Waters Biopharmaceutical Training Kit - Manual (Page 4)

4
[ CARE AND USE MANUAL ]
Biopharmaceutical Training Kit
2.5 pmol/µL Optional Analyte Enolase digest (Comparative)
sample preparation:
1. Using a 200 µL pipette, transfer remaining 100 µL
of Enolase digest to a Waters Max Recovery vial.
2. Using a 20 µL pipette, add 10 µL of 3% hydrogen
peroxide solution.
3. Vortex.
4. Incubate at 40 °C for 30 minutes.
5. Quench after 30 minutes by using a 200 µL pipette
to add 80 µL of 0.2% formic acid to the vial.
6. Using a 20 µL pipette, add 10 µL of LC/MS grade
acetonitrile to the vial.
7. Vortex. Final concentration is 2.5 pmol/µL.
8. Label as “Analyte Enolase 2.5 pmol/µL.
9. Inject 5-10 µL for UPLC/MS
e
peptide map analysis,
depending on instrument sensitivity.
f. Reverse Peptide Mix (100 pg/µL):
186006921-5 1 vial
Reverse Peptide
Ser-Asp-Gly-Arg-Gly
1.0 mg
of each
4 mL Glass
Amber Bottle
with Screw Top
186006921-6 1 vial
Reverse Peptide
Gly-Arg-Gly-Asp-Ser
1.0 mg
of each
4 mL Glass
Amber Bottle
with Screw Top
290002010 2
30 mL Nalgene
fludics bottle
(with cap)
Empty
30 mL Nalgene
Fludics Bottle
(with cap)
1. Using a 1,000 µL pipette, add 1,000 µL of 50:50
water:acetonitrile + 0.1% formic acid to contents of 1 mg
Ser-Asp-Gly-Arg-Gly (186006921-7) in a 4 mL amber bottle.
2. Vortex and sonicate vial for 5 minutes.
3. Using a 1,000 µL pippette, add 1,000 µL of 50:50 water:
acetonitrile + 0.1% formic acid to contents of 1 mg Gly-
Arg-Gly-Asp-Ser (186006921-8) in a 4 mL amber bottle.
4. Vortex and sonicate vial for 5 minues.
5. Using a 200 µL pipette , transfer 200 µL of Ser-Asp-Gly-Arg-
Gly 1 mg/mL solution into one of the empty, clean 30 mL
Nalgene bottles provided in the kit.
6. Using a 200 µL pippette, transfer 200 µL of Gly-Arg-Gly-Asp-
Ser 1 mg/mL solution directly into the same 30 mL Nalgene bottle.
7. Using a 5,000 µL pipette, add 19.600 mL of 50:50
water:acetonitrile + 0.1% formic acid to the bottle.
Note: Add both peptide solution aliquots directly to the 30 mL
bottle, then add 19.600 mL 50:50 water:acetonitrile + 0.1% formic
acid to the 30 mL bottle, using a 5,000 µL pipette (eg. Add 3 x
5,000 µL aliquots, followed by a single 4,600 µL aliquot of diluent).
8. Vortex and sonicate for 5 minutes. Label this solution as
“10 ng/µL reverse peptide” standard.
9. Using a 200 µL pipette, transfer 200 µL of the 10 ng/µL
reverse peptide solution into one of the empty clean 30 mL
Nalgene bottles provided in the kit.
10. Pipette 19.800 mL of 50:50 water:acetonitrile + 0.1% formic
acid to the bottle.
11. Vortex and sonicate for 5 minutes.
12. Label this solution as “100 pg/µL reverse peptide” standard.
III. ADDITIONAL RESOURC ES
Additional literature for some of the products found in the kit can
be located on the waters website at www.waters.com.
Table 1. Kit Components
Description Part Number
MassPREP Enolase Digestion Standard
Care and Use Manual
715001598
Intact mAb Mass Check Standard Care
and Use Manual
720004420EN
Reverse Peptide Kit Care and Use Manual 720004421en