Strategies and Methods for Proteome Analysis
Fig. 1.6. CD3-~ peptide
(SAETAANLQDPNQLp YNELNLGR) taken from spot
3 in Fig. 1.4 was subjected to
!lLC A) in source cm where
VAPICID = -40V and B) tandem MS as described in Fig.
1.5. In both cases 800 fmoles
or 10 % of spot 3 was used
Q)
U
C
(U
-0
c
::::l
.D
«
Q)
-~
ro
C5
0:::
Q)
U
c:::
(U
'D
c
::::l
.D
«
Q)
>
ro
Q)
0:::
A
b4 y5
500
b2
200
700
900
(yS)2+
1100
(y11)2+
400
600
m/z
11
y12
2+ Ion
13)0
1:00
800
1000
ficient sample is available in-source cm is a route for MS/MS/MS experiments on
a triple quadrupole mass spectrometer. This would be necessary if the cm spectrum did not lead to unambiguous assignment of the phosphorylation site. A
given fragment ion produced by in-source CID is selected by quadrupole one for
cm in quadrupole 2. A MS/MS/MS spectrum is produced of ions that formed
during in-source cm, were selected by Ql and fragmented in Q2.
In the absence of a radiolabel diagnostic ions generated by in-source cm can
be monitored to identify phosphopeptides eluting from HPLC columns (Carr et
al. 1996; Huddelston et al. 1993; Hunter and Games 1994). For phosphopeptides
these diagnostic ions are HZP04- (97u) which results from beta-elimination,
HZP03- (79u) and P03- (63u). When in-source fragmentation (Cm) is combined
with on-line HPLC a chromatographic trace identifying where a phosphopeptide
eluted is produced.
An example of how a diagnostic scan can be useful to identify phosphopeptides in peptide mixtures is shown in Fig. l.7. After SDS-PAGE of whole yeast lysate
a 32 kDa silver stained band was excised and digested with trypsin. To this was
added one picomole of phosphorylated angiotensin II (DRApYIHPF). The mixture was pressure injected on a 50!-tm id capillary column packed with CIS
beads and eluted with a linear gradient of acetonitrile. The triple quadrupole
mass spectrometer was set up in negative ion mode and peptides fragmented by
in-source CID (Hunter and Games 1994). The mass spectrometer was programmed to automatically cycle between three scan types, which collectively
detected the phosphopeptides eluting from the HPLC column and determined
Fig. 1.6. CD3-~ peptide
(SAETAANLQDPNQLp YNELNLGR) taken from spot
3 in Fig. 1.4 was subjected to
!lLC A) in source cm where
VAPICID = -40V and B) tandem MS as described in Fig.
1.5. In both cases 800 fmoles
or 10 % of spot 3 was used
Q)
U
C
(U
-0
c
::::l
.D
«
Q)
-~
ro
C5
0:::
Q)
U
c:::
(U
'D
c
::::l
.D
«
Q)
>
ro
Q)
0:::
A
b4 y5
500
b2
200
700
900
(yS)2+
1100
(y11)2+
400
600
m/z
11
y12
2+ Ion
13)0
1:00
800
1000
ficient sample is available in-source cm is a route for MS/MS/MS experiments on
a triple quadrupole mass spectrometer. This would be necessary if the cm spectrum did not lead to unambiguous assignment of the phosphorylation site. A
given fragment ion produced by in-source CID is selected by quadrupole one for
cm in quadrupole 2. A MS/MS/MS spectrum is produced of ions that formed
during in-source cm, were selected by Ql and fragmented in Q2.
In the absence of a radiolabel diagnostic ions generated by in-source cm can
be monitored to identify phosphopeptides eluting from HPLC columns (Carr et
al. 1996; Huddelston et al. 1993; Hunter and Games 1994). For phosphopeptides
these diagnostic ions are HZP04- (97u) which results from beta-elimination,
HZP03- (79u) and P03- (63u). When in-source fragmentation (Cm) is combined
with on-line HPLC a chromatographic trace identifying where a phosphopeptide
eluted is produced.
An example of how a diagnostic scan can be useful to identify phosphopeptides in peptide mixtures is shown in Fig. l.7. After SDS-PAGE of whole yeast lysate
a 32 kDa silver stained band was excised and digested with trypsin. To this was
added one picomole of phosphorylated angiotensin II (DRApYIHPF). The mixture was pressure injected on a 50!-tm id capillary column packed with CIS
beads and eluted with a linear gradient of acetonitrile. The triple quadrupole
mass spectrometer was set up in negative ion mode and peptides fragmented by
in-source CID (Hunter and Games 1994). The mass spectrometer was programmed to automatically cycle between three scan types, which collectively
detected the phosphopeptides eluting from the HPLC column and determined