Quantification of Transferrin in Human Serum Using Both QconCATand Synthetic Internal StandardsTyler A. Zimmerman,*,†,‡ Meiyao Wang,†,‡ Mark S. Lowenthal,† Illarion V. Turko,†,‡

and Karen W. Phinney†

†Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899 United States‡Institute for Bioscience and Biotechnology Research, 9600 Gudelsky Drive, Rockville, Maryland 20850 United States

*S Supporting Information

ABSTRACT: Transferrin, an iron transport protein, is aclinically important biomarker in diseases such as iron-deficiencyanemia. Current diagnostic methods for transferrin levels lackquantitative accuracy, suggesting the need for alternativeapproaches like LC-MS with isotope-labeled peptides as internalstandards. Besides solid-phase synthesis, isotope-labeled peptidesare also generated by a method called QconCAT where peptidesare expressed from DNA in the presence of heavy isotope media.After evaluation of the expressed QconCAT, this study comparestransferrin levels obtained by synthetic peptides versusQconCAT peptides as internal standards. Transferrin levelsobtained by both internal standards give overlapping, or nearly overlapping, uncertainty values and are near ≈200 mg/dL oftransferrin in human serum. Close agreement between the two methods suggests that the quantitative values are reasonable.Using QconCAT and synthetic peptides in parallel gives a refined focus on method development, and the resulting methodsshould be applicable to other clinically relevant proteins.

Transferrin (≈80 kDa) is a clinically relevant glycoproteinthat transports iron in the circulatory system,1 and its

levels are affected in iron deficiency anemia,2 iron overloaddiseases,3 iron poisoning,4 and hemochromatosis type 1(excessive iron absorption into tissues).5 Transferrin levelsincrease to compensate for lack of iron in iron-deficiencyanemia6 but are conversely lower in iron overload disorders.7

When symptoms of such disorders are present, physiciansspecifically test for transferrin saturation in blood using the totaliron-binding capacity (TIBC) test8,9 that indirectly and ofteninaccurately10−12 infers transferrin levels by colorimetricdetection of iron. The accuracy of the TIBC test is furtherhindered by nonspecific binding of iron to other proteins likealbumin. In comparison, direct measurement of transferrinlevels was judged to be a better biomarker6 and is alsodiagnostically more robust than levels of ferritin, an iron storageprotein. Therefore, transferrin levels in human serum have beenmeasured by chemiluminescence,6 immunochemistry,9 andother clinical techniques.13 The accuracy of these routineclinical measurements exceeds that of the TIBC test, but theystill report large standard deviations of about 40 to 50 mg/dL,which is not ideal when comparing normal levels of transferrin(≈200 mg/dL) to levels in iron-deficiency anemia (≈310 mg/dL)6 and levels in iron overload hemochromatosis (≈112 mg/dL).7 Given the limitations of existing methods, theinvestigation of an alternative approach is warranted. Massspectrometry is attractive for this purpose because it removesthe need for antibodies and gives low detection limits and good

selectivity.14−17 While transferrin has recently been quantifiedin serum by LC-MS,17 the reported work utilized a singlepeptide for quantification. In the current study, we utilize avariety of labeled peptides and compare different labelingapproaches.To quantify proteins in serum using mass spectrometry, the

protein is first digested with trypsin and the tryptic peptides arequantified as measures of protein concentration. The isotope-labeled peptides as internal standards are generated in severalways, including digestion of labeled intact proteins expressed inculture,18 solid-phase peptide synthesis,19 or QconCATmethods.20,21 First, digestion of intact proteins is advantageousin that the protein internal standard is almost chemicallyidentical to the analyte. Implementation of this method ishindered by expression difficulty and by variable post-translational modifications including disulfide linkages.22

Second, isotope incorporation by solid-phase synthesis yieldsisolated peptides that are selected to avoid disulfide linkage sitesand other PTMs. The disadvantage is that synthetic peptides donot account for digestion variability, and adherence of the smallpeptides to container walls has significant effects, leading tounexpected lower quantification values.19,23 Third, theQconCAT approach provides greater functionality by generat-ing many peptides for a given protein at once, where multiple

