Phosphopeptide Enrichment

Phosphorylation analysis provides many highly interesting insights into the cell's inner workings under different conditions. By today's estimations 30% of all proteins get phosphorylated to some extent. Therefore an effective and affordable enrichment method for these phosphopeptides is key for scientific advances in this field. Cube Biotech provides multiple different IMAC agarose resins and magnetic beads for this purpose. They are all suited for phosphopeptide enrichment for mass spectrometry as well as phosphopeptide purification.
Their key features are:

Overview of Cube Biotech's phosphopeptide enrichment MagBeads

Fe-NTA Magnetic Beads

Al-NTA Magnetic Beads

Zr-NTA Magnetic Beads

Ti-NTA Magnetic Beads

MAgBeads by Cub Biotech for phosphopeptide enrichment

Features

Usage	Enrichment of phosphorylated biomolecules for mass spectrometry analysis
Specifity	Phosphorylated biomolecules (e.g. peptides)
Binding efficiency of phosphorylated bimolecules	See figure 2
pH stability	2-14
Other stabilities	100% methanol, 100% ethanol, 100% Isopropanol (v/v) acetonitrile, Ammonium Hydroxide (2,5%), Deoxycholate For about one hour: TFA (1%), Formic acid (1%)
Bead Ligand	Fe- / Zr- / Al- or Ti-NTA

Phosphopeptide enrichment products by Cube Biotech were used in the following publications:

Protein	Year	Author	Product
Enriching numerous phosphopeptides	2019	Searle B.C., Lawrence R.T., MacCoss M.J., Villén J.⁶	Fe NTA MagBeads
Enriching numerous phosphopeptides	2018	Searle B.C., Pino L.K., Egertson J.D., Ting Y.S., Lawrence R.T., Villén J., Macoss M.J.⁷	Fe NTA MagBeads
Numerous phosphopeptides	2019	Leutert M., Rodríguez-Mias R.A., Fukuda N.K., Villén J.⁸	Fe NTA MagBeads
Enriching numerous phosphopeptides	2020	Smith I.R.m Hess K.N., Bakhtina A., Valente A.S., Rodriguez-Mias R.A., Villen J.⁹	Fe NTA MagBeads
Enriching numerous phosphopeptides	2020	Calejman C.M., Trefley S., Entwisle S.W., Luciano A., Jung S.M., Hsiao W., Torres A., Hung C.M., Li H., Snyder N.W., Villén J., Wellen K.E., D.A. Guertin¹⁰	Fe NTA MagBeads
Enriching numerous phosphopeptides	2020	Fan. Z., Delvin J.R.m Hogg S.J., Doyle M.A., Harrison P.F., Todorovski I., Cluse L.A., Knight D.A., Sandow J.J., Gregory G., Fox A., Beilharz T.H., Kwiatkowski N., Scott N.E., Vidakovic A.T., Kelly G.P., Svejstrup J.Q., Geyer M., Gray N.S., Vervoort S.J., Johnstone R.W.¹¹	Fe NTA MagBeads
Enriching numerous phosphopeptides	2020	Vervoort S., Welsh S., Delvin J.R., Barbieri E., Knight D.A., Costacurta M., Todorovski I., Kearney C.J., Sandow J.J., Bjelosevic S., Fan Z., Vissers J.H.A., Pavic K., Martin B.P., Gregary G., Kong I.Y., Hawkins E.D., Hogg S.J., Kelly M.J., Newbold A., Simpson K.J., Kauko O., Harvey K.F., Ohlmeyer M., Westermarck J., Gray N., Gardini A., Johnstone R.W.¹²	Fe NTA MagBeads

Phosphopeptide enrichment for mass spectrometry.

IMAC materials, in particular Fe-NTA, have been widely used to enrich phosphopeptides as part of the sample preparation for mass spectrometry (1,3,4). Under certain circumstances other transition metals, such as zirconium or aluminium have also been loaded on NTA or IDA matrices for enrichment (5). Magnetic beads have proven to be useful for this kind of sample preparation (2) in a similar manner to their agarose resin counterparts.

Automatic Phosphoproteomics for high-throughput projects:

Fe-NTA magnetic beads by Cube Biotech have been proven to increase the speed of a high throughput experiment drastically. Leutert et al. (2019) presented the application of PureCube Fe-NTA MagBeads in a procedure that they named R2-P2, which is short for Rapid-Robotic PhosphoProteomics. They used a KingFisher^TM Flex for their robotic runs to fully automize the phosphopeptide enrichment process.

