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instruments are still important for solid phase sequencing to identify post-translational

modifications; in particular, sites of phosphorylation and a combination of micro-

sequencing and mass spectrometry techniques are now commonly employed for

complete covalent structure determination of proteins.

Example 4PEPTIDE SEQUENCING (I)

Question An oligopeptide obtained by tryptic digestion was investigated by ESI–MS and ion
trap MS–MS both in positive mode, and gave the followingm/zdata:

ESI 223.2 297.3

Ion trap 146 203 260 357 444 591 648 705 802 890

(i) Predict the sequence of the oligopeptide. Use the amino acid residual mass

values in Table 9.2.

(ii) Determine the average molecular mass.

(iii) Identify the peaks in the ESI spectrum.

Note: Trypsin cleaves on the C-terminal side of arginine and lysine.

Answer (i) The highest mass peak in the ion trap MS spectrum ism/z¼890, which

represents (MþH)þ.

HenceM¼889 Da.

m/z 146 203 260 357 444 591 648 705 802 889

D 57 57 97 87 147 57 57 97 87

aa Gly Gly Pro Ser Phe Gly Gly Pro Ser

The mass differences (D), between sequence ions, represent the amino acid (aa)

residue masses. The lowest mass sequence ion,m/z¼146, is too low for arginine and

must therefore represent LysþOH. The sequence in conventional order from the

N-terminal end would be:

Ser-Pro-Gly-Gly-Phe-Ser-Pro-Gly-Gly-Lys

(ii) The summation of the residues¼889 Da, which is a check on the mass

spectrometry value forM.

(iii) Them/zvalues in the ESI spectrum represent multiply charged species and may

be identified as follows:

m/z¼223.2(Mþ4H)^4 þfrom 889/223.2¼3.98

m/z¼297.3(Mþ3H)^3 þfrom 889/297.3¼2.99

Remember thatzmust be an integer and hence values need to be rounded to the

nearest whole number.

384 Mass spectrometric techniques