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Researchers Submit Patent Application, "Isolation of Phosphoproteins, Glycoproteins and Fragments Thereof", for Approval

August 5, 2014

By a News Reporter-Staff News Editor at Life Science Weekly -- From Washington, D.C., NewsRx journalists report that a patent application by the inventors Rainer, Matthias (Grinzens, AT); Polato, Fabio (Innsbruck, AT); Gjerde, Douglas T. (Saratoga, CA); Bonn, Guenther (Zirl, AT), filed on January 17, 2013, was made available online on July 24, 2014 (see also Patents).

No assignee for this patent application has been made.

News editors obtained the following quote from the background information supplied by the inventors: "This invention relates to methods for the isolation of phosphoproteins and glycoproteins and fragments of proteins for the analysis of biological samples, including biological fluids, biological cultures, cell cultures, cell lysates, cell-free cultures, yeast, HeLa cells, food, blood, urine, tissue and human cerebrospinal fluid.

"Protein phosphorylation is the enzymatic process performed by kinases of adding phosphate group(s) to a protein. Phosphorylation is a reversible post translational modification that is important in many different cellular pathway processes including those involving enzymatic activities. It is estimated that up to a third of the proteins contained in a cell can be phosphorylated at some point in the life cycle of any particular protein (Hubbard, M. J. et al. (1993) Trends Biochem. Sci. 5, 172-177). Phosphorylation of proteins that are contained in eukaryotic cells occurs mainly on serine, threonine, and tyrosine residues with serine being the most frequently modified amino acid. One study performed on HeLa cell phosphorylation site distribution reported the relative concentration of the phosphoserine, phosphothreonine, and phosphotyrosine sites at 86.4, 11.8, and 1.8%, respectively (Olsen, J. et al. (2006) Cell. 127, 635-648).

"Substantial work has been devoted to the development and improvement of methods for selective enrichment of phosphopeptides (Trojer, L., et al. (2005) J. Chromatogr. A. 1079, 197-207; Feuerstein, I. et al. (2006) J. Am. Soc. Mass Spectrom. 17, 1203-1208; Valiant, R. M. et al. (2007) Anal. Chem. 79, 8144-8153; Feuerstein, I. et al. (2005) Proteomics. 5, 46-54) but not phosphoprotein enrichment. The isolation of low concentrations of a phosphorylated protein fragment from its non-phosphorylated counterpart and a mixture of polypeptides has been shown to be difficult. Even more difficult is separating a phosphorylated protein from a mixture containing its non-phosphorylated counterpart and other proteins. The reason for this is unknown but may be because the number of phosphorylated sites on a given protein is small.

"Two common strategies are currently employed for the isolation of phosphopeptides: immobilized metal ion affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC). IMAC is the most widely used strategy for phosphopeptide enrichment. This technique was initially developed in 1975 and was originally used to separate His-tagged proteins (Porath, J. et al. (1975) Nature. 258, 598-599). IMAC resins containing iron and gallium are the commonly used metals for the enrichment of phosphorylated species (Bonn, G. K. et al. (1990) Chromatographia. 30, 9/10; Aprilita, N. H. et al. (2005). J. Proteome Res. 4, 2312-2319; Sykora, C. et al. (2007) Protein & Peptide Letters. 14, 489-496). The other chemical strategy, MOAC (Wolschin, F. et al. (2005) Proteomics. 5, 4389-4397) employs metal oxides such as titanium dioxide (TiO.sub.2) (Mazanek, M. et al. (2007) Nat. Protoc. 2, 1059-1069), zirconium dioxide (ZrO.sub.2) (Zhou, H. et al. (2007) Electrophoresis. 28, 2201-2215; Kweon, H. K. et al. (2006) Anal. Chem. 78, 1743-1749) or mixed TiO.sub.2/ZrO.sub.2 nanoparticles embedded in a monolithic polymer (Rainer, M. et al. (2008) Proteomics. 8, 4593-4602) to selectively retain phosphopeptides from complex biological samples. In several studies, aluminum hydroxide and aluminum oxide were shown to exhibit a high and selective attraction to phosphorylated proteins (Chang, M. F. et al. (1997) J. Pharm. Sci. Technol. 51, 25-29; Lyer, S. et al. (2003) Pharm. Dev. Technol. 8, 81-86). After capture and isolation of the phosphopeptides, analysis is often performed by mass spectrometry.

"More recently, a two-step phosphopeptide enrichment method was reported in which calcium cations are used to help isolate the phosphopeptides and then IMAC was used for final purification of the precipitated peptides (Zhang, H. et al. (2007) Molec. and Cell. Prot. 6.11, 2032-2042). However, the researchers found that calcium cation did not precipitate the phosphopeptides contained in a mixture. When calcium cation was added to the mixture, no precipitate was formed. Instead, a phosphate ion solution was added to the trypsin digested sample first. Then, with the phosphate present in the digested sample, calcium cation was added to the digested sample solution to precipitate calcium phosphate. Using this procedure, the calcium phosphate precipitate also pulled down the phosphopeptides that became associated with the calcium phosphate precipitate. After this process, it was further discovered that IMAC was needed for additional purification and enrichment to remove non phosphopeptides that also became associated with the calcium phosphate precipitate. Phosphoproteins, glycoproteins and glycopeptides could not be recovered by these researchers.

