for 
 cystic fibrosis ( cf ) , the most common autosomal recessive genetic 
 disease , there is a recent paradigm shift from treating disease symptoms 
 toward developing drugs that repair fundamental defects in the anion 
 channel of cystic fibrosis transmembrane conductance regulator ( cftr ) .
cftr is a member of the atp - binding cassette ( abc ) transporter superfamily 
 and functions as a camp - dependent , protein kinase - activated cl channel in the plasma membrane ( pm ) .
it has five structural 
 domains : two transmembrane domains ( tmd1 and tmd2 ) , two nucleotide - binding 
 domains ( nbd1 and nbd2 ) , and a unique regulatory domain ( rd ) .
deletion 
 of a phenylalanine at position 508 ( f508del ) is the most frequent 
 mutation , accounting for 90% of the cf population .
the f508del 
 mutation is located in nbd1 , putatively interfacing with tmds , and 
 impairs coupled domain folding , pm expression , ion channel function , 
 and protein stability .
the misfolded mutant is retained 
 inside cells at the endoplasmic reticulum ( er ) , and is degraded by 
 the proteasome .
evidence suggests that , 
 during biosynthesis and trafficking of cftr , different domains of 
 the protein interact with various chaperone systems , facilitating 
 the maturation of wild - type cftr ( wtcftr ) and the recognition of the 
 f508del mutant .
thus , f508del expression in the apical pm is minimal ; 
 consequently , f508del cf patients do not have sufficient cftr channel 
 activity in epithelial cells in their airways , intestine , pancreas , 
 sweat ducts , testes , and other fluid - transporting tissues .
two major categories of new cftr modulator drugs are currently 
 under extensive research and clinical investigations .
corrector drugs repair the biogenesis , trafficking , and 
 ultimately the apical pm expression of the protein , and potentiator 
 drugs enable the channel function of mutant cftr proteins in the pm . 

corrector drugs like vx-809 ( lumacaftor ) repair folding and trafficking 
 of cftr mutants and enhance the apical pm expression of f508del .
the rescue of pm expression of 
 mutant cftr , like f508del , by corrector drugs is the first step toward 
 restoring cl channel activity .
however , vx-809 
 has limited clinical benefit for f508del cf patients , and its mechanism 
 of action has yet to be fully understood . in 
 addition
, it is generally agreed that the first generation of cf drugs 
 has reached an apparent therapeutic ceiling .
in - depth analysis of how current 
 investigational drugs work will provide mechanistic insights for developing 
 future cf medicines .
these studies would be greatly enhanced by accurate 
 measurements of cftr and its mutants in cells , especially in the pm , 
 where the protein performs its vital ion channel function .
difficulties 
 in the analysis of cftr are among the major challenges 
 for molecular investigations of cf drug action .
cftr is a large , glycosylated , 
 multidomain , and low - abundance integral membrane protein . in order 
 to evaluate the efficacy of drug compounds ,
historically , 
 western blot analysis of cftr has been the central cftr quantitation 
 method in cf research ; newly developed 
 antibodies have greatly improved the sensitivity of this method . however , the reproducibility , precision , accuracy , 
 and robustness of the method are less than ideal , providing only semiquantitative 
 measurements , with a relatively large coefficient of variance and 
 a narrow dynamic range . furthermore , cftr aggregates and degrades 
 during sample preparations , leading to large measurement variations . 
 these critical problems with membrane proteins present
major , intrinsic 
 challenges for analysis . other methods for quantifying 
 surface expression of cftr require engineered cells [ e.g. , green fluorescent 
 fusion proteins ( gfp ) ] and are not directly applicable to primary 
 cells , which are better systems for testing new experimental drugs .
technology innovations , from genomics to proteomics , have played important roles in 
 advancing cf research and therapy .
we have developed the first quantitative 
 method to mitigate the intrinsic analytical problems with full - length 
 cftr and use cftr signature peptides as measurement surrogates . cftr is digested into peptides that are relatively 
 easy to prepare for mass spectrometry ( ms ) quantitation .
