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Quantification of protein phosphorylation by liquid chromatography-mass spectrometry.

Previs MJ, VanBuren P, Begin KJ, Vigoreaux JO, LeWinter MM, Matthews DE - Anal. Chem. (2008)

Bottom Line: The method also improves the retention and elution of hydrophilic peptides.The method defines phosphorylation without having to measure the phosphorylated peptides directly or being affected by variable miscleavage.Measurement of phosphorylation is shown to be linear (relative standard error <5%) with a detection limit of <10%.

View Article: PubMed Central - PubMed

Affiliation: Cell and Molecular Biology Program and Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405, USA.

ABSTRACT
The identification and quantification of specific phosphorylation sites within a protein by mass spectrometry has proved challenging when measured from peptides after protein digestion because each peptide has a unique ionization efficiency that alters with modification, such as phosphorylation, and because phosphorylation can alter cleavage by trypsin, shifting peptide distribution. In addition, some phosphorylated peptides generated by tryptic digest are small and hydrophilic and, thus, are not retained well on commonly used C18 columns. We have developed a novel C-terminal peptide (2)H-labeling derivatization strategy and a mass balance approach to quantify phosphorylation. We illustrate the application of our method using electrospray ionization liquid chromatography-mass spectrometry by quantifying phosphorylation of troponin I with protein kinase A and protein kinase C. The method also improves the retention and elution of hydrophilic peptides. The method defines phosphorylation without having to measure the phosphorylated peptides directly or being affected by variable miscleavage. Measurement of phosphorylation is shown to be linear (relative standard error <5%) with a detection limit of <10%.

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Demonstration of linearity of phosphorylation measurement. The degree of phosphorylation was determined for peptides SSANYR (●) and ISASR (○) from the samples shown in Figure 5. A linear response was observed for SSANYR, p(%) = (0.937 ± 0.017) pRx(%) + (5.6 ± 6.4); r2 = 0.998 and for ISASR, p(%)= (0.438 ± 0.036) pRx(%) + (8.7 ± 9.5), r2 = 0.961, where p is the fraction of peptide that is phosphorylated and pRx is the degree of PKA treatment.
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fig6: Demonstration of linearity of phosphorylation measurement. The degree of phosphorylation was determined for peptides SSANYR (●) and ISASR (○) from the samples shown in Figure 5. A linear response was observed for SSANYR, p(%) = (0.937 ± 0.017) pRx(%) + (5.6 ± 6.4); r2 = 0.998 and for ISASR, p(%)= (0.438 ± 0.036) pRx(%) + (8.7 ± 9.5), r2 = 0.961, where p is the fraction of peptide that is phosphorylated and pRx is the degree of PKA treatment.

Mentions: Figure 6 shows the degree of phosphorylation calculated for the SSANYR and ISASR peptides in each of the PKA-treated sample mixtures. We determined that 99% of the SSANYR peptide and 49% ISASR peptide were phosphorylated by PKA treatment. A linear decrease was observed for the degree of both SSANYR and ISASR phosphorylation when the PKA-treated sample was diluted with unphosphorylated peptides. These results demonstrate the linearity and precision of our method for determining phosphorylation.


Quantification of protein phosphorylation by liquid chromatography-mass spectrometry.

Previs MJ, VanBuren P, Begin KJ, Vigoreaux JO, LeWinter MM, Matthews DE - Anal. Chem. (2008)

Demonstration of linearity of phosphorylation measurement. The degree of phosphorylation was determined for peptides SSANYR (●) and ISASR (○) from the samples shown in Figure 5. A linear response was observed for SSANYR, p(%) = (0.937 ± 0.017) pRx(%) + (5.6 ± 6.4); r2 = 0.998 and for ISASR, p(%)= (0.438 ± 0.036) pRx(%) + (8.7 ± 9.5), r2 = 0.961, where p is the fraction of peptide that is phosphorylated and pRx is the degree of PKA treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3050605&req=5

fig6: Demonstration of linearity of phosphorylation measurement. The degree of phosphorylation was determined for peptides SSANYR (●) and ISASR (○) from the samples shown in Figure 5. A linear response was observed for SSANYR, p(%) = (0.937 ± 0.017) pRx(%) + (5.6 ± 6.4); r2 = 0.998 and for ISASR, p(%)= (0.438 ± 0.036) pRx(%) + (8.7 ± 9.5), r2 = 0.961, where p is the fraction of peptide that is phosphorylated and pRx is the degree of PKA treatment.
Mentions: Figure 6 shows the degree of phosphorylation calculated for the SSANYR and ISASR peptides in each of the PKA-treated sample mixtures. We determined that 99% of the SSANYR peptide and 49% ISASR peptide were phosphorylated by PKA treatment. A linear decrease was observed for the degree of both SSANYR and ISASR phosphorylation when the PKA-treated sample was diluted with unphosphorylated peptides. These results demonstrate the linearity and precision of our method for determining phosphorylation.

Bottom Line: The method also improves the retention and elution of hydrophilic peptides.The method defines phosphorylation without having to measure the phosphorylated peptides directly or being affected by variable miscleavage.Measurement of phosphorylation is shown to be linear (relative standard error <5%) with a detection limit of <10%.

View Article: PubMed Central - PubMed

Affiliation: Cell and Molecular Biology Program and Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405, USA.

ABSTRACT
The identification and quantification of specific phosphorylation sites within a protein by mass spectrometry has proved challenging when measured from peptides after protein digestion because each peptide has a unique ionization efficiency that alters with modification, such as phosphorylation, and because phosphorylation can alter cleavage by trypsin, shifting peptide distribution. In addition, some phosphorylated peptides generated by tryptic digest are small and hydrophilic and, thus, are not retained well on commonly used C18 columns. We have developed a novel C-terminal peptide (2)H-labeling derivatization strategy and a mass balance approach to quantify phosphorylation. We illustrate the application of our method using electrospray ionization liquid chromatography-mass spectrometry by quantifying phosphorylation of troponin I with protein kinase A and protein kinase C. The method also improves the retention and elution of hydrophilic peptides. The method defines phosphorylation without having to measure the phosphorylated peptides directly or being affected by variable miscleavage. Measurement of phosphorylation is shown to be linear (relative standard error <5%) with a detection limit of <10%.

Show MeSH