Received: July 26, 2013Accepted: September 27, 2013

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peptides give confidence to the quantitative results. The shorterprotein sequence of the QconCAT avoids the higher orderstructure of intact proteins, while still providing structuralsimilarity to the analyte protein.24 Thus, the QconCATapproach accounts for some digestion variability, but whetherthe QconCAT is digested in the same manner as the analyteprotein will depend on the peptides chosen as internalstandards. Optimizing QconCAT peptides as internal standardsrequires an evaluation process, to determine if they accuratelymirror the behavior of the analyte.The QconCAT approach is so named because peptides are

expressed from a custom DNA sequence, where DNAsequences corresponding to the peptides are concatenated.Then, the expressed protein construct contains isotope-labeledpeptides concatenated to each other, which are then released bytrypsin digestion. The “Q” in QconCAT refers to quantitationor to the produced Q-peptides that act as internal standards.21

The QconCAT technology was originally designed for globalproteomics where the expressed construct contains peptidesfrom many different proteins. In this study, we alter this processso that the QconCAT construct contains peptides only fromthe target protein, transferrin. We also add flanking regions tothe expressed construct. Flanking regions extend the aminoacid sequence of each peptide by four residues in eachdirection, as the sequence would have occurred in the originalprotein.25−27 The amino acids directly surrounding the trypsincleavage site are known to influence digestion efficiency, and aprevious experiment on a QconCAT construct both with andwithout flanking regions showed the advantages of flankingregions.26 Flanking regions help the digestion efficiency of theQconCAT to more effectively mirror the digestion efficiency ofthe analyte, by helping trypsin to recognize its binding sites asthey would occur in the intact protein. Trypsin digestion thenseparates the peptides from the flanking regions, releasing thelabeled peptides as internal standards.This study compares traditional solid-phase synthesis to the

QconCAT approach. While such a comparison was previouslymade,19 we update this comparison by adding flanking regionsto the QconCAT and apply a quality control process to thecalibration curves. We also alter the synthetic peptide protocolby adding concentrated bovine serum albumin to coat thecontainer walls, which is known to prevent sample loss.23 Thesealterations allow updated comparisons between synthetic andQconCAT approaches.For either type of internal standard, eq 1 relates the

chromatographic peak area ratio to the mole ratio, where A isanalyte and S is internal standard. This equation is used toconstruct calibration curves and to quantify transferrin levels inhuman serum samples.

=pmolpmol

peak areapeak area

A

S

A

S (1)

While previous studies used QconCAT peptides to measureproteins in homogenized tissue samples,24,25,28 we present whatseems to be the first application of QconCAT peptides tomeasurements of proteins in human serum. This studyquantifies transferrin in human serum using QconCAT andsynthetic peptides in parallel. If the obtained transferrin levelsare in agreement between the two methods, this will addconfidence to the quantitative values.29 The same experimentprovides updated comparisons between the two isotope-labeling methods. While there are advantages to each labeling

method, both methods are used here in concert to providefurther insight into method development.

■ EXPERIMENTAL SECTIONOptimization of Multiple Reaction Monitoring (MRM).

Purified human transferrin (Sigma Aldrich, St. Louis, MO) wasdiluted at 1.0 mg/mL in 50 mol/L ammonium bicarbonatebuffer at pH 7.8. Trypsin digestion was done starting withaddition of RapiGest surfactant (Waters Corp. Milford, MA).Before trypsin digestion, samples were alkylated and reduced toremove disulfide bonds, using 5 mmol/L dithiothreitol at 60 °Cand 15 mmol/L iodoacetamide at room temperature. Sampleswere incubated overnight at 37 °C with porcine sequencinggrade trypsin (Promega Corp., Madison, WI). Acid treatmentwas done at a 100 mmol/L final concentration of HCl. Aftercentrifugation and vacuum drying to remove solvent, eachsample was reconstituted in 0.1% (v/v, volume fraction) formicacid in water and placed in an autosampler vial for subsequentLC-MS measurements.A series of LC-MS runs were performed on the transferrin

digest. The LC-MS system was an Agilent Technologies 1200series coupled to an Applied Biosystems API 5000 triplequadrupole mass spectrometer with a QJet ion guide. Thechromatographic mobile phase consisted of 0.1% formic acid inwater mixed with 0.1% formic acid in acetonitrile (Burdick &Jackson, Muskegon, MI). The column was equilibrated over 2 hat a flow rate of 0.2 mL/min at 5% organic phase and thenramped to 95% over 65 min and then back to 5% over 5 minusing a Supelco C18 HPLC column (2.1 mm × 150 mm, 3 μmpacking) at a column temperature of 45 °C.The first LC-MS run was done in MS mode on the