cftr quantified 
 in this work ranges from a few tens of picograms to low nanograms 
 per million baby hamster kidney cells overexpressing wtcftr ( bhk - wtcftr ) 
 or human colorectal adenocarcinoma cells ( ht-29 ) .
a signature peptide is selected for quantitation by a method 
 of liquid chromatography  stable isotope dilution  multiple 
 reaction monitoring ms ( lc  sid  mrm ms ) .
it is the method 
 of choice for quantifying target proteins in complex biomatrices , which is regarded as the ms version of western blot analysis , and 
 has comparable sensitivity to that of western blot analysis but superior 
 specificity .
mrm ms monitors gas - phase dissociation reactions of target 
 analytes , which requires sequential detection of an analyte precursor 
 ion as the reactant followed by one or several analyte fragment ions 
 as the products ; therefore , it is highly specific and can be applied 
 to complex samples with minimal component separation .
the sensitivity 
 and specificity afforded by the mrm ms method removes the dependence 
 on high - quality antibodies for conventional immunoassays of protein 
 targets like cftr .
however , later 
 addition of peptide quantitation reference standards 
 can not be used to normalize ( 1 ) differential sample loss during cftr 
 enrichment ( cftr has low concentration in the pm , and it is essential 
 to enrich the protein to obtain the needed quantitation limit ) and 
 ( 2 ) variations in proteolytic digestion of cftr ( membrane proteins 
 are difficult to digest completely ) .
thus , 
 peptide - level quantitation reference standards are less than ideal 
 for the absolute quantitation of cftr .
more reliable absolute quantitation 
 of pm cftr will enable accurate and precise sample - to - sample , day - to - day , 
 analyst - to - analyst , and lab - to - lab comparisons of results , similar 
 to that shown for lc 
mrm ms quantitation of plasma 
 proteins , and significantly facilitate 
 the development of new cf drugs .
herein , we report a general workflow 
 ( scheme 1 ) for the enrichment and absolute 
 quantitation of large , low - abundance cftr protein ( and other membrane 
 proteins ) in the apical pm ( broadly referred to as the fraction of 
 pm that is not in contact with the culture support and thus is accessible 
 to cell surface biotinylation ) of bhk - wtcftr and cfbe cells . with 
 a variation of this method , cftr turnover in the apical pm of bhk - wtcftr 
 cells is measured for the first time without using radioisotopes .
urea , tris base , iodoacetamide , 
 acrylamide / bis - acrylamide solution ( 40% , 29:1 ) , n , n , n,n-tetramethylethylenediamine , l - leucine , l - lysine , l - arginine , methotrexate , doxycycline , protease 
 inhibitor cocktail , sodium orthovanadate , 1 m tris - hcl buffer solution , 
 phosphate buffer saline ( pbs , 10 , ph 7.4 ) , triton x-100 , and 
 the anhydrides were purchased from sigma - aldrich ( st .
louis , mo ) . 
 pierce cell surface protein isolation kit , dithioerythritol ( dte ) , 
 -mercaptoethanol ( -me ) , trifluoroacetic acid ( tfa ) , 
 ammonium bicarbonate , ammonium persulfate , formic acid , and high - performance 
 liquid chromatography ( hplc ) grade acetonitrile were purchased from 
 fisher scientific ( fair lawn , nj ) .
the peptide cftr01 was synthesized 
 by anaspec ( san jose , ca ) or peptide 2.0 ( chantilly , va ) .
isotopic 
 cftr01 peptide ( nsiltet[l - c6n]hr was synthesized by cambridge research biochemicals ( cleveland , 
 uk ) .
o - water ( > 97% ) was purchased from cambridge isotope 
 laboratories ( andover , ma ) or given as a gift from olinax ( hamilton , 
 on , canada ) .
water was obtained from a milli - q ultrapure water purification 
 system ( millipore , billerica , ma ) .
proteomics grade recombinant trypsin 
 was purchased from roche applied science ( indianapolis , in ) .
human 
 embryonic kidney cell line ( hek293f ) stably transfected with fused 
 cdna for wtcftr and gfp ( hek293f - d042-jk ) , hek293f - wtcftr , was a gift 
 from dr .
john c. kappes ( university of alabama at birmingham ) . a cystic 
 fibrosis bronchial - derived cell line complimented with a 4.7 kb wild - type 
 cftr cdna ( cfbe 41o-/pcep - cftr n 4.7 kb ) and a cystic fibrosis bronchial - derived 
 cell line complimented with a 4.7 kb f508del cftr cdna ( cfbe 41o-/pcep - cftr 
 f508del 4.7 kb ) were from dr .
the ht-29 cell line 
 was purchased from american type culture collection ( manassas , va ) . 

