Limits...
Targeted proteomic quantitation of the absolute expression and turnover of cystic fibrosis transmembrane conductance regulator in the apical plasma membrane.

McShane AJ, Bajrami B, Ramos AA, Diego-Limpin PA, Farrokhi V, Coutermarsh BA, Stanton BA, Jensen T, Riordan JR, Wetmore D, Joseloff E, Yao X - J. Proteome Res. (2014)

Bottom Line: It was found that CFBE 41o- cells expressing wild-type CFTR (wtCFTR), when cultured on plates, had 2.9 ng of the protein in the apical PM per million cells; this represented 10% of the total CFTR found in the cells.The absolute quantitation and turnover measurements of CFTR in the apical PM can significantly facilitate understanding the disease mechanism of CF and thus the development of new disease-modifying drugs.Absolute CFTR quantitation allows for direct result comparisons among analyses, analysts, and laboratories and will greatly amplify the overall outcome of CF research and therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Connecticut , Storrs, Connecticut 06269, United States.

ABSTRACT
Deficient chloride transport through cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes lethal complications in CF patients. CF is the most common autosomal recessive genetic disease, which is caused by mutations in the CFTR gene; thus, CFTR mutants can serve as primary targets for drugs to modulate and rescue the ion channel's function. The first step of drug modulation is to increase the expression of CFTR in the apical plasma membrane (PM); thus, accurate measurement of CFTR in the PM is desired. This work reports a tandem enrichment strategy to prepare PM CFTR and uses a stable isotope labeled CFTR sample as the quantitation reference to measure the absolute amount of apical PM expression of CFTR in CFBE 41o- cells. It was found that CFBE 41o- cells expressing wild-type CFTR (wtCFTR), when cultured on plates, had 2.9 ng of the protein in the apical PM per million cells; this represented 10% of the total CFTR found in the cells. When these cells were polarized on filters, the apical PM expression of CFTR increased to 14%. Turnover of CFTR in the apical PM of baby hamster kidney cells overexpressing wtCFTR (BHK-wtCFTR) was also quantified by targeted proteomics based on multiple reaction monitoring mass spectrometry; wtCFTR had a half-life of 29.0 ± 2.5 h in the apical PM. This represents the first direct measurement of CFTR turnover using stable isotopes. The absolute quantitation and turnover measurements of CFTR in the apical PM can significantly facilitate understanding the disease mechanism of CF and thus the development of new disease-modifying drugs. Absolute CFTR quantitation allows for direct result comparisons among analyses, analysts, and laboratories and will greatly amplify the overall outcome of CF research and therapy.

Show MeSH

Related in: MedlinePlus

LC–SID–MRM MS chromatograms of signaturepeptidesfor quantifying CFTR in the apical PM of CFBE cells grown on filters.Peptides are denoted as in Table 1.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4227562&req=5

fig3: LC–SID–MRM MS chromatograms of signaturepeptidesfor quantifying CFTR in the apical PM of CFBE cells grown on filters.Peptides are denoted as in Table 1.

Mentions: The CFBE cell line is a commonmodel for CF research and drug development. We performed absolutequantitation of apical PM CFTR in CFBE 41o- cells grown on platesand snapwell filters via LC–MRM MS of signature peptides. Threesignature peptides, CFTR01, CFTR02, and CFTR04, were monitored byLC–SID–MRM MS (Figure 3), butonly CFTR01 and CFTR02 peptides were consistently able to be quantifiedfor measuring their precursor CFTR protein in the PM together withCFTR in the cytoplasm (i.e., CFTR in the Triton X-100 extract afterremoving PM CFTR). Quantitation results based on CFTR01 and CFTR02were comparable (Table 2). Each sample wasrepeated with at least three different biological preparations. Resultsare summarized in Table 4. As expected, surfaceexpression of CFTR in CFBE cells (14.1% of CFTR in the whole celllysate) grown in snapwell filters is higher than that of CFBE cellsgrown in flasks (10.1%). This is in agreement with semiquantitativewestern blot analysis (Supporting Information Figure S4). CFBE cells grown on snapwell filters mimic airway epithelialcells, with the air–liquid interface creating a polarized cellenvironment, increasing the PM expression of CFTR. The absolute quantitationmethod reported in this work will also enable accurate measurementsof CFTR in primary cells. Absolute quantitation of PM CFTR will setthe basis for the comparison of results and thus will greatly amplifythe overall outcome of CF research and therapy. In reference, highreproducibility for LC–SID–MRM MS quantitation of proteintargets in complex matrices has recently been shown for sample-to-sample,day-to-day, analyst-to-analyst, and lab-to-lab comparisons.26


Targeted proteomic quantitation of the absolute expression and turnover of cystic fibrosis transmembrane conductance regulator in the apical plasma membrane.

