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Example 2PEPTIDE MASS DETERMINATION (I)

Question A peptide metabolite and an enzyme digest of it were analysed by a combination
of mass spectrometric techniques giving the data listed below:
(i) The peptide showed two signals at 3841.5 and 1741 in the MALDI–TOF.
(ii) Five signals could be discerned when the peptide was introduced into a mass
spectrometer via an electrospray ionisation source:

m/z 498.2 581.1 697.1 871.2 1161.2

(iii) HPLC-MS of the digest indicatedfourcomponents, the (MþH)þdata for the

components beingm/z¼176, 625, 1229 and 1508. The ions corresponding to

the MS of the ‘625’ component appeared atm/z¼521, 406, 293, 130 and 113.

(iv) HPLC-MS–MS of them/z¼406 ion of the ‘625’ component identified two

ions atm/z¼378 and 336, and that of them/z¼113 ion gavem/z¼85 and 57,

in the product ion spectra.

Use the above data to compare and contrast the different ionisation methods, deduce

a molecular mass for the peptide and determine a sequence for the ‘625’ component.

Use the amino acid residue mass values in Table 9.2.

AnswerThe data in (i) arem/z¼3481.5 andm/z¼1741. These data could represent either of

the following possibilities:

(a)m/z¼3481.5(MþH)þ

whenm/z¼ 1741 (MþH)^2 þ, givingM¼3480.5

(b)m/z¼3481.5(2MþH)þ

whenm/z¼ 1741 (MþH)þ, givingM¼ 1740

Consideration of the data in (ii) allows a choice to be made between these two

alternatives, usingn 2 ¼(m 1 1)/(m 2 m 1 ) andM¼n 2 (m 2 1).

m 1  1 m 2 m 1 n 2 m 2  1 M(Da) z

870.2 290 3.0006 1160.2 3481.2 3

696.1 174.1 3.9982 870.2 3479.3 4

580.1 116 5.0000 696.1 3481.1 5

497.2 82.9 5.9975 580.1 3479.2 6

SM¼13920.8 Da

MeanM¼3480.2 Da

The meanMresult confirms set (a) of the conclusions above concerning the data

obtained from the MALDI experiments.

371 9.3 Mass analysers