transferrin digest to detect the most intense tryptic peptides.The peptide sequences of the detected parent masses were usedto create a list of possible multiple reaction monitoring (MRM)transitions. This calculation was done using in-house Rlanguage software. The second LC-MS run was set in MRMmode to detect possible fragmentation transitions (30 ms dwelltime). The 2 to 3 most intense MRM transitions for eachpeptide were collated in a method. A series of MRM runs weredone on the transferrin digest over a gradient of collisionenergies (in volts) of 10, 12, 15, 20, 25, 30, 35, 40, 50, and 60 tooptimize the collision energy for each MRM transition. Thiswas followed by another series of MRM runs at the optimizedcollision energies but over a gradient of declustering potentialsof 40, 50, 60, 70, 80, 90, 100, 110, and 120 (in volts) tooptimize this parameter.

Isotope-Labeled Internal Standards. Synthetic isotope-labeled peptides were obtained commercially (GenScript,Piscataway, NJ). These two synthetic peptides are sufficientto provide comparisons to QconCAT measurements. Thefollowing peptides (HSTIFENL*ANK and DSAHGFL*K)were synthetically labeled with six 13C atoms on a leucineside chain (L*) to create a 6 Da mass shift. Stock solutions ofthese peptides were made in 50 mmol/L ammoniumbicarbonate and aliquoted. The amino acid sequence of theQconCAT construct was designed to contain six trypticpeptides of transferrin (HSTIFENLANK, DSAHGFLK,SASDLTWDNLK, DGAGDVAFVK, APNHAVVTR,KPVDEYK; see Figure 1), along with flanking regions25,26 offour amino acids on each side.The QconCAT amino acid sequence was coded into the

corresponding DNA sequence and incorporated into thepET21a expression vector, with codon optimization for E. coli

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cells. The DNA sequences corresponding to the above peptideswere synthesized and placed into plasmids commercially(Biomatik, Cambridge, Ontario) and were reconstituted to 5ng/μL in nuclease-free water. A total of 10 ng of the plasmidwas added to E. coli cells and placed on ice. To express DNAinto protein, the protein was overexpressed using the One ShotBL21 (DE3) Competent E. coli Kit (Invitrogen, Grand Island,NY). The M9 minimal media was used containing 1 g/L of15NH4Cl (Cambridge Isotope Laboratories, Andover, MA) asthe only nitrogen source for E. coli culture. Initial inoculationwas done for 25 mL of media, and the cell culture was grownfor 12 to 14 h at 37 °C. Cells were collected by centrifugationat 5000g for 15 min and then washed 3 times in 10 mL of fresh15NH4Cl-containing M9 media. Cells were then transferred to500 mL of fresh 15NH4Cl-containing M9 media and keptgrowing at 37 °C until the UV−vis optical density reached 0.6to 0.8 at 600 nm. Protein overexpression was induced by 0.5mmol/L of IPTG. After 4 h of growth, the cells were harvestedby centrifugation at 5000g for 15 min and resuspended in BugBuster extraction reagent with 0.05% (v/v) benzonase (EMDBiosciences, Darmstadt, Germany). After sonication, theinclusion body containing the QconCAT protein was collectedby centrifugation at 7500g for 30 min. The inclusion bodycontaining the QconCAT protein was dissolved in 8 mol/Lurea, 50 mmol/L sodium phosphate buffer, pH 8.0, containing0.3 mol/L NaCl. Protein purification based on a 6xHis-Tag wasachieved on a Ni-NTA Agarose column (Qiagen, Valencia,CA). The washing and elution buffers were 8 mol/L urea and50 mmol/L sodium phosphate (pH 8.0)/0.3 mol/L NaCl,containing 40 and 300 mmol/L imidazole, respectively.To verify protein expression of the QconCAT, a 15% SDS-

