Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Jun 18;85(12):5699-706.
doi: 10.1021/ac400198n. Epub 2013 May 28.

Phosphopeptide enrichment with TiO2-modified membranes and investigation of tau protein phosphorylation

Affiliations

Phosphopeptide enrichment with TiO2-modified membranes and investigation of tau protein phosphorylation

Yu-Jing Tan et al. Anal Chem. .

Abstract

Selective enrichment of phosphopeptides prior to their analysis by mass spectrometry (MS) is vital for identifying protein phosphorylation sites involved in cellular regulation. This study describes modification of porous nylon substrates with TiO2 nanoparticles to create membranes that rapidly enrich phosphopeptides. Membranes with a 22-mm diameter bind 540 nmol of phosphoangiotensin and recover 70% of the phosphopeptides in mixtures with a 15-fold excess of nonphosphorylated proteins. Recovery is 90% for a pure phosphopeptide. Insertion of small membrane disks into HPLC fittings allows rapid enrichment from 5 mL of 1 fmol/μL phosphoprotein digests and concentration into small-volume (tens of microliters) eluates. The combination of membrane enrichment with tandem mass spectrometry reveals seven phosphorylation sites from in vivo phosphorylated tau (p-tau) protein, which is associated with Alzheimer's disease.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Cartoon of a membrane containing TiO2 for phosphopeptide enrichment and a miniaturized membrane holder with a syringe pump.
Figure 2
Figure 2
Cross-sectional SEM images of nylon membranes before (a) and after (c) coating with a PSS/TiO2 nanoparticle film. Images b and d show the EDX Ti maps for the images in (a) and (c), respectively. In (b) and (d) Ti signals are proportional to the intensity of the red color, and image b indicates the noise of the measurement.
Figure 3
Figure 3
Breakthrough curve for the passage of 20 μM phosphoangiotensin (in loading buffer I, 0.1% TFA) through a 22 mm-diameter nylon membrane modified with a (PSS/TiO2)2 film. The flow rate through the membrane was 2 mL/min. Angiotensin served as an internal standard, and the large error bars (standard deviation of 40%), result from the uncertainty inherent in quantitative analysis based on MALDI-MS.
Figure 4
Figure 4
(a) MALDI mass spectrum of 1 μL of a loading solution containing 30 fmol (each) of digested α-casein, β-casein and ovalbumin and 3 pmol of BSA in 260 mM urea. (b) Mass spectrum of the phosphopeptides enriched from 500 μL of the above loading solution by adsorption on a TiO2-modified nylon membrane, rinsing with 3 mL washing buffer II, and recovery with 10 μL of elution buffer. One μL of the eluate was spotted on the MALDI plate prior to addition of matrix.
Figure 5
Figure 5
MALDI mass spectra of 1-μL eluates from (a) a Titansphere Spin-Tip loaded with 1 mL of 5 fmol/μL digested proteins, (b) a TiO2-modified membrane loaded with 1 mL of 5 fmol/μL digested proteins and (c) a TiO2-modified membrane loaded with 5 mL of 1 fmol/μL digested proteins. Assuming full recovery, each 1-μL eluate contains 500 fmol of phosphopeptides from digested α-casein, β-casein and ovalbumin. Total eluate volumes were 10 μL.
Figure 6
Figure 6
(a) MALDI mass spectrum of a digested FPLC fraction that contained approximately 800 fmol of GSK-3β-p-tau complex along with associated proteins; (b) The corresponding mass spectrum of peptides enriched from the above solution by adsorption on a TiO2-modified nylon membrane and subsequent elution. The figure shows the sequences of 4 phosphopeptides, one of which, P2, results from in-source P3 dephosphorylation. Four major unphosphorylated peptides are labeled with n.
Figure 7
Figure 7
ESI-Orbitrap ETD-MS/MS spectrum of the [M+3H]3+ precursor ion of 386TDHGAEIVYKSpPVVSpGDTSpPR (m/z 819.34): The sequence shows cleavage sites for formation of c and z ions.

Similar articles

Cited by

References

    1. Lee M, Goodbourn S. In: Regulation of Gene Expression. Chapman KE, Higgins SJ, editors. Portland Press; London, UK: 2001. pp. 71–86.
    1. Cohen P. Trends Biochem Sci. 2000;25:596–601. - PubMed
    1. Graves JD, Krebs EG. Pharmacol & Ther. 1999;82:111–121. - PubMed
    1. Hunter T. Cell. 2000;100:113–127. - PubMed
    1. Aebersold R, Mann M. Nature. 2003;422:198–207. - PubMed

Publication types

LinkOut - more resources