Questions for MS mock exam

1. You are going to measure a mixture of peptides originating from a digested mixture of proteins
and have to decide which ionization technique to use. Available you have EI, MALDI and ESI.
Which one do you pick and why?

Answer: EI is too harsh a fragmentation technique for peptides. MALDI and ESI are would be
suitable as they are soft ionization techniques. Having a complex mixture of peptides one would
preferably pick an ESI instrument as it can be combined with a chromatographic device prior to
peptides being introduced into the MS.

2. Are there any indications in the below EI spectrum as to whether or not we are looking at an
aliphatic or aromatic compound? Explain.

Answer: First, identify the molecular ion: look in the high end of the MS spectrum. m/z 94 would
make sense as the molecular ion as we then see a logical loss of 17 (loss of an OH group, one of the
common losses, see appendix 2). EI is a harsh ionization technique. In the spectrum above the
molecular ion (m/z 94) is the most intense peak in the entire spectrum indicating a very stable
molecular ion (i.e. relatively little fragmentation). Aromatic compounds tends to show high stability,
i.e. having strong molecular on peaks. The compound is suspected to be aromatic.
This also fits with peaks 39, 65, 77, 91 which are typically seen for aromatics (see appendix 2).
3. You are going to measure the peptide “AVERA” on an ESI-MS. Calculate the b2 ion and y3 of
“AVERA” (it is written N to C terminus) for a charge state of +1.

Answer:

Using the residues mass of each peptide (see appendix 1) and:

Mass of b-ions = ∑ (residue masses) + 1 (H+)
Mass of y-ions = ∑ (residue masses) + 19 (H2O+H+)
(in the below calculation H = 1, and H2O = 18 (i.e. rounded off values))

Seq # B Y #
A 1 72.03711 545.2863 5
V 2 170.10552 474.2492 4
E 3 300.14811 375.1808 3
R 4 456.2492 246.1382 2
A 5 527.2863 90.03711 1

I.e. the answer is:

b2 = 170.10552 and y3 = 375.1808

In the above example the entire ion series was calculated for show. You just need to calculate as
much as you need to answer the question.

4. What is monoisotopic mass?

Answer: monoisotopic mass is the sum of masses of atoms in a molecule using the most abundant
isotope of each element.

5. The following EI spectrum is of an aromatic compound. Which substitution can be found on the
aromatic ring? One Br. Isotopic pattern of the intact molecule. See appendix.
6. What does the term “mass defect” describe? Explain.
Answer: In mass spectrometry, it often refers to the difference between compounds exact mass
and its nominal mass. Generally, it refers to the discrepancy between the masses of single atoms
and their molecules.

7. In ppm please calculate the mass error if Mtheo = 770.434 Da and Mexp = 770.432 Da.

Answer:

770.432 Da−770.434 Da 6
MA= ∙10 ppm=-2.6ppm
770.434 Da

8. Does the base peak always correspond to the monoisotopic ion? Explain.
Answer: No. For smaller compounds the base peak (the most intense peak) is often the
monoisotopic ion. For larger compounds, such as peptides the base peak does not necessarily
correspond to the monoisotopic peak.

9. What is the difference between base peak and molecular ion?

Answer: The base peak is the most intense ion in a mass spectrum. The molecular ion is the m/z of
the intact ionized molecule. If the molecular ion is the most intense peak in a spectrum it is also the
base peak.

10. Explain the difference between accuracy and precision in the context of mass spectrometry.
Answer: Accuracy is a measure of how well the mass spectrometer is able to hit a reference value
(systematic error), whereas precision relates to the reproducibility of the machine (random error):

11. What resolving power/resolution is required to separate m/z 50000 from m/z 50003.5?
Answer:
Using the 10% valley definition:
 M 50000
RP= = =14286
∆ M 50003.5−50000
12.
12. What is the proper nomenclature for protonated molecule in mass spectrometry?
Answer:
[M+H+]

13. A pure hypothetical peptide has been analyzed by ESI-MS in positive mode and you got the
following peak table:

m/z Intensity
501 10000
501.5 4000
502 1000

(a) Draw an appropriate mass spectrum (schematic) -- nothing special, but label the axis
(b) Label the monoisotopic peak -- first peak
(c) Define the charge – z = 2
(d) What is the mass of the peptide? – m = 1000
(e) Do you expect an odd or even number of nitrogens? – Even mass, even number of nitrogens
(f) Why do you observe more than one m/z value for this pure compound? – natural abundance of
isotopes (mainly 13C in this case)
Appendix 1
Appendix 2