Fig 1: Schematic depiction of the setup of a R2-P2 assay using a KingFisher^TM Flex. The robotic configuration allows for loading of eight different 96-well plates. Each plate can be rotated into position under a 96-pin magnetic head that drops down inside the 96-well plate to release, bind, or agitate the magnetic microspheres in solution. In the first robotic run, peptides are captured from lysates by carboxylated magnetic beads, purified, and eluted by digestion at 37°C. Eluted peptides are dried down and can be resuspended for total proteome analysis by LC-MS/MS and/or for automatic phosphopeptide enrichment. Phosphopeptides are enriched using a second robotic run on the KingFisher^TM Flex, using Fe-IMAC, Ti-IMAC, Zr-IMAC, or TiO₂ magnetic microspheres, and analyzed by LC-MS/MS to obtain the phosphoproteome.
Source: Leutert et al. (2019)

Superiority over other phosphopeptide enrichment methods:

Leutert et al. compared three different types of IMAC beads (including our PureCube Fe-NTA) and TiO₂ microspheres. As it can be seen in figure 2 out PureCube Fe-NTA magnetic beads presented themselves to be the best option for phosphopeptide enrichment. With our Fe-NTA MagBeads the most unique phosphopeptides (Fig. 2 A and C) were enriched with the highest efficiency (Figure 2 B).

Different phosphopeptide enrichment products compared

Fig. 2: Comparison of phosphopeptide enrichment performance between four different products/methods. A: Number of unique phosphopeptides identified by the different enrichments (mean +/- SD, n = 3). B: Phosphopeptide enrichment efficiency shown as the fraction of phosphorylated peptides over total peptides (mean +/- SD, n = 3). C: Venn diagram of identified phosphopeptides by the different phosphopeptide enrichment methods.
Source: Leutert et al. (2019)

The best product for the best prize:

Comparing products from different manufacturers and suppliers can be tedious. Different concentrations and volumes from different suppliers can be misleading on the search for the best cost-benefit relationship. Therefore we at Cube Biotech compiled the prices of the most commonly used phosphopeptide enrichment products to give some overview about the actual prize situation on the market.

Prize Compairson of different phosphopeptide products

Fig. 3: Cube Biotech does not only offer the best single product for phosphopeptide enrichment, but also the most prize efficient one. Competitor T and G are ranging at about half the volume of beads you get for 200 USD in comparison to Cube Biotech. The extreme difference between competitor R and Cube Biotech is a ratio of 1 : 24.

References

Albuquerque, C.P. et al. A multidimensional chromatography technology for in-depth phosphoproteome analysis. Mol Cell Proteomics (2008), 7(7), 1389-1396.
Herskowitz, J., et al. Phosphoproteomic analysis reveals site-specific changes in GFAP and NDGR2 phosphorylation in frontotemporal lobar degeneration. J. Proteome Res (2010), 9(12):6368-6379.
Yu, P. et al. Global analysis of neuronal phosphoproteome regulation by chondroitin sulfate proteoglycans. PLoS One (2013), 8,3, e59285.
Aryal, U.K. et al. Optimization of immobilized Gallium (III) ion affinity chromatography for selective binding and recovery of phosphopeptides from protein digests. Journal of Biomolecular Techniques (2008), 19:296-310.
Block et. al. Immobilized-metal affinity chromatography (IMAC) a review. Methods Enzymol. (2009), 463:439-73.
Searle, B. et. al. (2019). Thesaurus: quantifying phosphopeptide positional isomers. Nature Methods. 16. 703-706. 10.1038/s41592-019-0498-4.
Searle, Brian et al. (2018). Chromatogram libraries improve peptide detection and quantification by data independent acquisition mass spectrometry. Nature Communications. 9. 10.1038/s41467-018-07454-w.
Leutert, M. et al. (2019). R2-P2 rapid-robotic phosphoproteomics enables multidimensional cell signaling studies. Molecular Systems Biology. 15. e9021. 10.15252/msb.20199021.
Smith, I. et al. (2020). Identification of phosphosites that alter protein thermal stability. 10.1101/2020.01.14.904300.
Calejman, C.M. et al. (2020). mTORC2-AKT signaling to ATP-citrate lyase drives brown adipogenesis and de novo lipogenesis. Nature Communication 11. doi.org/10.1038/s41467-020-14430-w.
Fan, M. et al. (2020). CDK13 cooperates with CDK12 to control global RNA polymerase II processivity. Science Advances. 6. eaaz5041. 10.1126/sciadv.aaz5041.
Vervoort, S. et al. (2020). A PP2A-Integrator complex fine-tunes transcription by opposing CDK9. 10.1101/2020.07.12.199372.