PAGE gel was run on the expressed QconCAT construct,which for verification purposes was expressed without usingheavy isotope media. This was followed by in-gel trypsindigestion of the gel spot and MALDI-MS detection on a 4700Proteomics Analyzer MALDI TOF/TOF instrument (ABSciex, Framingham, MA). After expression was confirmed, theexpression was repeated on a larger scale in the presence of 15Nmedia. Incorporation of 15N into the QconCAT wasdetermined to be >99% using a MALDI-TOF spectrum of arepresentative QconCAT peptide 15N-DSAHGFLKVPPRwhich matched the isotopic distribution for >99% incorpo-ration of 15N in this peptide as calculated by the NIST IsotopeEnrichment Calculator v1.1 (http://www.nist.gov/mml/bmd/bioanalytical/isoenrichcalc.cfm).30

Buffer exchange was done from 8 mol/L urea by an Amiconmolecular weight centrifugal filter (Millipore, Billerica, MA) sothat the QconCAT construct was dissolved in 0.5 mL of 50mmol/L ammonium bicarbonate buffer. To determine a roughconcentration value of total protein, a Bradford protein assaywas done using a DC Protein Colorimetry Assay kit (Bio-RadLaboratories, Hercules, CA). The UV−vis optical density wastaken and gave the total protein level at 0.2 mg/mL. The

QconCAT construct was aliquoted and used below as aninternal standard for transferrin.

Sample Preparation. To prevent sample losses to thecontainer, a concentrated bovine serum solution was added tocoat the walls of the centrifuge tubes when preparingcalibration solutions. It was not necessary to add bovineserum albumin to the human sera that already contain albumin.Using the same isotope-labeled peptides, two human serum

samples were prepared, one from a single individual(Bioreclamation, Westbury, NY) and a pooled serum sample(NIST Standard Reference Material 909c). These sera weretaken from healthy donors and should have normal transferrinlevels. A 100-fold dilution of 20 mg of each serum wasperformed gravimetrically in 50 mol/L ammonium bicarbonatebuffer.

Constructing Calibration Curves. Purified human trans-ferrin (Sigma Aldrich) as the calibrant was diluted so that theLC-MS injected transferrin concentrations were 4, 2, 1, 0.4, 0.2,and 0.04 pmol/μL to create a calibration curve. The isotope-labeled peptides were then added at a constant finalconcentration of 0.4 pmol/μL to all samples and six calibrantsolutions.For the QconCAT internal standards, a separate calibration

curve was created in the same way. The precise concentrationof the QconCAT construct was not known, so a fixed mass ofthe QconCAT construct solution (20 mg) was added to each ofthe calibrants and serum samples. All the above dilutions weredone gravimetrically to account for pipetting error. Therecorded masses of pipetted volumes were used to correctthe final quantitative calculations.Trypsin digestion was done on the sera and calibrants as

described above. To fully dissolve the resulting serum digest,sonication was done in an ultrasonic bath for 20 min. This wasfollowed by sample cleanup using C18 ZipTip pipet tips(Millipore Corp., Billerica, MA) and vacuum drying to removesolvent. The serum samples were reconstituted in 0.1% formicacid in water and placed in autosampler vials.

LC-MS/MS Analysis. Finally, LC-MS/MS analysis wasperformed on the calibrants and serum samples using the aboveLC conditions and optimized MRM transition parameters forthe unlabeled analyte, QconCAT, and synthetic internalstandards.

Data Analysis and Calculations. Peak area integrationswere done manually on each MRM peak using Analyst 1.5software (Applied Biosystems). A spreadsheet was used tocalculate the peak area ratio and plot this against the mole ratioof transferrin in the calibrants. This created a calibration curveof six points for each MRM transition, which was fitted with alinear trendline. The equation of this trendline was used todetermine transferrin concentration in the serum samples.

■ RESULTS AND DISCUSSIONTo verify QconCAT expression, an SDS-PAGE gel was runafter purification and shows the 11.8 kDa QconCAT constructat the expected gel band location (Figure S-1, SupportingInformation). After in-gel digestion of this gel spot, a MALDI-MS spectrum shows masses that match predicted masses from atheoretical digest of the QconCAT construct (Figure S-2,Supporting Information). An accompanying table identifiesseveral of the Q-peptide peaks in the MALDI-MS spectrum.Also, the 15N isotope incorporation into the QconCAT wasdetermined by MALDI-MS to be >99% (Figure S-3,Supporting Information). Thus, expression of the QconCAT

Figure 1. Amino acid sequence of the QconCAT construct. The six Q-peptides are highlighted in red. The intervening sequences are flankingregions of four amino acids on both sides of each Q-peptide. Thegreen methionine corresponds to a start codon in the DNA sequence,which is necessary for gene expression.

