The principle of the TiO2 method described in this chapter is illustrated using a peptide mixture originating from tryptic digestions of 12 standard proteins (Model proteins). The TiO2 purification method is a simple and straightforward method. It is fast and efficient for enrichment of phosphopeptides even from highly complex samples.
1 Model Proteins and Peptide Mixture
a) Dissolve each protein in 50 mM ammonium bicarbonate, pH 7.8, 10 mM DTT and incubated at 37°C for 1 h. After reduction, add 20 mM iodoacetamide and incubate the samples at room temperature for 1 h in the dark. After incubation, quench each reaction with 10 mM DTT.
b) Digest each protein using trypsin (1–2% w/w) at 37°C for 12 h.
2 Packing the TiO2 Micro-Column
a) When working with less complex samples use TiO2 microcolumns of ~3 mm (~0.4 μg TiO2 beads). Use longer columns for more complex samples. The capacity of TiO2 towards phosphorylated peptides is very high so even a small column will retain significant amount of phosphorylated peptide.
b) Suspend ∼2 mg TiO2 beads in 500 μL 100% acetonitrile.
c) Stamp out a small plug of C8 material from a 3 M Empore™ C8 extraction disk using a HPLC syringe needle, and place this plug in the constricted end of a GELoader tip using e.g., a LC capillary. The C8 material will prevent the TiO2 beads from leaking, while still allowing for liquid to pass through.
d) Wash the membrane using 100% acetonitrile to reduce the level of polymers in the membrane plug: Load 20 μL 100% acetonitrile into the GELoader tip and apply gentle air pressure using a plastic syringe.
e) Prepare a TiO2 micro-column by packing the TiO2 beads on top of the C8 membrane disk in the GELoader tip. Load an aliquot of the TiO2 suspension (depending on the amount of the sample) into the GELoader tip and apply gentle air pressure as in last step.
3 Loading the Sample Onto the TiO2 Micro-Column
a) It is important that buffers used for loading or washing of the TiO2 micro-column contain 50–80% acetonitrile to abrogate adsorption of peptides to the C8 membrane and the TiO2 beads due to hydrophobic interactions.
b) Dilute the peptide sample at least five times in the TiO2 Loading buffer (v/v) and mix well. Load the sample onto the TiO2 micro-column by applying air pressure.
c) Wash the TiO2 micro-column using 5 μL TiO2 Loading buffer and subsequently with 30 μL TiO2 washing buffer.
4 Elution of Phosphorylated Peptides from the TiO2 Micro-column
a) Elute the phosphopeptides bound to the TiO2 micro-column using a minimum of 25 μL of TiO2 elution buffer 1 (depending on the size of the column).
b) Since peptides will have binding affinity towards the C8 membrane plug, some phosphopeptides may have bound to the C8 membrane during the previous elution step (step 1). Elute these using 1 μl of TiO2 elution buffer 2 and pool this with the eluate from step 1.
b) Acidify the pooled eluates using 1 μL of 100% formic acid per 10 μL eluate. Make sure that the eluate obtains a pH value of 2–3. At this stage the purified phosphopeptides can be analyzed by MS. It is, however, advisable to desalt/concentrate the samples first using reversed phase chromatography.
5 Packing a Reversed Phase (RP) Micro-column for Desalting/Concentrating the Sample
a) Use RP micro-columns of ~3–6 mm (0.4–0.8 μg) depending on the amount of material.
b) Suspend ~2 mg POROS Oligo R3 reversed phase (RP) material in 200 μL of 50% acetonitrile.
c) Prepare a RP micro-column by stamping out a small plug of C18 material from a 3 M Empore™ C18 extraction disk and place it in the constricted end of a GELoader tip.
d) Pack the RP beads in the GELoader tip as described for the TiO2 beads in Subheading 2, step e.
e) Load the acidified sample slowly onto the RP micro-column (~1 drop/s).
f) Wash the RP micro-column using 30 μL RP washing buffer.
6 Loading the Sample Onto a the Reversed Phase (RP) Micro-column
a) Load the acidified sample slowly onto the RP micro-column (~1 drop/sec).
b) Wash the RP micro-column using 30 μL 0.1% TFA.
7 Elution of Phosphorylated Peptides from the RP Micro-column
a) Elute the phosphopeptides from the RP micro-column using 20 μL of RP elution buffer, followed by lyophilization of the phosphopeptides. (N.B. For MALDI MS analysis the peptides can be eluted off the RP micro-column directly onto the MALDI target using 1 μL DHB solution.
b) LC-ESI-MSn analysis: Redissolve the dried phosphopeptides in 0.5 μL 100% formic acid and immediately dilute to 10 μL with UHQ water.
LC-ESI-MSn analysis of multi-phosphorylated peptides is improved by re-dissolving the phosphopeptides by sonication in an EDTA containing buffer prior to LC-ESI-MS/MS analysis.
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Reference
- de Graauw, M. (2009). Phospho-Proteomics. Humana Press.