intermediate,
it was reduced with sodium borohydride.
b. Calculate the expected, overall mass
difference of the derivative product
after sodium borohydride reduction.
(The theoretical, average atomic
masses are: C = 12.0110, H = 1.0079,
O = 15.9994, P = 30.9738). Compare
the final structure at the bottom of
the scheme with the initial protein
structure (top left). (3)
The modified Class I aldolase was digested with a protease to give a mixture of peptides
which were purified by HPLC. Mass measurements of the HPLC fractions indicated that the
peptide containing residues 194 to 239 of the aldolase contained the expected modified
lysine:
194- VT VLWAYLRNSA FKKDGVDYHV SADLTGOANH LAATIGADIV KQKM -239
This fragment has 4 lysine residues. Lysines 207 and 208 were ruled out as being derivatised
by using amino acid sequencing from the N-terminus. The N-terminal sequencing did not get
as far as the other two lysines. Two mutants were created, one with K236A and the other
with K238A, and their molecular masses were confirmed by MS. Both were treated
independently with DHAP/NaHBH4, with subsequent digestion and mass measurement of
the same peptide. The MS data for the two mutants together with the wild-type aldolase
are summarised in the Table:
Aldolase
Calculated mass
Measured mass
Mass after DHAP/NaBH4 treatment
Wild Type 37978.2
37979.3
38134.3
K236A
37921.1
37923.8
37921.9
K238A
37921.1
37923.5
38076.3
c. From the table above, which aldolase mutant reacts with DHAP? (1)
d. Which lysine is responsible for enzyme activity, K236 or K238? (1)
e. Suggest another method of determining which lysine is the active site in the protein.
Include some mass spectrometry in your analysis. (2)
Question 8 – structural proteomics (20 marks total)
You are involved in a project to develop a new antibody drug against SARS-CoV-2 infection.
For this to be effective, the antibody needs to bind at or near the SARS-CoV-2 spike which is
key for infection of host cells.
Suggest a method based on mass spectrometry that can be used to determine the binding
site of this novel antibody drug to the spike protein.
Explain your choice of method and its specific advantages and disadvantages for this task,
in no more than 5 sentences (5).
Explain the principle of your chosen method and what type of information it provides,
in no more than 5 sentences (5).
Explain how you would use this method here, and include a brief outline of some key steps,
in no more than 5 sentences (5).
You can include up to 2 figures and legends here; they can be made by you but if they are
taken from literature or the internet, they must be correctly referenced (5).
(80 total)
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