StageTip-Based HAMMOC, an Effi cient and Inexpensive Phosphopeptide Enrichment Method for Plant Shotgun Phosphoproteomics - Plant Proteomics: Methods and Protocols - page 575

3.2 StageTip
and Spin Adaptor
Preparation

This protocol is adapted from [ 18 ].

1. Place the Empore C8 or C18 extraction disks on a clean dis-
posable plastic petri dish. The C8 disk is for a HAMMOC
StageTip. The C18 disk is for a desalting StageTip.
2. Wet the membrane disks with methanol.
3. Punch out small disks using 90° bevelle needles with proper
inner diameters ( see Note 9 ).
4. Push the disk out of the needle and fi x it into tapering of a
pipet tip using plunger assemblies ( see Note 10 ).
5. Drill a hole in the lids of micro-centrifuge tubes and 15 ml
conical tubes, and use as spin adaptors for the 200 μ l and 1 ml

StageTips, respectively.

3.3 Desalt the
Digests with C18 1 ml
StageTip ( See Note 11 )

1. Prepare a C18 1 ml StageTip.

2. Place the C18 1 ml StageTip on a 15 ml conical tube using the
spin adaptor.
3. Add an equal volume of 2 % TFA to acidify the prepared digest
( see Note 12 ). Acidifi cation is required for peptide binding to
the C18 membrane disks.
4. Conditioning of the C18 1 ml StageTip with Solution A. Load
1 ml Solution A onto the C18 1 ml StageTip. Spin the C18
1 ml StageTip to force Solution A through.
5. Conditioning of the C18 1 ml StageTip with Solution B. Load
1 ml Solution B onto the C18 1 ml StageTip. Spin the C18
1 ml StageTip to force Solution B through.
6. Load the acidifi ed sample onto the conditioned C18 1 ml
StageTip ( see Note 13 ). Spin the C18 1 ml StageTip to bind
peptides to the C18 disks and force the rest through.
7. Washing of the C18 1 ml StageTip with Solution B. Load 1 ml
Solution B on the C18 1 ml StageTip. Spin the C18 1 ml
StageTip to force Solution B through.
8. Elution of the bound peptides from the C18 disks with
Solution A. Replace the waste conical tube with a new tube.
Load 800 l Solution A on the C18 1 ml StageTip. Spin the

C18 1 ml StageTip to elute peptides into a clean tube.

μ

9. Dry the eluent in a SpeedVac. Dried peptide sample can be
stored at −20 °C.

3.4 HAMMOC
StageTip Preparation

1. Prepare a C8 200 l StageTip ( see Note 14 ).

2. Weigh TiO 2 beads and add to the disk in the C8 200

μ

l
StageTip ( see Note 15 ). To prepare many columns, TiO 2 beads
can be suspended in methanol to load on C8 200

μ

l StageTips
( see Note 16 ). Most importantly, TiO 2 beads have to be kept
under dry conditions to keep specifi city against phosphopeptides
( see Note 2 ).

μ