McShane AJ, Bajrami B, Ramos AA, Diego-Limpin PA, Farrokhi V, Coutermarsh BA, Stanton BA, Jensen T, Riordan JR, Wetmore D, Joseloff E, Yao X - J. Proteome Res. (2014)

LC–SID–MRM MS chromatograms of signaturepeptidesfor quantifying CFTR in the apical PM of CFBE cells grown on filters.Peptides are denoted as in Table 1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4227562&req=5

fig3: LC–SID–MRM MS chromatograms of signaturepeptidesfor quantifying CFTR in the apical PM of CFBE cells grown on filters.Peptides are denoted as in Table 1.
Mentions: The CFBE cell line is a commonmodel for CF research and drug development. We performed absolutequantitation of apical PM CFTR in CFBE 41o- cells grown on platesand snapwell filters via LC–MRM MS of signature peptides. Threesignature peptides, CFTR01, CFTR02, and CFTR04, were monitored byLC–SID–MRM MS (Figure 3), butonly CFTR01 and CFTR02 peptides were consistently able to be quantifiedfor measuring their precursor CFTR protein in the PM together withCFTR in the cytoplasm (i.e., CFTR in the Triton X-100 extract afterremoving PM CFTR). Quantitation results based on CFTR01 and CFTR02were comparable (Table 2). Each sample wasrepeated with at least three different biological preparations. Resultsare summarized in Table 4. As expected, surfaceexpression of CFTR in CFBE cells (14.1% of CFTR in the whole celllysate) grown in snapwell filters is higher than that of CFBE cellsgrown in flasks (10.1%). This is in agreement with semiquantitativewestern blot analysis (Supporting Information Figure S4). CFBE cells grown on snapwell filters mimic airway epithelialcells, with the air–liquid interface creating a polarized cellenvironment, increasing the PM expression of CFTR. The absolute quantitationmethod reported in this work will also enable accurate measurementsof CFTR in primary cells. Absolute quantitation of PM CFTR will setthe basis for the comparison of results and thus will greatly amplifythe overall outcome of CF research and therapy. In reference, highreproducibility for LC–SID–MRM MS quantitation of proteintargets in complex matrices has recently been shown for sample-to-sample,day-to-day, analyst-to-analyst, and lab-to-lab comparisons.26

Bottom Line: It was found that CFBE 41o- cells expressing wild-type CFTR (wtCFTR), when cultured on plates, had 2.9 ng of the protein in the apical PM per million cells; this represented 10% of the total CFTR found in the cells.The absolute quantitation and turnover measurements of CFTR in the apical PM can significantly facilitate understanding the disease mechanism of CF and thus the development of new disease-modifying drugs.Absolute CFTR quantitation allows for direct result comparisons among analyses, analysts, and laboratories and will greatly amplify the overall outcome of CF research and therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Connecticut , Storrs, Connecticut 06269, United States.

ABSTRACT
Deficient chloride transport through cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes lethal complications in CF patients. CF is the most common autosomal recessive genetic disease, which is caused by mutations in the CFTR gene; thus, CFTR mutants can serve as primary targets for drugs to modulate and rescue the ion channel's function. The first step of drug modulation is to increase the expression of CFTR in the apical plasma membrane (PM); thus, accurate measurement of CFTR in the PM is desired. This work reports a tandem enrichment strategy to prepare PM CFTR and uses a stable isotope labeled CFTR sample as the quantitation reference to measure the absolute amount of apical PM expression of CFTR in CFBE 41o- cells. It was found that CFBE 41o- cells expressing wild-type CFTR (wtCFTR), when cultured on plates, had 2.9 ng of the protein in the apical PM per million cells; this represented 10% of the total CFTR found in the cells. When these cells were polarized on filters, the apical PM expression of CFTR increased to 14%. Turnover of CFTR in the apical PM of baby hamster kidney cells overexpressing wtCFTR (BHK-wtCFTR) was also quantified by targeted proteomics based on multiple reaction monitoring mass spectrometry; wtCFTR had a half-life of 29.0 ± 2.5 h in the apical PM. This represents the first direct measurement of CFTR turnover using stable isotopes. The absolute quantitation and turnover measurements of CFTR in the apical PM can significantly facilitate understanding the disease mechanism of CF and thus the development of new disease-modifying drugs. Absolute CFTR quantitation allows for direct result comparisons among analyses, analysts, and laboratories and will greatly amplify the overall outcome of CF research and therapy.

Show MeSH
Related in: MedlinePlus