Mass spectrometry - Exercises
Exercise 1
Determine the isotopic profile (nominal mass / exact mass / relative abundance of the following compound:
a) CH2 = CH - Cl b) CH2BrCl
Nb: one can admit that the relative abundance of the isotope 2H is negligible.
Element Symbol Nominal mass Exact mass Natural Abundance (%)
Average mass
Hydrogen 1H 1 1,0078 99,989
1,0079
2H 2 2,0141 0,115
Carbone 12C 12 12 98,93
12,0107
13C 13 13,0034 1,07
Oxygen 16O 16 15,9949 99,757
15,9994
17O 17 16,9991 0,038
18O 18 17,9992 0,205
Nitrogen 14N 14 14,0031 99,632
14,0067
15N 15 15,0001 0,368
Bromide 79Br 79 78,9183 50,69
79,904
81Br 81 80,9162 49,31
Chloride 35Cl 35 34,9688 75,78
35,453
37Cl 37 36,9659 24,22
Exercise 2
Calculate the instrumental resolution required in order to distinguish the molecular ions corresponding to the following compounds (use the average masses):
a) CO et C2H4
b) C10H21CHO et C12H26
Exercise 3
The atrazine is a pesticide belonging to the herbicide category. Its use has been forbidden in the European Union since 2003, however it is still in use in various countries such as the US.
Atrazine can be analysed using either gas chromatography coupled to mass spectrometry (GC-MS) or liquid chromatography coupled to mass spectrometry (LC-MS).
Atrazine
(2-Chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine)
A sample containing traces of atrazine was analysed independently using the following conditions:
Conditions 1:
Instrument GC-MS Hewlett-Packard 5890 Series II
Column DB-1701, 30 m × 0.25 mm id, 0.15 μm film thickness Carrier gas : Helium (5 mL/min)
MS analysis : EI-MS
Conditions 2:
Instrument HPLC-MS/MS Perkin Elmer Series 200 – API 2000 MS Column Hypersil BDS C18, (250 × 2.1 mm, 5µm)
Phase mobile A: MeOH – H2O (10 : 90) 0.1% acetic acid Phase mobile B: MeOH + 0.1% acetic acid
MS/MS analysis : ESI-MS/MS (fragmentation CID ; triple quadripole)
a) For each conditions, define the type of ions generated during the ionisation process to atrazine and the m/z ratio corresponding
b) For the LC-MS/MS analysis, the signal measured is demonstration a m/z ratio of 216.663. What is the mass accuracy obtained? Is it in agreement with the type of instrument employed.
Exercise 4
We want to confirm the identification of a protein sample using high resolution MS. Therefore, the following MS spectra was obtained by direct infusion of the sample using the following conditions:
- Sample concentration 1 µM (in H2O / ACN ; 0.1% FA) - Sample direct infusion 5 µL/min
- MS instrument : Bruker Maxis 4G (ionisation source ESI – QTOF mass analyser)
a) Attribute the charge states exhibited in the MS spectrum and calculate the mass of the protein contained in the sample
b) Attribute the identification of the protein using the mass calculated previously and deduce the mass accuracy obtained (in Da)
Cytochrome C (M = 11 749 Da) ; Myoglobine (M = 16 950 Da) ; Infliximab (M = 144 190 Da) ; Albumine (M = 66 469 Da)
Exercise 5
Here is the MS/MS spectrum obtained from the LC-MS/MS analysis of a peptide mixture. During the experiments, collision induced dissociation (CID) was performed to generate the fragmentation.
The precursor ion was measured at 542.7752 demonstration a charge state (2+)
Using the y ions series, determine the amino acid sequence of this peptide
1-letter code
3-letter code
Chemical
formula Monoisotopic Average
A Ala C3H5ON 71.03711 71.0788 R Arg C6H12ON4 156.10111 156.1875 N Asn C4H6O2N2 114.04293 114.1038 D Asp C4H5O3N 115.02694 115.0886 C Cys C3H5ONS 103.00919 103.1388 E Glu C5H7O3N 129.04259 129.1155 Q Gln C5H8O2N2 128.05858 128.1307 G Gly C2H3ON 57.02146 57.0519 H His C6H7ON3 137.05891 137.1411
I Ile C6H11ON 113.08406 113.1594 L Leu C6H11ON 113.08406 113.1594 K Lys C6H12ON2 128.09496 128.1741 M Met C5H9ONS 131.04049 131.1926 F Phe C9H9ON 147.06841 147.1766 P Pro C5H7ON 97.05276 97.1167 S Ser C3H5O2N 87.03203 87.0782 T Thr C4H7O2N 101.04768 101.1051 W Trp C11H10ON2 186.07931 186.2132 Y Tyr C9H9O2N 163.06333 163.1760 V Val C5H9ON 99.06841 99.1326