purification of the later full - length cftr sample was 
 performed according to a procedure developed by dr .
the absolute concentration 
 of this highly purified cftr sample was obtained by amino acid analysis , 
 and this sample was used as the reference standard to quantify stable 
 isotope labeled cftr samples prepared in - house ( see the following 
 section ) .
amino acid analysis for the highly purified , native cftr 
 was performed at the keck biotechnology resource laboratory at yale 
 university .
dmem , ham s f-12 , dme / low media deficient in l - arginine , l - leucine , and l - lysine , and fetal 
 bovine serum were purchased from thermo scientific hyclone ( logan , 
 ut ) .
dialyzed fetal bovine serum was purchased from life technologies 
 ( thermo , carlsbad , ca ) .
bhk - wtcftr cells were cultured in dmem / ham s 
 f-12 with 550 m methotrexate and 10% of each of the following 
 reagents : fetal bovine serum , pen  strep , nonessential amino 
 acids , and sodium pyruvate . the heavy isotope labeled , full - length 
 cftr was obtained by the method of stable isotope labeling by amino 
 acids in cell culture ( silac ) from bhk - wtcftr 
 cells cultured in dme / low deficient in l - lysine , l - leucine , and l - arginine supplemented with 550 m 
 methotrexate , 0.4 mm l - argininehcl , 0.8 mm l - lysinehcl , 0.8 mm l - leucine-1,2-c ( cambridge 
 isotopes , andover , ma ) , and 10% dialyzed fetal bovine serum . cells 
 grown in this labeled media were allowed at least 78 doublings 
 prior to harvesting .
cfbe 41o-/pcep - cftr n 4.7 kb cells were grown 
 in mem medium with 0.3 g / l l - glutamine , 1 g / l glucose , 2.2 
 g / l nahco3 , 10% fetal bovine serum , 20 mm sodium pyruvate , 
 10  000 g / ml streptomycin , and 10  000 units / ml 
 penicillin .
the cfbe cells were grown in plates or snapwell filters 
 ( sigma - aldrich , st .
louis , mo ) that were coated with human fibronectin 
 ( fisher scientific , fair lawn , nj ) .
the hek293f - wtcftr cells stably 
 transfected with wtcftr - gfp fused cdna were cultured using dmem with 
 10% fetal bovine serum , and doxycycline was added at 0 , 0.25 , 0.75 , 
 or 1.00 g / ml the night before cell harvesting .
surface biotinylation was performed on cells using the surface protein isolation kit from 
 pierce , following the manufacturer s instructions .
briefly , 
 cells were first labeled with a thiol - cleavable , amine - reactive biotinylation 
 reagent [ sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate 
 ( sulfo - nhs - ss - biotin ) ] and quenched .
the biotinylated cells were dislodged 
 by trypsinization or scraping from the plates or filters and subsequently 
 lysed with buffer . the lysis buffer ( 800 l ) containing 1% ( v / v ) 

triton x-100 , 150 mm nacl , 20 mm tris - hcl at ph 7.2 , and 1% ( v / v ) 
 protease inhibitors was used for the lysis of 1214 million 
 bhk - wtcftr cells or 68 million cfbe cells .
the labeled proteins 
 were captured on avidin agarose and released using triton x-100 buffer 
 containing 50 mm dithiothreitol ( dtt ) .
gel electrophoresis was 
 performed using a resolving gel consisting 
 of 8% acrylamide and 0.2% bis(acrylamide ) .
