Waters MassPREP Phosphopeptide Enrichment Kit - Manual (Page 3)

[ Care and Use ManUal ]
IV. PhoSPhoPEPtIdE EnrIchMEnt KIt uSagE guIdElInES
Part A - How to Use the MassPREP ™ Phosphopeptide
Enrichment µElution Plate to Condition, Bind, Wash and
Elute the Phosphopeptides.
1. Place MassPREP ™ Phosphopeptide Enrichment µElution plate on a
vacuum manifold.
2. Wash the wells using 200 µL of Condition Solution I. (vacuum setting:
12 inch Hg).
3. Wash the wells using 200 µL of Condition Solution II. (vacuum setting:
0.5 inch Hg).
4. Prepare the sample by constituting or mixing it with the Loading
Solution. The final volume of the sample should be between 200
to 500 µL.
5. To obtain maximum recovery, use gravity flow, which means vacuum
is turned off. A total of 15 minutes is needed to completely flow
200 µL of the loading solution through a well. Vacuum can be turned
on briefly at the end to force residue liquid through the well.
User can also load the sample directly on the well and turn on the
vacuum manifold to remove the non-phosphorylated peptides. (vacuum
setting: 0.5 inch Hg)
The loading capacity for each well is ~ 100μg of protein digests.
Samples greater than this amount must be split into multiple wells.
6. Wash the well with 200 µL of Wash Solution I, repeat (vacuum setting:
0.5 inch Hg).
7. Wash the well with 200 µL of Wash Solution II (vacuum setting:
7 inch Hg).
8. Elute the phosphopeptides with 100-400 μL of Elution Solution,
repeat. Combine the two elutions (vacuum setting: 7 inch Hg).
9. Neutralize the eluent with pure formic acid
10. Lyophilize the eluent and reconstitute the pellet with a desirable solvent.
Part B - How to Use the MassPREP ™ Enolase/Phosphopeptide
Standard as a Test Sample
1. Solubilize 1 nmol Enolase/Phosphopeptide standard in 1 mL Milli-Q ®
water to a final concentration of 1 pmol/µL.
2. Transfer 10 µL of the 1 pmol/ µL standard to 200 µL of the loading
solution: 0.2-0.5% TFA in 80% acetonitrile. Store the remaining
standards at 4 ˚C.
3. Proceed with the MassPREP ™ Phosphopeptide Enrichment µElution
protocol to enrich the phosphopeptides (Part A).
4. Reconstitute the lyophilized pellet with 20 µL of Milli-Q ® water.
5. Proceed with capillary or nano-LC-MS analysis (see Figure 1).
Figure 1. NanoLC/MS analysis of: A) Control sample (before affinity
enrichment) that is made of 1:1 MassPREP
™ Enolase/ Phosphopeptide
Standard. B) The four phosphopeptides are selectively enriched using the
affinity SPE without using enhancer. The phosphopeptides are marked
with symbols T43_1P and T43_2P are observed coeluting. Total sample
injected on nanoLC column was about 500 fmol. A trap column is used
to remove the excess EDTA in the sample.