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Protocols for The Use of Titanium Dioxide Micro-Columns to Selectively Isolate Phosphopeptides from Proteolytic Digests

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.

Reference

de Graauw, M. (2009). Phospho-Proteomics. Humana Press.

* For Research Use Only. Not for use in diagnostic procedures.

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