Limits...
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

Related in: MedlinePlus

Linearity and precision of isotopologue ratio measurements. Data shown are the measured ion current (I0/I4) ratios for each peptide listed in Table 1 for samples prepared with defined mole d0/d4 ratios ranging from 0:1 to 1:1 d0- to d4-labeled peptides. A linear response in the measured ion current ratio, I0/I4 = (0.956 ± 0.005) (d0/d4) + (0.025 ± 0.003) with r2 = 0.999 was observed as a function of mole ratio of unlabeled (d0) to deuterated (d4) peptides.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3050605&req=5

fig4: Linearity and precision of isotopologue ratio measurements. Data shown are the measured ion current (I0/I4) ratios for each peptide listed in Table 1 for samples prepared with defined mole d0/d4 ratios ranging from 0:1 to 1:1 d0- to d4-labeled peptides. A linear response in the measured ion current ratio, I0/I4 = (0.956 ± 0.005) (d0/d4) + (0.025 ± 0.003) with r2 = 0.999 was observed as a function of mole ratio of unlabeled (d0) to deuterated (d4) peptides.

Mentions: The peptides generated from the digestion of the isolated troponin protein-complex were differentially labeled with propyl-d0 or -d4 and then mixed with a mole ratio ranging from 0:1 to 1:1 d0-labeled peptides to d4-labeled peptides and measured by ESI-LC−MS/MS. Figure 4 shows the measured ion current ratios for each set of isotopologues listed in Table 1. This graph demonstrates linearity and precision of the d0/d4 peptide ratio measurement for a range of peptides.


Quantification of protein phosphorylation by liquid chromatography-mass spectrometry.

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

Linearity and precision of isotopologue ratio measurements. Data shown are the measured ion current (I0/I4) ratios for each peptide listed in Table 1 for samples prepared with defined mole d0/d4 ratios ranging from 0:1 to 1:1 d0- to d4-labeled peptides. A linear response in the measured ion current ratio, I0/I4 = (0.956 ± 0.005) (d0/d4) + (0.025 ± 0.003) with r2 = 0.999 was observed as a function of mole ratio of unlabeled (d0) to deuterated (d4) peptides.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Linearity and precision of isotopologue ratio measurements. Data shown are the measured ion current (I0/I4) ratios for each peptide listed in Table 1 for samples prepared with defined mole d0/d4 ratios ranging from 0:1 to 1:1 d0- to d4-labeled peptides. A linear response in the measured ion current ratio, I0/I4 = (0.956 ± 0.005) (d0/d4) + (0.025 ± 0.003) with r2 = 0.999 was observed as a function of mole ratio of unlabeled (d0) to deuterated (d4) peptides.
Mentions: The peptides generated from the digestion of the isolated troponin protein-complex were differentially labeled with propyl-d0 or -d4 and then mixed with a mole ratio ranging from 0:1 to 1:1 d0-labeled peptides to d4-labeled peptides and measured by ESI-LC−MS/MS. Figure 4 shows the measured ion current ratios for each set of isotopologues listed in Table 1. This graph demonstrates linearity and precision of the d0/d4 peptide ratio measurement for a range of peptides.

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
Related in: MedlinePlus