TitanSphere Particles

Protein phosphorylation is a fundamental process and influences cell differentiation, growth and migration. However, analysing phosphorylated proteins is very complicated due to the low concentrations and low ionisation efficiency in MS analyses. It is therefore important to concentrate the phosphorylated proteins and reduce the non-phosphorylated proteins in order to improve the analysis of protein digest studies using LC/MS.

The Titansphere products from GL-Sciences have been specially developed for this application. Titansphere products are more efficient than IMAC (Immobilised metal affinity chromatography) purification, whereby both methods can be combined for optimal results. The phosphate group of a protein is absorbed on the surface of the Titansphere particles under acidic conditions and eluted under basic conditions. Non-phosphorylated proteins do not absorb and are therefore not retained. This means that purification can take place under acidic conditions. The Titansphere material is available in bulk for various applications. The advanced form Titansphere Phos-TiO is available in bulk as well as Spin Tips and Titansphere Phos-TiO MP Kit.

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Technical Data

Titansphere bulk material

Description	Titansphere TiO
Particle size	5, 10 µm
Particle shape	Spherical
Absorption point	Titanium dioxide crystals
Pore size	100 Å
pH range	2.0 - 12.0

Description	Particle size	Quantity	Art. No.
Titansphere TiO	5 µm	500 mg	5020-75000
Titansphere TiO	10 µm	500 mg	5020-75010

Titansphere Phos-TiO bulk material

Description	Particle size	Quantity	Art. No.
Titansphere Phos-TiO Bulk	10 µm	500 mg	5010-21315

Titansphere Phos-TiO Kit

Description	5010-21309	5010-21310	5010-21311	5010-21312
Titansphere Phos-Tio Kit	24 pcs	96 pcs	24 pcs	96 pcs
Titansphere Sorbent Mass/Tip Volume	1mg/10µl	1mg/10µl	3mg/200µl	3mg/200µl
Spin Tip Quantity	24 pcs	96 pcs	24 pcs	96 pcs
Waste Tube quantity	24 pcs	96 pcs	24 pcs	96 pcs
Recovery tube quantity	24 pcs	96 pcs	24 pcs	96 pcs
Instructions	1 pc	1 pc	1 pc	1 pc

Titansphere Phos-TiO MP Kit

Description	5010-21282	5010-21283
Titansphere Phos-TiO MP Kit	24 pcs	24 pcs
Titansphere Sorbent Mass/Tip Volume	1 mg / 200 μL	3 mg / 200 μL
Spin Tip Quantity	24 pcs (6 x 4 packs)	24 pcs (6 x 4 packs)
Waste Fluid Tube Quantity	24 pcs	24 pcs
Recovery Tube (2.0 mL) Quantity	24 pcs	48 pcs
Recovery Tube (1.5 mL) Quantity	48 pcs	48 pcs
Solution B (Lactic acid) Quantity	2 mL	2 mL xb14
Instruction Manual	1/pk	1/pk

Titansphere Phos-TiO MP Kit Protocol

Applications

Efficient purification of HeLa Cell lysate with Titansphere TiO

The HeLa Cell Lysate consists mainly of non-phosphorylated peptides. Titansphere TiO shows excellent selectivity. 90% of the bound peptides are phosphorylated.

Probe: HeLa Cell Lysat, 5µg

Titansphere TiO: 1mg

Comparison of Titansphere TiO with IMAC

Compared to the IMAC method, Titansphere TiO shows a much higher capacity and a significantly higher number of isolated phosphopeptides.

Probe: Arabidopsis Cell Extrakt, 100µg

Titansphere TiO: 1mg

Comparison of Titansphere Tio Bulk and Titansphere Phos-TiO Kit

The Titansphere Phos-Tio kit uses the optimised Titansphere Phos-TiO bulk material. The Phos-TiO material was developed with a view to better absorption and capacity of phosphopeptides. The Titansphere Phos-TiO kit shows a 2.6 times higher peak area and 1.6 times more identified phosphopeptides.

Probe: HeLa Cell Lysat, 50µm

Titansphere Media: 1mg

Produkt	Phosphoryliert	Nicht Phosphoryliert
Titansphere Phos-TiO Kit	996	185
Titansphere TiO Bulk	635	71

New TiO2-HAMMOC method vs. SIMAC method with the Titansphere Phos-TiO MP Kit

The working group led by Wakabayashi et al. (2015) has demonstrated the highly efficient recovery of single and multiply phosphorylated peptides using HeLa cells 100µg in contrast to the Sequential Elution from IMAC (SIMAC) method.

By using basic elution buffers, multiply phosphorylated peptides can be recovered.

Wakabayashi M, Kyono Y, Sugiyama N, Ishihama Y (2015): Extended coverage of singly and multiply phosphorylated peptides from a single titanium dioxide microcolumn. Analytical Chemistry 87 (20), 10213-10221.

Conclusion

The new Titansphere Phos-TiO MP Kit enables a higher recovery of single and multiply phosphorylated peptides. This can reduce ion suppression in subsequent LC-MS/MS detection. This kit shows higher capacities for phosphorylated proteins and peptides than the previous Titansphere Phos-TiO kit.

Titansphere