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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%.

Show MeSH
Analytic Scheme Used for Phosphorylation AnalysisThe protein sample is split into two fractions. One protein fraction is treated with phosphatase and serves as an internal standard. Each fraction is digested with trypsin. The peptides in each fraction are differentially labeled with propylation reagent-d0 or -d4, and then the fractions are recombined. Ion current ratios are measured for each pair of labeled isotopologues.
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sch1: Analytic Scheme Used for Phosphorylation AnalysisThe protein sample is split into two fractions. One protein fraction is treated with phosphatase and serves as an internal standard. Each fraction is digested with trypsin. The peptides in each fraction are differentially labeled with propylation reagent-d0 or -d4, and then the fractions are recombined. Ion current ratios are measured for each pair of labeled isotopologues.


Quantification of protein phosphorylation by liquid chromatography-mass spectrometry.

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

Analytic Scheme Used for Phosphorylation AnalysisThe protein sample is split into two fractions. One protein fraction is treated with phosphatase and serves as an internal standard. Each fraction is digested with trypsin. The peptides in each fraction are differentially labeled with propylation reagent-d0 or -d4, and then the fractions are recombined. Ion current ratios are measured for each pair of labeled isotopologues.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

sch1: Analytic Scheme Used for Phosphorylation AnalysisThe protein sample is split into two fractions. One protein fraction is treated with phosphatase and serves as an internal standard. Each fraction is digested with trypsin. The peptides in each fraction are differentially labeled with propylation reagent-d0 or -d4, and then the fractions are recombined. Ion current ratios are measured for each pair of labeled isotopologues.
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