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DNA into protein was successful, and the six Q-peptides(Figure 1) can be evaluated for use as internal standards fortransferrin.The Q-peptide evaluation starts by verifying reasonable levels

of detection. Of the six Q-peptides, five were sufficientlydetectable in representative mass chromatograms (Figure S-4,Supporting Information), with the peptide elution ordermatching theoretical predictions (Figure S-5, SupportingInformation).31 The sixth Q-peptide of the sequenceKPVDEYK had no QconCAT signal present at the lowesttwo concentration points on the calibration curve. Possibleexplanations for absence of signal for this peptide include a lowdigestion efficiency due to a proline residue next to the trypticcleavage site (Figure 1), which is known to inhibit trypsin frombinding.32,33 This peptide was included in the QconCATbecause it showed good signal at higher concentrations in theinitial optimization procedure. On the basis of the absence ofsignal at lower concentrations, it is apparent that such peptidescontaining a proline N-terminal to the basic site should beexcluded when designing a QconCAT. For the remainingdetectable Q-peptides, the calibration curves must be evaluatedto determine if they result in linear calibration curves within thetargeted concentration range.Calibration curves were constructed in the same way for both

QconCAT and synthetic internal standards. The midpoint waschosen at 0.4 pmol/μL because this is an approximatetransferrin concentration in human serum, 40 pmol/μL6 at100-fold dilution, which gives an appropriate concentration forLC-MS injections. The end points of the calibration curveencompass 2 orders of magnitude between 0.04 and 4 pmol/

μL. Using the QconCAT internal standards, calibration curvesresulted from several different Q-peptides in Figure 2. To savespace, the fifth peptide DSAHGLFK is not included in Figure 2but is shown later in Figure 3. In Figure 2, representativecalibration curves from three different Q-peptides show similarbehavior and give linear trendlines (R2 > 0.98), whereas the

Figure 2. Calibration curves of representative MRM transitions from the QconCAT data. Panels A, B, and C show good linearity, while Panel Dshows poor linearity and a vastly different y-intercept. Thus, the HSTIFENLANK peptide in panel D was determined as unsuitable for quantification,but the other three peptides give ideal calibration curves.

Figure 3. Overlay of MRM calibration curves, which were constructedfrom four different Q-peptides. The two MRM transitions ofDSAHGLFK (abbreviated DSA) give calibration curves with lowerslope values than the other peptides, suggesting a low digestionefficiency of this peptide from the QconCAT construct. Thus, onlyAPNHAVVTR, DGAGDVAFVK, and SASDLTWDNLK were usedfor quantification.

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fourth Q-peptide HSTIFENLANK in Figure 2D gives acalibration plot that is not sufficiently linear (R2 = 0.86) anda y-intercept of −2.1. This y-intercept differs from the othercalibration plots that have y-intercepts near the origin. Aseparate MRM transition from the same HSTIFENLANKpeptide produced a y-intercept of −6.4 (Figure S-6, SupportingInformation), and such variability points to the unsuitability ofthis peptide for quantification. Given that other QconCATpeptides in the same experiment show linear behavior and thesynthetic version of HSTIFENLANK gives linear plots (FigureS-7, Supporting Information), it is unclear why the QconCATversion gives a variable calibration curve. Because largedifferences are seen between two MRM transitions of thesame Q-peptide, the cause might relate to the structure of theQconCAT construct. In fact, an aspartic acid residue at positionP2′ relative to the cleavage site,34 as found in the C-terminalflanking region of HSTIFENLANK, is known to inhibit trypsinbinding.17 So far, of the six Q-peptides, KPVDEYK waseliminated due to absence of signal and HSTIFENLANK waseliminated because of nonlinear calibration curves and variabley-intercepts, leaving four Q-peptides. These four remaining Q-peptides must be evaluated to determine if their calibrationcurves give reasonable slope values.A plot overlaying two MRM transitions from each of the four