biorad glass plates with 
 1.0 mm spacer plates and a 10-well comb were used for analysis of 
 overexpressed cftr ; a single well allowing for a 20 l loading 
 volume was sufficient for analysis of overexpressed cftr in about 
 8 g of total biotinylated protein . for endogenous cftr , 1.5 
 mm spacer plates and a 2-well comb consisting of 1 preparative well 
 for sample loading ( 680 l , 500 g of total biotinylated 
 proteins ) and 1 well for loading the standard molecular weight protein 
 marker or purified cftr were used . for the turnover study , 1.5 mm 
 spacer plates and a 5-well ( 120 l )
cftr samples 
 were mixed with aemmli sample buffer containing 20% ( v / v ) -me 
 and incubated at 30 c for 30 min .
gel electrophoresis was carried 
 out at 200 v constant voltage for 45 min or until the dye front reached 
 the bottom of the gel .
gel 
 pieces at the interface 
 between the resolving and stacking gels ( 1 mm ) 
 were excised for further sample preparation .
gel pieces were washed 
 ( 25 mm ammonium bicarbonate in h2o / acetonitrile ) , reduced 
 ( 10 mm dte ) , and alkylated ( 20 mm iodoacetamide ) before trypsinization 
 ( 20 ng/l ) .
the resulting digestion solution was dried via speedvac 
 ( savant , farmingdale , ny ) and overnight lyophilization ( labconco , 
 kansas city , mo ) .
the optimization 
 and data analysis of this work were assisted by the skyline software .
the preparation of stable isotope reference o(4)-cftr01 and lc  sid  mrm ms quantitation 
 were performed following previously reported procedures .
some exceptions were those experiments where 
 cftr protein quantitation was accomplished in cfbe and bhk - wtcftr 
 cells . in those cases ,
an eksigent nanolc - ultra 2d ( redwood 
 city , ca ) was used at a flow rate of 400 nl / min with a self - pack picofrit 
 column ( new objective , woburn , ma ) filled with 2.7 m diameter , 
 160  pore halo resin ( macmod , chadds ford , pa ) , and the length 
 of resin bed was around 15 cm .
solvent a was composed of 98.8% h2o , 1.0% acetonitrile , and 0.2% formic acid ( v / v ) , and solvent 
 b was composed of 98.8% acetonitrile , 1.0% water , and 0.2% formic 
 acid ( v / v ) .
a typical running gradient was 1% b at 0 min  
 3% b at 5 min  30% b at 50 min  80% b at 59 min  
 90% b at 69 min  1% b at 80 min , with a 10 min equilibration . 

samples were loaded on the trap column at flow rate of 4 l / min 
 at 1% solvent b for 10 min .
a triple quadruple mass spectrometer was 
 used , 4000 qtrap from absciex ( foster city , ca ) .
transitions for the 
 native and stable isotope labeled peptides are summarized in table 1 .
x denotes 
 a mass increase , i.e. , 4 means a mass increase of 4 da for 
 the isotope - labeled peptide compared with the native counterpart . following in - gel digestion of the hek293f - wtcftr cell lysate
, 
 the n - termini of the peptide mixtures were derivatized with anhydrides 
 ( acetic , propionic , butyric , valeric , or succinic anhydride ) . to each 
 sample
were added 70 l of acetonitrile , 70 l of 1 m 
 ammonium bicarbonate , and 10 l of the appropriate anhydride , 
 and samples were incubated on ice for 1 h. following the incubation , 
 another addition of 100 l of acetonitrile , 100 l of 
 1 m ammonium bicarbonate , and 10 l of the appropriate anhydrate 
 were added .
samples were then incubated on ice for an additional 2 
 h. after the chemical derivatization , the modified peptides were dried 
 and desalted using hydrophilic  lipophilic - balanced sorbent 
 ( hlb , oasis ) and lyophilized .
