Targeted Proteomic Quantitation of the Absolute Expression and Turnover of Cystic Fibrosis Transmembrane Conductance Regulator in the Apical Plasma Membrane

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.

Development of Structural Marker Peptides for Cystic Fibrosis Transmembrane Conductance Regulator in Cell Plasma Membrane by Reversed-Footprinting Mass Spectrometry

“…In contrast, transporter proteins like CFTR exist in small amounts in the cellular plasma membrane, which makes the application of current FPOP-MS practices very challenging, if even possible. We used 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 three strategies in combination to overcome the quantitation challenges for lowabundance membrane proteins treated with FPOP: (1) targeting the remaining, unoxidized peptides for relative quantitation, (2) using highly sensitive and specific multiple reaction monitoring (MRM) MS for targeted quantitation, [30][31][32] and (3) adding the internal protein standard at an early stage of the sample preparation for high quantitation precision. 31,32 Unoxidized peptides are advantageous analytes, versus oxidized peptides, because each oxidized peptide often represents only a tiny fraction of the oxidation product pool.…”

“…32 The chromatogram acquired by data dependent analysis (DDA) observed two major classes of oxidation products-CFTR02+16 Da (addition of an oxygen atom) and CFTR02+14 Da (e.g., addition of one oxygen and deletion of two H atoms to form a C=O bond)-as well as the remaining unoxidized CFTR02 ( (Figure 1e). In striking contrast and expectedly, the unoxidized CFTR02 eluted as a single sharp peak with an intensity to be 32 times higher than the strongest oxidized peptide (extracted ion chromatogram or XIC, solid green in Figure 1d).…”

“…1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 cell culture (SILAC)-wtCFTR BHK cells was used as the internal standard (IS) for quantitation (S6 and S7 in SI). 32 Also a western blot analysis was conducted before addition of SILAC-IS, for confirming the biotinylated CFTR enrichment quality (S8 in SI).…”

Nonisotopic Reagents for a Cost-Effective Increase in Sample Throughput of Targeted Quantitative Proteomics

“…Deficiency in plasma membrane expression and function of the protein results in diminished chloride ion transport, consequently developing into the pathological progression of cystic fibrosis. 28 Peptidyl reagents were activated in situ using DMTMM/ NMM mediation (Scheme S1) forming triazine esters before derivatizing a synthetic peptide mixture ( Figure S1b). The mixture of CFTR01 (1 nmol) and SVI (1 nmol), together with eight other synthetic peptides constituting a total of 20 nmol of peptidyl amine groups, was derivatized with 2.5 μmol (theoretical) of each of the reagent mixtures.…”

“…Additionally, the uMRM technology can also be used in pair with isotopic proteins as quantitation references, which can further eliminate quantitation variations originated from protein-level sample preparations. 28 Another investigation examined the practicality of using reagent candidates for the uMRM MS quantitation of PSA at concentrations that are clinically relevant. 21,30 This investigation used aliquots of a master digest, and each aliquot had an absolute amount of SVI that was equivalent to 1 ng of PSA and a relative concentration of a mixture of 1 ng/mL PSA in 1/ 10 nondepleted female serum (further details in the footnote for Figure S5).…”