Springer Protocols: Abstract: Identification of Protein Phosphorylation Sites by Advanced LC-ESI-MS/MS Methods

Identification of Protein Phosphorylation Sites by Advanced LC-ESI-MS/MS Methods

By: Christoph Weise³, Christof Lenz⁴

Abstract

Full Text

Download PDF (392K)

Phosphorylation, the process by which a phosphate group is attached to a pre-existing protein, is an evolutionarily and metabolically cheap way to change the protein's surface and properties. It is presumably for that reason that it is the most wide-spread protein modification: an estimated 10–30% of all proteins are subject to phosphorylation.

MS-based methods are the methods of choice for the identification of phosphorylation sites, however biochemical prefractionation and enrichment protocols will be needed to produce suitable samples in the case of low-stoichiometry phosphorylation. Using emerging MS-based technology, the elucidation of the “phosphoproteome,” a comprehensive inventory of phosphorylation sites, will become a realistic goal. However, validating these findings in a cellular context and defining their biological meaning remains a daunting task, which will inevitably require extensive and time-consuming additional biological research.

Affiliation(s): (3) Institut für Chemie und Biochemie, Freie Universität Berlin, Berlin, Germany
(4) Applied Biosystems, Darmstadt, Germany

Book Title: Post-translational Modifi cations of Proteins: Tools for Functional Proteomics

Series: Methods in Molecular Biology | Volume: 446 | Pub. Date: Apr-04-2008 | Page Range: 33-46 | DOI: 10.1007/978-1-60327-084-7_3

Subject: Protein Science

Key Words: Phosphorylation - LC-ESI-MS/MS - phosphoproteome

References

Download References

1.	1. Hunter, T. (1995) Protein kinases and phosphatases: the yin and yang of protein phosphoryla tion and signaling. Cell 80, 225–236.

2.	2. Cohen, P. (2002) The origins of protein phosphorylation. Nat Cell Biol. 4, E127–30.

3.	3. Reinders, J. and Sickmann, A. (2005) State-of-the-art in phosphoproteomics. Proteomics 5, 4052–4061.

4.	4. Steen, H., Jebanathirajah, J. A., Rush, J., Morrice, N. and Kirschner, M. W. (2006) Phosphorylation analysis by mass spectrometry: myths, facts and the consequences for qualitative and quantitative measurements. Mol. Cell. Proteomics 5, 172–181.

5.	5. Annan, R. S., Huddleston, M. J., Verma, R., Deshaies, R. J. and Carr, S. A. (2001) A multidi mensional electrospray ms-based approach to phosphopeptide mapping. Anal. Chem. 73, 393–404.

6.	6. Zappacosta, F., Huddleston, M. J., Karcher, R. L., Gelfand, V. I., Carr, S. A. and Annan, R. S. (2002) Improved sensitivity for phosphopeptide mapping using capillary column hplc and microionspray mass spectrometry: comparative phosphorylation site mapping from gel-derived proteins. Anal. Chem. 74, 3221–3231.

7.	7. Steen, H., Köster, B. and Mann, M. (2001) Quadrupole time-of-flight versus triple-quadrupole mass spectrometry for the determination of phosphopeptides by precursor ion scanning. J. Mass Spectrom. 36, 782–790.

8.	8. Schlosser, A., Pipkorn, R., Bossemeyer, D. and Lehmann, W. D. (2001) Analysis of protein phosphorylation by a combination of elastase digestion and neutral loss tandem mass spec-trometry. Anal. Chem. 73, 170–176.

9.	9. Hager, J. W. (2002) A new linear ion trap mass spectrometer. Rapid Commun. Mass Spectrom. 16, 512–526.

10.	10. Le Blanc, J. C. Y, Hager, J. W., Illisiu, A. M. P., Hunter, C., Zhong, F. and Chu, I. (2003) Unique scanning capabilities of a new hybrid linear ion trap mass spectrometer (Q TRAP) used for high sensitivity proteomics applications. Proteomics 3, 859–869.

11.	11. Williamson, B. F., Marchese, J. and Morrice, N. A., (2006) Automated identification and quantification of protein phosphorylation sites by lc/ms on a hybrid triple quadrupole linear ion trap mass spectrometer. Mol. Cell. Proteomics 5, 337–346.

12.	12. Kottwitz, D., Kukhtina, V., Dergousova, N., Alexeev, T., Utkin, Y., Tsetln, V. and Hucho, F. (2004) Intracellular domains of the δ-subunits of Torpedo and rat acetylcholine receptors– expression, purification, and characterization. Protein Expr. Purif. 38, 237–247.

13.	13. Cox, D. M., Zhong, F., Du, M., Duchoslav, E., Sakuma, T. and McDermott, J. C. (2005) Multiple reaction monitoring as a method for identifying protein posttranslational modifications. J. Biomol. Tech. 16, 83–90.

14.	14. Unwin, R. D., Griffiths, J. R., Leverentz, M. K. Grallert, A., Hagan, I. M. and Whetton, A. D. (2005) Multiple reaction monitoring to identify sites of protein phosphorylation with high sensitivity. Mol. Cell. Proteomics 4, 1134–1144.

15.	15. Covey, T., Shushan, B., Bonner, R., Schröder, W. and Hucho, F. (1991) In Methods in Protein Sequence Analysis (Jörnvall, H., Höög, J.-O. and Gustavsson, A.-M., eds.) Birkhäuser Verlag, Basel, Switzerland, pp. 249–256.

16.	16. DeGnore, J. P. and Qin, J. (1998) Fragmentation of phosphopeptides in an ion trap mass spectrometer. J. Am. Chem. Soc. Mass Spectrom. 9, 1175–1188.

17.	17. Krüger, R., Hung, Ch.-W., Edelson-Averbukh, M. and Lehmann, W. D. (2005) Iodoacetamide-alkylated methionine can mimic neutral loss of phosphoric acid from phosphopeptides as exemplified by nano-electrospray ionisation quadrupole time-of-flight parent ion scanning. Rapid Commun. Mass Spectrom. 19, 1709–1716.

18.	18. Steen, H., Küster, B, Fernandez, M., Pandey, A. and Mann, M. (2001). Detection of tyrosine phosphorylated peptides by precursor ion scanning quadrupole tof mass spectrometry in posi tive ion mode. Anal. Chem. 73, 1440–1448.

19.	19. Schroeder, M. J., Shabanowitz, J., Schwartz, J. C., Hunt, D. F. and Coon, J. J. (2004) A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry. Anal. Chem. 76, 3590–3598.

Comments (Loading...)

Post comment/View all comments