the resulting five derivatized digests 
 were combined for a single umrm ms experiment . in a typical 
 experiment , bhk - wtcftr cells for each time point were grown in triplicate 
 in nonisotopic media . at time zero , the media was aspirated , the cells 
 were washed twice with pbs , and isotopically labeled media was added 
 as follows : ( 1 ) bhk - wtcftr media minus arginine , leucine , and lysine 
 and supplemented with 0.4 mm l - argininehcl , 0.8 mm l - lysinehcl , and 0.8 mm l - leucine-1,2-c2 or ( 2 ) bhk - wtcftr media minus arginine , leucine , and 
 lysine and supplemented with 0.4 mm l - arginine - c6hcl ( cambridge isotopes , andover , ma ) , 0.8 mm l - lysine - c6n2hcl ( cambridge isotopes , andover , ma ) , and 0.8 mm l - leucine . at the appropriate time , the cells were biotinylated , combined , 
 and fractionated as previously detailed .
after protein digestion , 
 signature peptides for native and stable isotope labeled cftr were 
 quantified by lc 
membrane proteins are critical for cells to communicate with the 
 extra - cellular environment .
receptors and transporters in the pm thus 
 form the most dominant protein class for developing drugs to treat 
 human diseases .
preparation 
 of membrane proteins and proteome samples is a major analytical bottleneck 
 in analysis .
we have thus designed a novel , 
 broadly applicable workflow to enrich low - abundance , large membrane 
 proteins by sequential use of ( 1 ) the established method of cell surface 
 biotinylation and ( 2 ) a method of gel electrophoretic enrichment ( gee ) , 
 which is a variant of gel electrophoresis ( scheme 1 ) . in this work ,
we have established a workflow for enriching 
 pm cftr and evaluated experimental variations in major steps of the 
 workflow ( see supporting information text 
 s1 , figure s1 , and tables s1 and s2 ) .
gee makes special use 
 of sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) 
 for separation of protein mixtures . in the gee method ,
nongradient 
 gels are used for protein fractionation , and the gel compositions 
 are formulated in such a way that proteins larger than a minimum molecular 
 weight do not migrate appreciably in the resolving gel .
it is a simple 
 but effective approach for enriching high - molecular - weight proteins 
 ( e.g. , > 150 kda ) in a complex mixture ( figure 1 ) . for enriching cftr ,
the composition for the resolving gel 
 is 8% 
 acrylamide and 0.2% bis(acrylamide ) .
this composition slows the migration 
 of large proteins and limits their location to the interface region 
 of the resolving and stacking gel while allowing for the electrophoretic 
 migration of smaller proteins . with or without gel staining ,
a protein 
 fraction with a predictable lower cutoff of molecular weight ( i.e. , 
 proteins that have molecular weights higher than a designed value ) 
 can be reproducibly sampled by excising gel slices at a fixed location 
 near the interface ( figure 1 ) for subsequent 
 in - gel digestion and ms analysis .
the large proteins are sampled by slicing the gel piece at the interface 
 of the stacking gel and the resolving gel ( a ) .
samples loaded are 
 as follows : 1 , protein ladder ; 2 , -galactosidase ; 3 , 500 ng 
 of cftr ; 4 , 1 g of cftr ; 5 , 10 g of bhk - wtcftr 
 biotinylated fraction ; 6 , 1 g of bhk - wtcftr biotinylated fraction ; 
 7 , 30 g of bhk - wtcftr triton x-100 extract ; and 8 , 3 g 
 of bhk - wtcftr triton x-100 extract . the gel piece ( red box )
was cut 
 for in - gel digestion ( b ; other bands were relatively low - molecular - weight 
 membrane proteins removed by gee ) , and the resulting peptides were 
 extracted ( c ) and analyzed by lc 
expression of cftr in the pm is 
 low , and the tandem enrichment 
 strategy made cftr quantitation possible by lc 
mrm 
 ms , without using antibody enrichment 
 of the protein ; this is due to both the 
 increase in cftr concentration and the decrease in the sample complexity 
 for ms quantitation .
mutant cftr proteins , even after therapeutic 
 intervention , exist in even lower amounts in the pm ; thus , enrichment 
 of pm cftr is necessary for quantifying the protein by ms .
mrm ms quantitation 
 of cftr signature peptides prepared by the tandem enrichment workflow 
 with those in the direct digestion mixture of the pm subproteome prepared 
 by surface biotinylation and avidin pull - down for ht-29 cells .
