9780521516358book.pdf

The reflector

Areflector(orreflectron) is a type ofion mirrorthat provides higher resolution

in MALDI–TOF. The reflector increases the overall path length for an ion and it corrects

for minor variation in the energy spread of ions of the same mass. Both effects improve

resolution. The device has a gradient electric field and the depth to which ions will

penetrate this field, before reversal of direction of travel, depends upon their energy.

Higher-energy ions will travel further and lower-energy ions a shorter distance.

The flight times thus become focussed, while neutral fragments are unaffected by

the deflection. Figure 9.16 shows a diagrammatic representation of a MALDI–TOF

Example 2(cont.)

The data in (iii) indicate that four products arise from the enzymatic digest of the

original peptide. As these products arise directly from the original, the sum of these

masses will be related to theMof the pepide.

Therefore

176 þ 625 þ 1229 þ 1508 ¼3538 Da

The difference between this mass and the M determined above is

3538  3480 : 2 ¼ 57 : 8 58 Da

The difference of 58 mass units is explained as follows.

Each of the enzyme digest products is protonated (to be ‘seen’ in the mass

spectrometer). Hence this accounts for 4 units. The remaining 54 unit increase arises

from the enzymic hydrolysis. From a linear peptide, four products arise from three

cleavage points (three cuts in a piece of string give four pieces). Each cleavage point

requires the input of one water molecule (hydrolysis, H 2 O¼18). Three cleavage

points require 3 18 ¼54.

Them/z¼625, (MþH)þ, peak was subjected to further mass spectrometry and

sequence ions were observed.

m/z 624 521 406 293 130 113

D 103 115 113 163 17

aa Cys Asp Ile/Leu Tyr Ile/Leu

The loss of 113 from them/z¼406 ion indicates either Ile or Leu. MS2 shows

consecutive losses of 28 (CO) and 42 (CH 2 ¼CH¼CH 3 ) which is indicative of Leu.

The loss of 17 (not a sequence ion) from 130 confirms this as the C-terminal

amino acid.

The predicted sequence from the N-terminal end is

Cys - Asp - Leu - Tyr - Ile

372 Mass spectrometric techniques