Abstract
Posttranslational modifications represent a major mechanism by which cells control molecular events. Sample preparation is one of the biggest keys in terms of proteomic analysis, and especially when looking at phosphoproteomics. Here we describe two techniques that are used to identify phosphopeptides. These are Titanium Dioxide and Immobilized Metal Affinity Chromatography (IMAC) which enrich for phosphopeptides. These enrichment techniques typically often identify ~35 % of the same and ~65 % unique phosphopeptides. In this chapter, we describe the use of an ion-trap mass spectrometer for the identification of phosphopeptides.
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References
Karas M, Bachmann D, Hillenkamp F (1985) Influence of the wavelength in high-irradiance ultraviolet-laser desorption mass-spectrometry or organic-molecules. Anal Chem 57:2935–2939
Griffiths J (2008) A brief history of mass spectrometry. Anal Chem 80:5678–5683
Baker MA et al (2010) Use of titanium dioxide to find phosphopeptide and total protein changes during epididymal sperm maturation. J Proteome Res 10:1004–1017
Larsen MR et al (2005) Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 4:873–886
Thingholm TE et al (2006) Highly selective enrichment of phosphorylated peptides using titanium dioxide. Nat Protoc 1:1929–1935
Thingholm TE, Larsen MR (2009) The use of titanium dioxide micro-columns to selectively isolate phosphopeptides from proteolytic digests. Methods Mol Biol 527:57–66
Schirmer EC, Yates JR III, Gerace L (2003) MudPIT: a powerful proteomics tool for discovery. Discov Med 3:38–39
Bodenmiller B et al (2007) Reproducible isolation of distinct, overlapping segments of the phosphoproteome. Nat Methods 4:231–237
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Baker, M.A. (2013). Phosphoproteomic Analysis of Peptides. In: Rose, R. (eds) Legume Genomics. Methods in Molecular Biology, vol 1069. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-613-9_18
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DOI: https://doi.org/10.1007/978-1-62703-613-9_18
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-612-2
Online ISBN: 978-1-62703-613-9
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