only 
 the former workflow resulted in confident quantitation , and the measurement 
 gave 40 fmol of apical pm cftr per million cells , equivalent to 23  000 
 molecules per cell ( figures 1 and 2 ) .
it should be noted that this amount is based 
 on a peptide quantitation reference standard [ o(4)-cftr01 ] and thus represents the lower range of pm cftr 
 in ht-29 cells ( see later for a discussion on protein vs peptide quantitation 
 reference standards ) .
although the gee method by itself can be used 
 for preparing cftr signature peptides from various protein ( mixture ) 
 samples containing cftr , it is necessary to have the surface biotinylation 
 enrichment in the workflow . thus , only matured full - length cftr ( commonly 
 referred to as band c by the cf research 
 and clinical community ) in the pm is sampled for quantitation ; the 
 gee method does not separate the nonglycosylated cftr ( commonly referred 
 to as band b ) from that which is fully 
 matured .
it is essential to use a tandem enrichment strategy for preparing 
 pm cftr samples .
mrm ion chromatograms are shown for the native signature 
 peptide ( solid line ) and the spiked stable isotope reference peptide 
 ( dotted line ) of digests of ht-29 cell lysate prepared by surface 
 biotinylation and gee ( a ) and surface biotinylation only ( b ) .
another analytical advantage for 
 the gee method is compatibility 
 with various solubilizing reagents used for preparing samples containing 
 cftr . to enhance solubilization of membrane proteins ,
however , 
 the presence of detergents can interfere with protease activity during 
 protein digestion and can also affect chromatographic separation and 
 suppress ionization of resulting peptides .
although the detergents 
 can be removed after digestion , cleanup of detergent - containing peptide 
 mixtures can cause significant sample loss and has varying efficiencies . 

in contrast , during the gel - based sample preparations , detergents 
 and other interfering reagents are removed by extensive washing before 
 digestion .
it has been reported that protein mixtures can be concentrated 
 to a thin band after a short - running sds - page ; the main purpose of 
 this practice has been the removal of detergents in membrane protein 
 preparations for enhanced ms - based proteomic analysis , not to enrich low - abundance proteins based on their size . compared 
 to the in - solution digestion , the in - gel digestion also increases 
 the digestion efficiency for membrane proteins ; this is possibly attributed to the decreased protein aggregation 
 during digestion . by immobilizing hydrophobic membrane proteins in 
 the gel matrix , aggregation of membrane proteins and large protein 
 fragments produced at the initial stage of the protein digestion is 
 likely to be minimized .
the full - length 
 cftr quantitation reference standard was prepared through metabolic 
 labeling using the silac method .
lysates 
 of bhk - wtcftr cells cultured with media containing leucine-1,2-c cftr ( seven doublings ) were prepared by a buffer containing 
 triton x-100 . the silac cftr amount in the triton x-100
extract was 
 quantified against a highly purified , full - length native cftr standard 
 ( this native cftr could be used as the master reference standard for 
 different laboratories ) using the gee method for sample preparation 
 and lc  sid  mrm ms for cftr quantitation .
concentration 
 of native cftr was determined by amino acid analysis to be 0.175 ( 0.025 ) 
 g/l .
the quantity of silac cftr was measured in triplicate 
 using two signature peptides ( cftr01 and cftr02 ) .
the protein concentration 
 in the silac triton x-100 extract was determined to be 4.72 ( 0.07 ) 
 ng/l ( table 2 ) .
it should be noted that 
 the silac cftr reference standard was used as a triton x-100 extract 
 directly for quantifying cftr samples .
the area ratios 
 between the native 
 peptides and heavy isotope labeled peptides were used to calculate 
 the concentration of silac cftr in cell lysate , which was labeled 
 with leucine-1,2-c .
highly - purified full - length cftr 
 ( 300 ng ) was used to calibrate the absolute amount of silac cftr . 

