Quantitative Performance of Internal Standard Platforms for Absolute

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Quantitative performance of internal standard platforms for absolute protein quantification using MRM-MS Kerry Bauer Scott, Illarion V. Turko, and Karen W. Phinney Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b00331 • Publication Date (Web): 26 Mar 2015 Downloaded from http://pubs.acs.org on March 31, 2015

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Analytical Chemistry

Quantitative performance of internal standard platforms for absolute protein quantification using MRM-MS

Kerry Bauer Scott1, 2, *, Illarion V. Turko1, 2, Karen W. Phinney1 1

Biomolecular Measurement Division, National Institute of Standards and Technology,

Gaithersburg, MD 20899 2

Institute for Bioscience and Biotechnology Research, Rockville, MD 20850

ABSTRACT Stable-isotope-labeling mass spectrometry involves the addition of known quantities of stableisotope labeled standards, which mimic native molecules, to biological samples. We evaluated three conventional internal standard platforms (synthetic peptides, QconCAT constructs, and recombinant proteins) for quantitative accuracy, precision, and inherent advantages and limitations. Internal standards for the absolute quantification of three human cytokine proteins (interferon gamma, interleukin-1 beta, and tumor necrosis factor alpha) were designed and verified. Multiple reaction monitoring assays, calibration curve construction, and regression analysis were used to assess quantitative performance of the internal standard platforms. We also investigated a strategy for methodological improvement to current platforms using natural flanking sequences. Data analysis revealed that full length protein standards have the broadest quantitative reliability with accuracy being peptide-dependent for QconCATs and synthetic peptides. Natural flanking sequences greatly improved the quantitative performance of both QconCAT and synthetic peptide standards.

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INTRODUCTION Protein quantification methods based on the principle of multiple reaction monitoring (MRM) with stable-isotope labeled standard peptides provide absolute protein quantification values. These methods are based on the addition of known quantities of isotope-labeled internal standards into biological samples. The internal standards are analogous to native molecules and quantification is achieved by comparing ion signals from isotope-labeled and native peptides. These methodologies are broadly applicable to clinical biomarkers, systems biology, and the pharmaceutical industry; which require accurate, precise, and reproducible protein abundance measurements. Several internal standard platforms exist for protein quantification, with the three conventional internal standard platforms including synthetic peptides, concatenated peptides (QconCAT)1, and recombinant proteins. Each method has its own strengths and limitations in terms of production, application, and analytical performance.2

A principle limitation of synthetic peptide and concatenated peptide standards is that amino acid sequences surrounding the tryptic cleavage sites are not identical between the standard and the native target protein. QconCATs are assembled with peptides from multiple different proteins. These peptides are ordered in the construct such that local primary sequences are preserved when possible. However, sequence context is not always able to be preserved. This difference between standard and analyte presents potential for quantitative error since the efficiency of the tryptic digestion is influenced by the amino acids neighboring the cleavage sites.3

Inclusion of natural flanking sequences around the cleavage sites of peptides, to better mimic the target protein, has become a new trend.4 Natural flanking sequences have been incorporated into QconCAT constructs5–8 and synthetic peptides9–12 by different researchers. A comparison of concatenated peptides with and without flanking sequences showed that the presence of flanking regions improved quantitative accuracy5 whereas a study comparing


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cleavable and non-cleavable synthetic peptides showed no difference in accuracy or precision.9 The length of natural flanking sequences in the literature has varied in the number of residues included in the flanking region, between two to six amino acids. There is a need to experimentally determine the optimal size of flanking sequence to match peptide release from the native protein. Furthermore, determining the minimal flanking length necessary to emulate native analyte signal is important as QconCAT constructs and synthetic peptides have size limitations for expression and synthesis.

Reviews have broadly discussed advantages and disadvantages of various stable isotope internal standards, but few experimental comparisons between the methods have been performed. Brun et al compared three standards including recombinant proteins, QconCAT, and synthetic peptides by LC-MS analysis.13 They reported the superior performance of recombinant protein standards for quantification. Two comparisons of QconCAT and peptide standards have been conducted; one study evaluated the fidelity of the standards in terms of production.14 The other study compared the performance of different peptides in the QconCAT from the synthetic peptides.15 Finally, a study evaluated both recombinant protein and synthetic peptide standards with improved accuracy and precision using the protein standard.16

This is the first systematic, parallel comparison between recombinant protein, QconCAT, and synthetic peptide standards designed to also assess the use of natural flanking sequences and peptide digestion kinetics. For this study, we randomly selected three clinically relevant proteins (interferon gamma (IFNG), interleukin beta (IL1B), and tumor necrosis factor alpha (TNFA)) to address conceptual questions regarding internal standard strategies. We developed recombinant proteins, QconCAT constructs, and synthetic peptides for the quantification of three human cytokine proteins. Calibration curve construction and regression analysis was used to assess the quantitative accuracy, precision, and inherent strengths and limitations. A



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methodological improvement using natural flanking sequences demonstrated improved performance for QconCAT standards and synthetic peptides.