remaining Q-peptides is in Figure 3. Good linearity is seen forall 8 calibration plots (R2 > 0.98), but six MRM transitions fromthe three Q-peptides APNHAVVTR, DGAGDVAFVK, andSASDLTWDNLK have similar slopes, whereas the slopes ofthe two MRM transitions from DSAHGFLK are consistentlylower. The interpretation of slope on this plot is that a higherslope means a higher digestion efficiency; see eq 1. Given thatall Q-peptides were present in equimolar amounts in theQconCAT construct before digestion, the lower slope forDSAHGFLK points to a low digestion efficiency for thispeptide. The lower digestion efficiency may result from adouble proline, PP, sequence in the C-terminal flanking regionfor this peptide (see Figure 1) or perhaps its location within theQconCAT sequence inhibits trypsin binding by someunidentified mechanism.33 Thus, of the six total Q-peptides,three were eliminated for the reasons listed in Table 1, whilethe other three appear to be suitable internal standards tomeasure transferrin.

The above evaluation of Q-peptides is comprehensive inthree stages, verification of detectability, sufficient linearity ofcalibration curves, and sufficient slope of calibration curves.While the DSAHGFLK peptide passes the first two testsbecause it is detectable and shows linear calibration curves, itfails the third test by having a significantly lower slope. TheQconCAT version of DSAHGLFK is thus determined to beunsuitable for quantitation. If it were the case that only the firsttwo tests were used, this peptide would prove deceptive and

would adversely affect the quantitative results. At the sametime, the QconCAT evaluation identified three Q-peptides thatare suitable to measure transferrin and pointed out factors thatshould be examined when using QconCAT peptides.In parallel with QconCAT development, the synthetic

isotope-labeled peptides DSAHGLFK and HSTIFENLANKwere used to measure transferrin in human serum. Thesesynthetic peptides provide a point of comparison against theresults obtained by the QconCAT peptides. Therefore, samplepreparation for LC-MS was done in a similar manner for bothsynthetic and QconCAT internal standards, as explained in theExperimental Section, to facilitate comparison of results. Table2 lists all MRM transitions used for quantification, for bothQconCAT and synthetic peptides. This table represents theoptimized MRM method and is filtered down to suitable Q-peptides as explained above and to synthetic peptides thatresulted in the best signal. Therefore, all MRM transitions inTable 3 were used to measure transferrin concentrations inserum.Transferrin levels were determined in two different serum

samples, a healthy individual serum and a healthy pooledserum, using both synthetic and QconCAT peptides. Theresults are in Table 3. Some outlier data points were removedby a Q-test and in all cases because the serum sample resultedin low signal for a particular MRM transition. There are threereasons for an outlier: it is erroneous because the measurementfailed to produce reasonable levels of signal, the regressionmodel is incorrect, or the outlying data point is real but is justan improbable occurrence.35 Evidence is presented in theSupporting Information that the removed outliers are under thecategory of low signal. Although a calibration curve may havepassed the above QconCAT evaluation, if the serum samplesignal is low and falls outside the calibration range for an MRMtransition, the data point must be removed. Of the original 26data points in Table 3, only four low signal points wereremoved, and of those four, the three lowest-signal data pointswere from the synthetic DSAHGFLK peptide, revealing thatsome MRM transitions of this peptide are detectable in serumsamples and others are not. Given that the main origin of lowsignal data points is the DSAHGFLK peptide, this suggests thatlarger numbers of serum samples can be quantified successfullyif one controls for a high percentage of outliers from thispeptide. After removal of low signal data, the remaining dataresulted in the transferrin levels in Table 3.Interestingly, the Q-peptide SASDLTWDNLK in Table 3

gives higher transferrin values than those from other Q-peptides. This occurs for both MRM transitions ofSASDLTWDNLK, so that interference from other ion speciessimultaneously occurring for both MRM transitions is anunlikely cause. Other sources of variation such as poorchromatography and inconsistent peak integration are alsounlikely causes, as higher quantitative values for this peptide areseen over different LC-MS runs from two serum samples.36

