Example 6PEPTIDE MASS DETERMINATION (III)
Question An unknown peptide and an enzymatic digest of it were analysed by mass
spectrometric and chromatographic methods as follows:
(i) MALDI–TOF mass spectrometry of the peptide gave two signals atm/z¼ 3569
and 1785;
(ii) MALDI–TOF of the hydrolysate showed signals atm/z¼766, 891, 953 and 1016;
(iii) the data obtained from analysis of the peptide using coupled HPLC–MS
operating through an electrospray ionisation source werem/z¼510.7, 595.7,
714.6, 893.0 and 1190.3;
(iv) when the hydrolysate was analysed by HPLC, four distinct components could be
discerned.
Explain what information is available from these observations and determine a
molecular mass, using the amino acid residue mass values in Table 9.2, for the
unknown peptide.
Answer (i) Signals from MALDI–TOF were observed atm/z¼3569 and 1785. These data
could represent either of the following possibilities:
(a)m/z¼ 3569 (MþH)þ
whenm/z¼ 1785 (Mþ2H)^2 þ, givingM¼ 3568
(b)m/z¼ 3569 (2MþH)þ,
whenm/z¼ 1785 (MþH)þ, givingM¼ 1784
(ii) It is possible to distinguish between these two options by considering the
MALDI–TOF of the products of hydrolysis. Fourm/zvalues were obtained: 766,
891, 953 and 1016.
Each is a protonated species and the sum of these masses, 3626, will be of the order of
theMof the original peptide. The value of this sum supports option (a) in (i) above.
(iii) Electrospray ionisation data represent multiply charged ions. Using the
standard formula the meanMmay be obtained.m 1 1 m 2 m 1 n 2 m 2 1 M (Da) z
892.0 297.3 3.0003 1189.3 3568.3 3
713.6 178.4 4.0000 892.0 3568.0 4
594.7 118.9 5.0016 713.6 3569.2 5
509.7 85.0 5.9964 594.7 3566.1 6SM¼14271.6 Da
MeanM¼3567.9 Da
This more precise value confirms the conclusions found above. For an explanation of
the mass difference betweenMrand the sum of the hydrolysate products, refer to the
answer to Example 2.
The data in (iv) are confirmatory chromatographic evidence that only four
hydrolysis products were obtained.387 9.5 Structural information by tandem mass spectrometry