Biology Reference
In-Depth Information
No matter the label position, the precursor
m
/
z
values of a HP-LP
pair (isotopologues) will differ, dependent on the labeled amino
acid. In principal, it doesn't matter which amino acid is chosen, if
15
N and
13
C labeling is applied, since delta M (the mass shift) will
be at least 4 Da (for Alanine) which can easily be separated by
modern mass spectrometers. However, it is recommended choos-
ing the C-terminal amino acid of the synthetic peptide to be labeled
with
15
N and
13
C. Y-ions are predominantly used for SRM transi-
tions in QqQ, due to their abundance and intensity but b-ions may
be chosen as well. Subsequently, the product
m
/
z
values of any
Y-ion will also differ for a HP-LP pair, resulting in highly selective
transition pairs. In theory, the selection of one transition is enough.
However, the more transitions per peptide selected, the higher the
certainty. More transitions may also increase signal intensity while
improving sensitivity, but increase the duty cycle at the same time.
3.4
Label Position
It is imperative to use identical types of transitions for a HP and
its LP counterpart (The charge state of the precursor of the HP
needs to be the same as for the LP. If a singly charged Y
3
-type-ion
is chosen for the HP, the same needs to be chosen for the LP.) A
similar ion fragmentation intensity pattern has been described
comparing HCD collision with QqQ fragmentation [
7
].
However, tuning of the mass spectrometers collision energy (CE)
by direct infusion (fl ow injection) of the synthetic peptides
enables maximum sensitivity for specifi c transitions. Atleast for
some mass spectrometers a semiautomatic ramping of CE and
selection of the most intense transitions is possible [
8
]. The
occurrence of other parameters such as Declustering Potential,
S-lens, Collision Exit Potential, Ion Transfer Capillary Offset
Voltage, etc. is vendor specifi c. Fine tuning of all possible param-
eters guarantees the highest possible sensitivity of the experiment.
Y-ions N-terminal to Proline frequently result in high signal
intensities, and are therefore preferably chosen. Selecting the
most abundant fragments aims at maximum sensitivity of the
assay (
see
Note 1
). High selectivity can usually be achieved by
choosing transitions whose product
m
/
z
values are higher than
their precursor
m
/
z
value (possible due to for example precursor
charge state 2, product charge state 1). A combination of sensi-
tive and selective transitions often results in the most effective
experimental setup. In general two transitions per peptide are
suffi cient for sensitivity and selectivity since the HP internal
standard includes the retention time as additional confi dence
identifi cation parameter (
see
Note 2
).
3.5 Choice of
Transitions
3.6 Calibration
Curves
Comparing HP and LP peak areas to deduce quantitative results
(single point calibration) is not recommended due to the well known
fact that peptide ionization effi ciency varies signifi cantly (Fig.
2
).
Except for nearly identical peak areas, quantitation will not be as
