Note: the structures, names, and codes of the amino acids are given in the Appendix. Tandem mass spe — Analytical Chemistry Chemistry Question
A fossilized peptide
Note: the structures, names, and codes of the amino acids are given in the Appendix.
Tandem mass spectrometry (MS-MS) provides a rapid approach for determining the sequence of polypeptides. This involves formation of a parent ion, which is then fragmented to form other smaller ions. In peptides fragmentation often occurs along the polypeptide backbone; the fragment ions are named depending on where fragmentation occurs and which atom retains the positive charge. Some of the ions formed in the fragmentation of an alanine-leucine-glycine peptide are shown below:
[VISUAL]
Fossilised bones potentially contain DNA and protein sequences that can be used to infer evolutionary links to modern species. Advances in mass spectrometry have made it possible to get sequence information from subpicomolar quantities of polypeptide, allowing analysis of material obtained from fossils. In reality, fossil polypeptide sequences typically have to be determined from mass-spectra using a combination of database searching and synthetic polypeptide standards. However for some younger fossils, where more material can be extracted, it is possible to determine the polypeptide sequence from the mass spectra once the ions have been identified.
The protein osteocalcin was extracted from a 42000 year old fossil bone found in Juniper Cave, Wyoming, USA. The MS-MS spectrum of a 19 amino acid polypeptide fragment of this protein is shown below:
[VISUAL]
ion m/z ion m/z ion m/z ion m/z
y1 175.1 b5 715.3 y8 986.5 b12 1400.7
a2 249.1 y6 726.4 b9 1069.5 y14 1508.8
y2 272.2 a6 800.4 y9 1083.5 b14 1612.7
y3 401.2 y7 823.4 b10 1140.5 a15 1681.8
a4 501.2 b6 828.4 a11 1209.6 y15 1694.9
b4 529.2 b7 885.4 y11 1267.6 y16 1831.9
y5 611.4 a8 928.4 y12 1338.7 y17 1946.9
a5 687.3 b8 956.5 y13 1395.7 b17 1951.9
Using the mass spectrum and the table of ion masses determine as far possible the sequence of this polypeptide. Where there is more than one possible amino acid at a position all possibilities should be listed. The first two amino acids in the polypeptide sequence are Tyr-Leu. The polypeptide sequence also contains the amino acid hydroxyproline, Hyp, which has a mass of 131.1:
[VISUAL]
Part of the polypeptide sequence of osteocalcin from a number of different modern species are shown below:
Carp DLTVAQLESLKEVCEANLACEHMMDVSGIIAAYTAYYGPIPY
Chicken HYAQDSGVAGAPPNPLEAQREVCELSPDCDELADQIGFQEAYRRFYGPV
Cow YLDHWLGAPAPYPDPLEPKREVCELNPDCDELADHIGFQEAYRRFYGPV
Horse YLDHWLGAPAPYPDPLEPRREVCELNPDCDELADHIGFQEAYRRFYGPV
Human YLYQWLGAPVPYPDPLEPRREVCELNPDCDELADHIGFQEAYRRFYGPV
Rabbit QLINGQGAPAPYPDPLEPKREVCELNPDCDELADQVGLQDAYQRFYGPV
Sheep YLDPGLGAPAPYPDPLEPRREVCELNPDCDELADHIGFQEAYRRFYGPV
Toad SYGNNVGQGAAVGSPLESQREVCELNPDCDELADHIGFQEAYRRFYGPV
Both hydroxyproline and proline are represented by P in the polypeptide sequences shown above.
Model Answer
The mass of ion y1 can be used to determine the identity of the last amino acid in the polypeptide. The y1 ion is one mass unit larger in size than the corresponding amino acid; therefore the last amino acid must be Arg.
The y-series of ions is the most complete, comparison of the masses of consecutive y-ions can be used to determine the sequence:
ion m/z Mass difference between b(n) and b(n-1) Corresponding amino acid Mass of amino acid
y1 175.1
y2 272.2 97.1 18 115.1
y3 401.2 129.0 17 147.0
y4
y5 611.4
y6 726.4 115.0 14 133.0
y7 823.4 97.1 13 115.1
y8 986.5 163.1 12 181.1
y9 1083.5 97.1 11 115.1
y10
y11 1267.6
y12 1338.7 71.0 8 89.0
y13 1395.7 57.0 7 75.0
y14 1508.8 113.1 6 131.1
y15 1694.9 186.1 5 204.1
y16 1831.9 137.1 4 155.1
y17 1946.9 115.0 3 133.0
From the y-series the sequence is:
Tyr-Leu-Asp-His-Trp-Leu/Ile/Hyp-Gly-Ala-xxx-xxx-Pro-Tyr-Pro-Asp-xxx-xxx-Glu -Pro-Arg
The identity of the 15th amino acid in the sequence can be determined from the difference in mass between b14 and a15:
Mr(amino acid 15) = mass(a15) – mass(a14) + Mr(C) + 2 Mr(O) + 2 Mr(H) = 115.0
Therefore amino acid 15 must be Pro.
The difference in mass between ions y3 and y5 gives the mass of the fragment corresponding to amino acids 15 and 16.
Mr(15-16 dipeptide) = mass(y5) – mass(y3) + Mr(H2O)
Mr(amino acid 16) = Mr(15-16 dipeptide) – Mr(amino acid 15) + Mr(H2O) = 131.1
Amino acid 16 must therefore be Ile, Leu or Hyp.
The mass of amino acid 10 can be determined from the difference in mass between b9 and b10:
Mr(amino acid 10) = mass(b10) – mass(b9) + Mr(H2O)
Amino acid 10 is Ala.
The difference in mass between ions y11 and y9 gives the mass of the fragment corresponding to amino acids 9 and 10.
Mr(9-10 dipeptide) = mass(y11) – massr(y9) + Mr(H2O)
Mr(amino acid 9) = Mr(9-10 dipeptide) – Mr(amino acid 10) + Mr(H2O)
Amino acid 9 has a mass of 131.1, so must be Ile, Leu or Hyp.
The sequence of the polypeptide is therefore:
Tyr-Leu-Asp-His-Trp-Leu/Ile/Hyp-Gly-Ala-Leu/Ile/Hyp-Ala-Pro-Tyr-Pro- Asp-Pro-Leu/Ile/Hyp-Glu-Pro-Arg
To which modern species does the protein from the fossil appear to be most closely related?
Model Answer
The sequence is most similar to that of the horse.