a quantitative comparison was performed to examine the difference 
 in cftr between using the silac cftr protein standard and using a 
 stable isotopic synthetic peptide , l(7)-cftr01 .
hek293f - wtcftr 
 cells , which overexpress wtcftr upon induction with doxycycline at 
 different concentrations ( 0 , 0.25 , 0.50 , 0.75 , and 1.0 g / ml ) , 
 were used .
apical pm cftr was prepared by the tandem enrichment workflow 
 ( scheme 1 ) .
one additional quantitation reference , 
 peptide l(7)-cftr01 , was added to the peptide mixtures resulting 
 from in - gel digestion .
furthermore , five different samples were derivatized 
 with different acid anhydrates ( acetic , propionic , butyric , valeric , 
 or succinic anhydride ) ; with this sample - specific coding procedure , 
 all five samples were quantified in a single lc 
the signature peptide 
 cftr01 was monitored in this study ( table 3 ; also see supporting information figures 
 s2 and s3 ) .
expression of native cftr in these doxycycline - inducible 
 hek293f - wtcftr cells was determined according to both l(4)-cftr01 
 and l(7)-cftr01 . while both data sets observed increased expression 
 of cftr , with the increase in the doxycycline concentration ,
cftr 
 amounts measured according to the peptide standard l(7)-cftr01 
 ( added after in - gel - digestion of cftr ) were only 1417% of 
 the corresponding measurements based on l(4)-cftr01 , produced 
 via in - gel digestion of silac cftr ( table 3 ) .
this study shows that for absolute quantitation of membrane proteins 
 like cftr , it is essential to use protein standards to obtain better 
 quantitation accuracy . area ratios 
 ( native / silac ) were 
 used to calculate expressed native cftr .
relative areas for 
 l(4 ) and 
 l(7 ) peptides , together with the known amounts of silac cftr 
 and l(7)-cftr01 , gave measurements for the digestion efficiency 
 for cftr .
these numbers should represent the lower limits for the 
 digestion efficiency because sample loss during gee and peptide preparation 
 could also be attributed to the calculated efficiency .
the use of an isotopic protein reference 
 allows for advantageous 
 addition of an internal standard at an early step of sample preparation . 
 for the pm cftr quantitation
, silac cftr was added as the reference 
 in the surface biotinylation fraction of membrane proteins before 
 further sample preparations . in comparison ,
mrm 
 ms uses isotopic reference peptides . for absolute quantitation of membrane proteins , peptide references 

membrane proteins are hard to digest ; in a typical 
 experiment , the yield of signature peptide cftr01 is only 1417% , 
 and this yield varies from one sample preparation to another ( table 3 ) .
furthermore , cftr is prone to aggregation and 
 degradation during sample preparation ; for instance , preparation of 
 cftr for sds - page is performed at 30 c instead of boiling temperature . 

addition of silac cftr before gee and protein digestion largely minimized 
 variations in sample preparation and afforded the absolute quantitation 
 of cftr in cfbe cells . the cfbe cell line is a common 
 model for cf research and drug development .
we performed absolute 
 quantitation of apical pm cftr in cfbe 41o- cells grown on plates 
 and snapwell filters via lc 
three 
 signature peptides , cftr01 , cftr02 , and cftr04 , were monitored by 
 lc  sid  mrm ms ( figure 3 ) , but 
 only cftr01 and cftr02 peptides were consistently able to be quantified 
 for measuring their precursor cftr protein in the pm together with 
 cftr in the cytoplasm ( i.e. , cftr in the triton x-100 extract after 
 removing pm cftr ) .
as expected , surface 
 expression of cftr in cfbe cells ( 14.1% of cftr in the whole cell 
 lysate ) grown in snapwell filters is higher than that of cfbe cells 
 grown in flasks ( 10.1% ) .
this is in agreement with semiquantitative 
 western blot analysis ( supporting information figure s4 ) .
cfbe cells grown on snapwell filters mimic airway epithelial 
 cells , with the air  liquid interface creating a polarized cell 
 environment , increasing the pm expression of cftr .
the absolute quantitation 
 method reported in this work will also enable accurate measurements 
 of cftr in primary cells .
absolute quantitation of pm cftr will set 
 the basis for the comparison of results and thus will greatly amplify 
 the overall outcome of cf research and therapy . in reference ,
mrm ms quantitation of protein 
 targets in complex matrices has recently been shown for sample - to - sample , 
 day - to - day , analyst - to - analyst , and lab - to - lab comparisons .
lc  sid  mrm ms chromatograms of signature 
 peptides 
 for quantifying cftr in the apical pm of cfbe cells grown on filters . 

