Biology Reference
In-Depth Information
turing conditions greatly increases effi ciency of the reaction,
and can inactivate unwanted enzymes which would otherwise
be active in trypsin-compatible buffers.
7. Incubate overnight when a sample will not proceed to the
trypsin digestion and a LysC digest is used for phosphopeptide
enrichment.
8. Trypsin retains most of its activity in 2.0 M urea.
9. We use Kel-F hub (KF) needles with a point style3 from
Hamilton. A 16 gauge needle for 200
l StageTip preparation
and 13/14 gauge needles are best for 1 ml StageTip
preparation.
10. We use plunger assemblies from Hamilton. A plunger (part
number 1122-01) for the 16 gauge needle and a plunger (part
number 1162-01) for the 13/14 gauge needles.
11. Any C18 columns can be used for desalting the digests.
12. When different buffers were used for extraction and digestion
steps, check pH with pH test paper.
13. We use a single C18 1 ml StageTip for desalting 400
μ
μ
g pro-
tein digest.
14. The membrane disk is only for keeping TiO
2
beads.
15. Specifi city and capacity of TiO
2
beads against phosphopeptides
may vary with samples. Test different ratios of TiO
2
to protein
digest amounts and fi nd the optimal conditions for your appli-
cation. We usually use 3 mg TiO
2
beads for a single column
and apply 400
g of protein digests from Arabidopsis.
16. Keep stirring the beads and dispense an equal amount of well-
suspended solution onto the C8 columns.
17. Pyrrolidine can be used instead of piperidine [
15
].
18. It is better to elute peptides into a vial or a 96-well plate for
LC-MS to minimize sample loss from transfers.
μ
References
1. Dissmeyer N, Schnittger A (2011) The age of
protein kinases. Methods Mol Biol 779:7-52
2. Nakagami H, Sugiyama N, Ishihama Y et al
(2012) Shotguns in the front line: phospho-
proteomics in plants. Plant Cell Physiol
53:118-124
3. Macek B, Mann M, Olsen JV (2009) Global
and site-specifi c quantitative phosphopro-
teomics: principles and applications. Annu Rev
Pharmacol Toxicol 49:199-221
4. Thingholm TE, Jensen ON, Larsen MR
(2009) Analytical strategies for phosphopro-
teomics. Proteomics 9:1451-1468
5. Stensballe A, Andersen S, Jensen ON (2001)
Characterization of phosphoproteins from
electrophoretic gels by nanoscale Fe(III) affi n-
ity chromatography with off-line mass spec-
trometry analysis. Proteomics 1:207-222
6. Ikeguchi Y, Nakamura H (1997) Determination
of organic phosphates by column-switching
high performance anion-exchange chromatog-
raphy using on-line preconcentration on tita-
nia. Anal Sci 13:479-483
7. Larsen MR, Thingholm TE, Jensen ON et al
(2005) Highly selective enrichment of
phosphorylated peptides from peptide mix-
