Samenvatting Les 7 'Tandem Mass spectrometry'

Les 7: 13/11
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è First one is false
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Tandem Mass spectrometry
Introduc)on
- You can determine the aminoacid sequence of the pep9de
- The first analyzer isolates precursor ion
- Precursor ion is fragmented in product ions and neutral fragments
o mpre+ -> mpro+ + mn
- Second analyser analyses product ions
- If the resolu9on of the instrument is sufficient, the first analyser selects for the highest
isotopic peak. A fragmenta9on spectrum, free of isotopic clusters, is then generated
- The mass of the precursor (protonated) will be dissociated in the mass for the product
ion and the mass of a neutral ion. Only if the pep9de has one charge. The second
analyzer analyses the product ions. If the resolu9on is sufficient the first analyzer select
for the highest isotopic peak. If the pep9de is long enough it will be a Gaussian peak of
…
Tandem MS in space and in Time
Tandem MS in Time
- See ion trap: analysis and fragmenta9on occur in the same quadrupole.

, - A precursor is selected, aNer which a fragment of this precursor is selected, aNer which
a fragment of this fragment is selected, etc.: MSn
Tandem MS in Space
- Two physically different analysers in a row.
- In between, a collision cell is posi9oned wherin ‘Collision Induced Dissocia9on’ or CID)
occurs: a precursor ion is fragmented in product ions and neutral fragments.
The 4 Major MS/MS modes of opera)on (!!!)
a. Product ion scanning. MS1 selects a specific precursor ion at a par9cular 9me point.
ANer CID, resul9ng fragments are analysed by MS2. In shotgun proteomics this proces
is preceded by an MS scan (e.g. in MS1) during which the most abundant ions are
selected as precursor ions for subsequent product ion scanning.
o Most abundant peaks are selected by a MS instrument. You can also ask to select
other peaks. In shotgun proteomics the most abundant ions are selected, gives the
best MS scan. MS1 is fixed aNer it has a done a scan.
b. Precursor ion scanning. MS2 selects for one specific ion fragment. MS1 is scanned in
order to detect all precursor ions that produce this fragment.
o It looks for one par9cular fragment that is present in the pep9de of on the pep9de.
The first one selects for many precursors pep9des. The second one checks of the
fragment is present in the pep9de
c. Neutral loss scanning. MS1 and MS2 scan in a synchronised way such that the mass
difference between MS1 and MS2 passing ions is constant. This difference corresponds
to a neutral fragment that is generated by the CID.
o MS1 lowers in its scanning process -> MS2 will do that to -> synchronized
o Scanning process in the first analyser which selects for a precursor ion -> precursor
ion is fragmented -> falls apart in a neutral fragment and in a charged fragment ->
mass of the blue pep9de will be different than the mass of the green pep9de. That
difference is bthe difference between MS1 and MS2.
o Scan all different precursor pep9des and check if they lost a certain fragment during
the CID
d. Selected Reac9on Monitoring (SRM). One precursor ion, characteris9c for a protein
(proteotypic pep9de), and one (or several) specific fragment(s) characteris9c for that
precursor, are selected by resp. MS1 and MS2 (‘a transi9on’). In a series of several SRM
experiments, different precursors can be selected (Mul9ple Reac9on Monitoring or
MRM).
o Two fixed MS analyzer. The first one is set for a specific pep9de. The MS will only
select that pep9de -> will be fragmented -> will be measured with the second
analyser (one is selected)