One potential cause is the presence of an unexpected PTM orfragmentation pathway. However, it was decided to includeSASDLTWDNLK in the results as the chromatography wasideal (Figure S-4, Supporting Information), no bias sourceswere evident, and the quantitative results reasonably agreedwith the other Q-peptides.Table 3 shows encouragingly close results in terms of

transferrin levels between QconCAT and synthetic peptides.For the pooled serum sample (NIST Standard ReferenceMaterial 909c), transferrin levels were obtained at (192.2 ±

Table 1. Summary of Which QconCAT Peptides Were Usedto Quantify Transferrin in Serum

QconCAT peptide used for quantitation reason

APNHAVVTR used good signal, linear calibrationDGAGDVAFVK used good signal, linear calibrationSASDLTWDNLK used good signal, linear calibrationHSTIFENLANK not used calibration not linearKPVDEYK not used not detected, low signalDSAHGFLK not used digestion not efficient

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16.2) mg/dL with QconCAT peptides and (202.2 ± 36.5) mg/dL with synthetic peptides. The reported uncertaintiescorrespond to variability over the MRM transitions and donot correspond to replicate measurements. The uncertaintyranges of these values show high overlap, and this mutualagreement between different types of internal standards pointsto good quantitative accuracy.For the individual serum sample, the transferrin levels in

Table 3 are similar between QconCAT and synthetic peptides,albeit not quite as close as for SRM909c. The two uncertaintyranges almost but not quite overlap for the individual donorsample. If the measurements were repeated, one expects thatthese values would converge. Considering the usual diagnosticranges for transferrin in a variety of clinical conditions,6,7 the

uncertainty of transferrin levels seen here for both types ofinternal standards is better than the uncertainties generallyreported by some clinical methods. When comparingQconCAT to synthetic peptides, one advantage of theQconCAT approach is that several Q-peptides are createdsimultaneously by gene expression, as opposed to syntheticinternal standards that are synthesized individually, one peptideat a time. Therefore, the QconCAT approach has the potentialto generate data from multiple peptides, adding confidence tothe quantitative results. For an overall comparison betweensynthetic and QconCAT peptides, both give reasonablequantitative values in this study. The disadvantages of theQconCAT approach are largely overcome by the QconCATevaluation process, which objectively narrows down the list ofsuitable peptides. For synthetic peptides, initial experimentswithout using BSA-coating to prevent sample losses failed toproduce usable signal, but the addition of BSA improved theutility of the synthetic peptides. Thus, in the context ofmeasuring a single analyte protein, the advantages betweenQconCAT and synthetic approaches remain related to cost,time, and effort.19 However, for both types of internal standard,trypsin digestion efficiency adds variability to the detectedamounts of unlabeled analyte. Given that each method hasassociated variability, further confidence is added by using otherorthogonal methods. In addition to the two methods presentedhere, synthetic and QconCAT labeling, a third method calledinductively coupled plasma (ICP) mass spectrometry was usedby our collaborators at NIST to measure transferrin.Transferrin levels were obtained by inductively coupled

plasma mass spectrometry (ICPMS) by our collaborators at theHollings Marine Laboratory (NIST). ICPMS indirectlymeasures transferrin levels by measuring iron content. Giventhat each transferrin molecule binds to two Fe3+ ions, thisstoichiometry infers transferrin levels in human serum. Theirresult is in a submitted journal article for transferrin inSRM909c (Nuevo-Ordonez, Y., Davis, W.C., 2013, submitted)and closely matches our results for the same sample (data notshown). Our results also show reasonable agreement withprevious transferrin measurements using LC-MS/MS and asingle synthetic peptide as an internal standard.17 This diverse

Table 2. MRM Transitions Used to Quantify Transferrin and Their Optimized Instrumental Parameters

QconCAT peptidetransitions

analyte(m/z)

analyte(m/z)

internal standard(m/z)

internal standard(m/z) ion ion

collisionenergy

declusterpotential

precursor product precursor product precursor product (volts) (volts)

