Biology Reference
In-Depth Information
Table 2
Retention time peptides, their transition and iRT values [
29
]
Peptide sequence
Precursor ion
Fragment ions
iRT
LGGNEQVTR
2
+
y8
+
, y4
+
, y7
+
−28.31
GAGSSEPVTGLDAK
2
+
y8
+
, y6
+
, y10
+
0.23
VEATFGVDESNAK
2
+
b8
+
, y9
+
, y6
+
13.11
YILAGVENSK
2
+
y8
+
, y6
+
, y7
+
22.38
TPVISGGPYEYR
2
+
y8
+
, y9
+
, y7
+
28.99
TPVITGAPYEYR
2
+
y8
+
, y7
+
, y9
+
33.63
DGLDAASYYAPVR
2
+
y7
+
, y8
+
, y5
+
43.28
ADVTPADFSEWSK
2
+
y9
+
, y9
++
, y10
+
54.97
GTFIIDPGGVIR
2
+
y6
+
, y7
+
, y8
+
71.38
GTFIIDPAAVIR
2
+
y6
+
, y8
+
, y7
+
86.72
LFLQFGAQGSPFLK
2
+
y9
+
, y10
+
, y4
+
98.09
Technologies Ireland Ltd). RapiGest™ SF itself is still not
compatible with LC-MS, however, RapiGest™ SF can easily be
precipitated by lowering the pH of the solution to pH 2-3. In
our hands, protein extraction is more than 10 times more effi -
cient using RapiGest™ SF as compared to 6 M urea/0.1 M
ammonium bicarbonate lysis buffer alone.
5. LC-SRM-MS Consideration
Regarding the liquid chromatography gradient, a shorter gra-
dient, e.g., 35 min linear gradient from 5 to 35 % acetonitrile,
is typically used for LC-SRM-MS [
35
]. The rationale behind a
shorter gradient is that for a particular peptide the area under
the curve is constant, regardless of the length of the gradient.
Hence, a shorter gradient will result in sharper and higher
peaks versus a longer gradient with wider peaks or elution pro-
fi le (which is advantageous for data dependent shotgun MS/
MS or SWATH-MS [
36
]). As the peak height is crucial for the
signal to rise above the noise, it is advantageous to use a shorter
gradient in LC-SRM-MS.
Another consideration regarding short versus long linear
LC gradients is the fact that in scheduled SRM a retention time
window has to be defi ned. The size of the window greatly
depends on the length of the gradient, for example a 2 min win-
dow of a 30 min linear LC gradient is equivalent to a 6 min
window of a 90 min linear LC gradient [
29
]. Hence, the three
times longer gradient does not allow for more transitions per
injection to be measured and only lowers the peak intensity.
