Waters Reverse Peptide Kit - Manual (Page 3)

Preparing 10 ng/µl reverse peptide stock solution
1. Add 10 mL of 50:50 acetonitrile:water (with 0.1% formic acid)
to a 20 mL volumetric flask.
2. Pipette 200 µL of the 1mg/ml reconstituted SDGAG sample
into a 20-mL volumetric flask.
3. Pipette 200 µL of the reconstituted GAGAS sample into the
flask and sonicate for 5 minutes.
4. Add enough of the 50:50 acetonitrile:water diluent to the
contents of the flask to make 20 mL of 10 ng/μL reverse
peptide stock solution standard solution.
5. Label as “10 ng/µL reverse peptide stock solution” as
appropriate. Store unused aliquots in freezer at -20 °C for no
longer than 1 week.
c. Reverse Peptide Standard Solution Working Concentration
Preparing 100 pg/µL Standard Solution
Prepare 100 pg/µL reverse peptide specification sample by
diluting the10 ng/µL peptide stock solution with 50:50
acetonitrile:water (with 0.1% formic acid). Store unused aliquots
20 mL standards solution at -20 °C for no more than 1 week.
1. Pipette 200 µL of the 10-ng/µL reverse peptide stock solution
into a 20-mL volumetric flask.
2. Add enough of the 50:50 acetonitrile:water diluent to the
contents of the flask to make 20 mL of 100 pg/μL reverse
peptide standard solution.
3. Sonicate the contents of the flask for 5 minutes.
4. Label as “100 pg/µL reverse standard solution”.
5. To prepare samples for use with built-in fluidics systems,
transfer the standards solution from the 20-mL volumetric
flask into a 30 mL nalgene bottle, label as “100 pg/µL reverse
standard solution” and store at -20 °C for no longer than
1 week.
IV. MOBILIT Y MODE COMMISSIONING T EST
a. Resolution Mode (Positive Ion)
The mobility separation of the m/z 246.1 ion species in nitrogen
gives two distinct arrival time peaks from which a mobility
resolution (Ω/ΔΩ) of >36 will be demonstrated using collision
cross-section (W values of 222.7 Å
2 and 211.7 Å 2 for the
ser-asp-gly-arg-gly and gly-arg-gly-asp-ser peptides
respectively. The high mass will be set to 600 Da.
This procedure requires the sample described in Table 3:
Table 3: Setup samples
Sample Reservoir Flow rate Sprayer
100 pg/μL each of
the peptides: ser-
asp-gly-arg-gly and
gly-arg-gly-asp-ser
A
5 μL/min
(0.5 μL/min for
NanoFlow)
Sample
Figure 1. Example display of the non-storage TOF and mobility TOF windows.
The screen-shot shows expected TIC and Mobility TIC for the
reverse peptide mixture, the MS/MS set mass should be m/z
246.1. The screen-shot shows on the left, the expected TOF TIC
and on the right the expected Mobility ToF trace for the reverse
peptide mixture is shown.
Drift times for the two peptides (indicated by the two peaks)
should be as observed, tune for maximum separation using
the IMS, Transfer and Trap DC tabs, parameters required are
instrument model specific. Refer to instrument operations manual
or applications support for assistance if required.
3
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Reliant HPLC Columns