APNHAVVTR(l) 322.2 375.2 327.2 381.2 3+ 1+, y3 15 50APNHAVVTR(2) 322.2 447.3 327.2 454.2 3+ 2+, y8 15 50APNHAVVTR(3) 322.2 474.3 327.2 481.3 3+ 1+, y4 15 50DGAGDVAFVK(l) 489.7 735.4 495.2 743.4 2+ 1+,y7 25 110DGAGDVAFVK(2) 489.7 563.4 495.2 569.3 2+ 1+, y5 25 120SASDLTWDNLK(l) 625.3 675.3 632.3 683.3 2+ 1+, y5 30 100SASDLTWDNLK(2) 625.3 776.4 632.3 785.4 2+ 1+, y6 30 90

synthetic peptidetransitionsa

analyte(m/z)

analyte(m/z)

internal standard(m/z)

internal standard(m/z) ion ion

collisionenergy

declusterpotential

precursor product precursor product precursor product (volts) (volts)

DSAHGFL*K(l) 292.2 336.7 294.2 339.7 3+ 2+,y6 12 70DSAHGFL*K(2) 437.7 464.3 440.7 470.3 2+ 1+, y4 20 110DSAHGFL*K(3) 292.2 464.3 294.2 470.3 3+ 1+, y4 25 60DSAHGFL*K(4) 292.2 601.3 294.2 607.4 3+ 1+, y5 25 60HSTIFENL*ANK(1) 425.2 445.3 427.2 451.3 3+ 1+, y4 15 100HSTIFENL*ANK(2) 425.2 559.3 427.2 565.3 3+ 1+,y5 15 100

aL* = 13C6.

Table 3. Transferrin Levels in Human Serum (in mg/dL)

QconCAT MRMtransition

SRM909C(pooledserum)

synthetic MRMtransition

SRM909C(pooledserum)

APNHAVVTR(l) 172.4 DSAHGFLK(2) 218.7APNHAVVTR(2) 191.8 DSAHGFLK(4) 243.1DGAGDVAFVK(l) 181.8 HSTIFENLANK(l) 187.5DGAGDVAFVK(2) 189.4 HSTIFENLANK(2) 159.3SASDLTWDNLK(l) 220.0SASDLTWDNLK(2) 198.0mean 192.2 mean 202.2st. dev. 16.2 st. dev. 36.5CV (%) 8.4 CV (%) 18.0QconCAT MRM

transitionserum

(individual)synthetic MRM

transitionserum

(individual)

APNHAVVTR(l) 241.7 DSAHGFLK(l) 211.8APNHAVVTR(2) 214.5 DSAHGFLK(2) 189.6APNHAVVTR(3) 224.5 DSAHGFLK(3) 194.0DGAGDVAFVK(l) 235.3 HSTIFENLANK(l) 206.3DGAGDVAFVK(2) 240.1 HSTIFENLANK(2) 216.1SASDLTWDNLK(l) 270.8SASDLTWDNLK(2) 260.3mean 241.0 mean 203.6st. dev. 19.4 st. dev. 11.4CV (%) 8.1 CV (%) 5.6

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array of methods (synthetic, QconCAT, and ICPMS)converging on similar transferrin levels suggests that theobtained values are reasonable, but further study is needed toreport quantitative values with certainty.Using synthetic and QconCAT peptides in parallel allows a

refined focus on method development. For example, thesynthetic version of HSTIFENLANK worked ideally, while theQconCAT version gave variable results, thus using both typesof peptides reveals that the variability stems from theQconCAT sequence and not the isolated peptide sequence.This also suggests the need for completely separate experimentsto uncover the physical mechanism behind the variable resultsof certain peptides, which is a future direction of this study.However, the above QconCAT evaluation was able to identifyand remove such variable-response peptides from thequantification, giving higher confidence in the reportedmeasurements. Such a QconCAT evaluation process could beused, along with synthetic peptides, to measure other clinicallyrelevant proteins.

■ ASSOCIATED CONTENT*S Supporting InformationAdditional material as described in the text. This material isavailable free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: tyler.zimmerman@northwestern.edu.Present AddressT.A. Zimmerman: Department of Chemistry, NorthwesternUniversity, Evanston, IL, U.S.A.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors thank Dr. Eric L. Kilpatrick and Alyssa Florwickfor assistance. T.A.Z. acknowledges a National ResearchCouncil (NRC)/NIST postdoctoral research associateship.Certain commercial materials, instruments, and equipmentare identified in this manuscript in order to specify theexperimental procedure as completely as possible. In no casedoes such identification imply a recommendation or endorse-ment by the National Institute of Standards and Technologynor does it imply that the materials, instruments, or equipmentidentified is necessarily the best available for the purpose.

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