we designed 
 and performed the first quantitative analysis of cftr 
 turnover in the pm using stable isotopes .
bhk - wtcftr cells were originally 
 cultured in native culture media . at time zero
, culture media were 
 switched to silac media containing stable isotope labeled lysine and 
 arginine or leucine ; thus , native cftr in the pm started to decrease 
 with time , and silac cftr labeled with the stable isotopes started 
 to appear and increase . at different time intervals ,
cells were harvested , 
 and the protein fraction containing pm cftr was processed according 
 to the tandem enrichment workflow ( scheme 1 ) .
native cftr in the pm was quantified against the newly synthesized 
 isotopic cftr by lc 
the time course for the newly produced cftr was followed for up to 
 60 or 84 h , through two experiments .
the average area ratio of the 
 signature peptides from the newly incorporated cftr versus the native 
 cftr ( isotopic / native ) was normalized to the maximum , and then the 
 percentage of remaining native cftr ( denoted as r in figure 4 ) was calculated .
the natural logarithm of r ( for 
 data points up to 48 h ; incorporation of isotope labels were saturated 
 beyond 48 h ) was plotted against time and fitted according to first - order 
 kinetics with an adjusted r of 0.9496 .
the half - life ( t1/2 ) of native cftr in the pm was calculated to be 29.0  2.5 h. turnover 
 of cftr in the pm of bhk - wtcftr cells .
r represents normalized 
 percentage degradation of native cftr ( see results 
 and discussion ) .
blue and red data points are from two separate 
 sets of experiments : isotopic leucine and isotopic arginine / lysine 
 cftr incorporation , respectively .
peptides cftr01 , 03 , and 04 were 
 monitored for the blue data points , and the individual transitions 
 were used for the error calculations .
peptides cftr0104 were 
 monitored for the red data points , and analytical triplicate measurements 
 were used for error calculations [ slope = 0.0239  0.0021 ; 
 adjusted r = 0.9496 ] .
rescuing cftr mutant proteins for expression in 
 the apical pm is 
 only the first step .
the rescued mutants have to be stable in the 
 membrane and perform the channel function for cl ions .
it has been recently reported that the cf drug vx-809 can 
 fully rescue the pm expression of f508del , but the lifespan of the 
 rescued mutant falls short compared with that of wtcftr .
conventional western blot analysis of cftr can not 
 be applied for turnover studies of pm cftr .
although metabolic pulse  chase 
 of radioisotopes provides a useful tool for this analysis , a more convenient method for accurate measurements 
 of the protein turnover in the pm can be greatly beneficial to cf 
 research and drug development .
the combination of metabolic stable 
 isotope labeling and quantitative proteomics is an emerging alternative 
 to radioisotope methods . under this experimental 
 framework ,
coupling the tandem enrichment of cftr with highly sensitive 
 mrm ms has made it possible to measure cftr turnover ( t1/2 = 29.0  2.5 h ) in the pm of bhk - wtcftr cells . 
 in comparison , a shorter half - life ( t1/2 14 h )
was recently reported for cftr in the whole - cell lysate 
 of the same cell line .
these results 
 are in agreement with the observation that pm proteins degrade more 
 slowly than proteins that do not reach the membrane .
membrane 
 receptors and transporters form the most important class 
 of protein targets for understanding intercellular communication and 
 developing new drugs .
recent advances in ms - based quantitative proteomics , 
 in combination with new sample preparation workflows , have made it 
 possible to obtain unprecedented accuracy and precision in membrane 
 protein measurements .
targeted quantitation of apical pm cftr reported 
 in this work exemplifies an important application of contemporary 
 proteomics technologies , and it will significantly help with understanding 
 the disease mechanism of cf and developing future medicine for its 
 treatment .
 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 . 
Introduction
Experimental Section
Results
and Discussion
Conclusions