"Glycoproteins play an essential role in the body. For instance, in the immune system, almost all of the key molecules involved in the immune response are glycoproteins. The analysis or determination of protein glycosylation has become an important goal for biomarker studies since it has long been known that cellular glycosylation profiles change significantly during oncogenesis, the process whereby normal cells are transformed to cancer cells (Hakomori, S. (1996) Cancer Res. 56, 5309-5318; Kobata, A. (1998) Glycoconj J. 15, 323-331). Similar to phosphoprotein and protein fragment isolation and analysis, the isolation and analysis of the glycoproteome presents a significant challenge for the bioanalytical chemist. The most common approach for the enrichment of glycosylated proteins is based on isolation by lectin affinity resins (Zhao, J. et al. (2006) J. Proteome Res. 5 (7), 1792-1802). A number of different resin types have been used to capture various types of glycoproteins. After enrichment and elution, the species are digested and then usually deglycosylated by protein- N-glycanase F. Finally, the proteins and the glycosylation sites are identified, primarily by mass spectrometry (Xiong, L. et al. (2003) J. Proteome Res. 2, 618-625; Alvarez-Manilla, G. et al. (2006) J. Proteome Res. 5, 701-708; Zhang, L. et al. (2005) Anal. Chem. 77, 7225-7231; Wang, Y. et al (2006) Glycobiology. 16, 514-523).

"Phosphoproteins and glycoproteins and their peptides are important for general biological research and are potentially important biomarkers that could be used for the diagnosis of disease. The ability to capture both species together or separately is desirable. There exists a need to be able to distinguish between phosphoproteins and glycoproteins. There exists a need to capture, at will, both phosphoproteins and glycoproteins together or separately and selectively.

"Valuable biological information can be obtained by capturing whole phosphorylated and glycosylated proteins rather than merely capturing their fragments. When enzymatic digestion is performed on a protein mixture, the resulting mix of polypeptides is very complex, composed of fragments originating from many different proteins. Analysis of this mixture requires attempting to determine the starting protein from which each particular polypeptide originated. Capturing whole phosphoproteins (or glycoproteins) and performing top down analysis simplifies the sample and provides additional insight into the position or positions that are phosphorylated (or glycosylated) on any particular protein. Once whole functionalized proteins are captured, chromatography or gel electrophoresis can be used to separate and collect individual proteins prior to enzymatic digestion.

"Therefore, there exists a need for the ability to isolate phosphoproteins and/or glycoproteins in a reproducible manner, especially when they are present at very low concentrations."

As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventors' summary information for this patent application: "The invention provides methods for the selective isolation of phosphorylated and glycosylated proteins and their fragments. Selective metal cations are used to precipitate phosphoproteins and/or glycoproteins from complex mixtures. Selective metal cations may be used to precipitate polypeptides containing phospho and/or glycan groups from complex mixtures. Lanthanide metals work particularly well for this purpose. For example, lanthanum (III) is used to preferentially complex into a precipitate the phosphate group and the glyco group, particularly N-linked glyco groups, contained on proteins or fragments of proteins allowing their isolation from complex mixtures. The invention also allows the controlled release and isolation of protein and protein fragments containing glycans from the initial precipitate leaving the phosphoproteins. Proteins containing phospho or glyco groups can be digested (and analyzed) while they are contained in the precipitation pellet or after the pellet containing the proteins has been dissolved. Proteins containing glyco groups can be digested and analyzed after the proteins containing these groups have been eluted from the pellet.

"The precipitated proteins may be digested with an enzyme directly on the pellets formed from the method or any of the precipitates may be dissolved and treated with an enzyme. The sample preparation method and device can be used for many different types of biological samples, including biological fluids, biological cultures, cell cultures, cell lysates, cell-free cultures, yeast, HeLa cells, fresh milk, blood, urine, tissue and human cerebrospinal fluid. The proteins are precipitated, recovered and ready for analysis by methods such as electrophoresis, chromatography, mass spectrometry, IR or UV spectrometry, ELIZA, protein array, SPR or other analytical methods. The sample can be ready for analysis in as little as ten minutes and may allow detection limits down to the femtomole level and lower. The method and device are valuable tools in the field of protein analysis and diagnostics.

"The application of lanthanide metals for the precipitation of phosphorylated and/or glycosylated proteins opens a new field of fast selective and sensitive biomarker analysis. The method can be used to determine biological pathways and mechanisms. The method was demonstrated for complex biological samples by being able to identify proteins present in the samples in low concentrations.


"FIG. 1: MALDI-TOF spectra showing the isolation method for phosphoproteins.

"FIG. 2: MALDI-TOF spectra showing the isolation method for phosphoproteins in milk.

"FIG. 3: MALDI-TOF spectra of tryptic digested HeLa cell lysate spiked with .alpha.-casein (1:100) before precipitation and after isolation.

"FIG. 4: MALDI-TOF spectra showing the precipitation and recovery of a phosphoprotein and glycoprotein.

"FIG. 5: MALDI-TOF spectra showing the precipitation, separation and recovery of a glycoprotein and a phosphoprotein.

"FIG. 6: Depicts embodiments of the workflow utilized for the isolation of phosphoproteins, phosphopeptides, glycoproteins and glycopeptides.

"FIG. 7: MALDI-TOF spectra showing phosphoprotein precipitation from a protein standard by trivalent europium, terbium and erbium."

For additional information on this patent application, see: Rainer, Matthias; Polato, Fabio; Gjerde, Douglas T.; Bonn, Guenther. Isolation of Phosphoproteins, Glycoproteins and Fragments Thereof. Filed January 17, 2013 and posted July 24, 2014. Patent URL:

Keywords for this news article include: Anions, Patents, Peptides, Hela Cells, Amino Acids, Glycoproteins, Glycoconjugates, Phosphoproteins, Phosphoric Acids, Calcium Compounds, Calcium Phosphates, Inorganic Chemicals, Phosphorus Compounds.

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Source: Life Science Weekly

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