EXPERIMENTAL SECTION Materials. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise noted. Synthetic Peptides. Synthetic peptides were synthesized by Biomatik (Cambridge, Ontario) in their unlabeled, natural isotope form. The purity was ≥98 % for all peptides, therefore peptide peak areas were not corrected for purity. Peptides were solubilized according to manufacture guidelines and stock peptide solutions were prepared in water at 1 µg/µL. Absolute concentrations were confirmed by amino acid analysis. The molecular weight of the peptides was verified by LC-MS and all peptides had their expected monoisotopic masses. QconCAT Design. Two QconCAT constructs were designed to contain 3 to 4 tryptic peptides of IL1B, IFNG, and TNFA concatenated with either no flanking sequences or flanking sequences of six amino acids on both the N-terminal and C-terminal sides. Tryptic peptides were selected following experimental evaluation of LC-MS detectability. Protein Expression and Purification. The amino acid sequence of IL1B, IFNG, TNFA, and two QconCAT constructs were coded into the corresponding DNA sequence and incorporated into the pET21a expression vector, with codon optimization for E. coli (Biomatik, Cambridge, Ontario). The plasmids were transformed into One Shot BL21 (DE3) competent E. coli cells (Invitrogen, Grand Island, NY) and grown in M9 minimal media at 37 °C until the optical density (OD) reached 0.6 to 0.8 at 600 nm. Protein expression was induced by 0.5 mmol/L isopropyl βD-1-thiogalactopyranoside (IPTG). After 3 h of growth, the cells were harvested by centrifugation at 5000 g for 10 min and resuspended in 0.1 mmol/L dithiothreitol (DTT) and sonicated. Following centrifugation at 35000 g for 30 min, supernatant containing IL1B was


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collected. Inclusion bodies containing IFNG, TNFA, and both QconCATs were resuspended in 8 mol/L urea and clarified by centrifugation at 10000 g for 10 min. Recombinant protein purification was achieved based on the 6xHis-Tag on a Ni-NTA agarose column (Qiagen, Valencia, CA). The binding, washing, and eluting buffers were 8 mol/L urea, 100 mmol/L Na2HPO4, 10 mmol/L Tris-HCl at pH 8.0, 6.0, and 4.5, respectively. Buffer exchange was done from 8 mol/L urea to 50 mmol/L ammonium bicarbonate (NH4HCO3) using an Amicon filter (3 kDa MWCO, Millipore, Billerica, MA). QconCAT proteins were purified using HisPur cobalt resin (Thermo Scientific, Waltham, MA) using the gravity-flow method. Columns were equilibrated, loaded, and washed with 50 mmol/L sodium phosphate, 300 mmol/L sodium chloride, and 10 mmol/L imidazole at pH 7.4. QconCAT proteins were eluted with 50mmol/L sodium phosphate, 300 mmol/L sodium chloride, and 150 mmol/L imidazole at pH 7.4. Eluted proteins were precipitated with methanol/chloroform/water and resuspended in 0.1 % RapiGest (Waters, Milford, MA). Protein concentration was determined using a BCA protein assay and bovine serum albumin as a standard (Thermo Scientific). Protein expression and purification were evaluated with SDS-PAGE and mass spectrometry analysis on a 4700 Proteomics Analyzer MALDI TOF/TOF (AB Sciex, Framingham, MA). 15

N Incorporation. Plasmid transformed DE3 cells were expressed in M9 minimal media with 1

g/L 15NH4Cl (Cambridge Isotope Laboratories, Andover, MA) as the sole nitrogen source and purified as described. Stable isotope incorporation into full length proteins was determined at the peptide level with multiple reaction monitoring (MRM) analysis. The pair transitions for the light (unlabeled) and heavy (15N labeled) form of each peptide were monitored in a sample containing 15N labeled IL1B, IFNG, and TNFA. Incorporation was calculated as the percentile of the area of the labeled peak to the sum of the labeled and unlabeled peaks. The final isotope incorporation is based on combined data for three peptides and was found to be greater than 99 % for all proteins, so no correction for protein concentration was made during data analysis.



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Digestion Optimization. Equal amounts of IL1B, IFNG, and TNFA were combined and digested with one of 8 methods: (1) 50 mmol/L NH4HCO3, pH 8.0/0.1 % RapiGest, (2) 50 mmol/L NH4HCO3, pH 7.0/0.1 % RapiGest, (3) 8 mol/L urea (urea samples were desalted using Zebra spin columns (7 kDa molecular weight cutoff, Thermo Scientific) prior to enzymatic digestion), (4) 80 % acetonitrile (acetonitrile was added prior to enzymatic digestion), or (5) 0.05 % SDS. In all cases, proteins were reduced with 5 mmol/L DTT at 56 °C for 60 min and alkylated with 15 mmol/L iodoacetamide in the dark for 30 min prior to enzymatic digestion. Samples were either subjected to digestion with trypsin at a 1:50 protein to enzyme mass ratio at 37 °C or (6) 48 °C. Digestions were also performed with (7) trypsin at a 1:5 protein to enzyme mass ratio or (8) trypsin/lys-C mix (Promega, Madison, WI) at a 1:25 protein to enzyme mass ratio. All digestion reactions were quenched after 16 h by addition of formic acid to 2.5 %. Equal amounts of 15N labeled IL1B, IFNG, and TNFA were digested following method (1) and used as a spike-in standard. Digestion Time Course. Equal amounts of mixed unlabeled and labeled IL1B, IFNG, and TNFA were used to perform a time course digestion. Sample digestion was quenched at (0.5, 1, 2, 4, 8, 16, 24, and 48) h after incubation with trypsin/lys-C at 37 °C for the 14N proteins and after a single 16 h incubation for the 15N protein mixture which was used as a spike-in standard. All digestions were performed in the presence of 0.1 % RapiGest. Calibration Curves. Calibrant sample preparation was completed by combining equimolar amounts of purified, 15N labeled IL1B, IFNG, and TNFA at a range of concentrations to give (10.0, 5.0, 2.5, 1.0, 0.5, and 0.25) pmol/µL of each. Unlabeled purified, full-length IL1B, IFNG, and TNFA, QconCAT standards, and synthetic peptide standards were spiked-in each calibrant sample at 2.5 pmol/µL. Calibrants and standards were combined prior to enzymatic digestion. The digestion conditions used for protein quantification are as follows: 0.1 % RapiGest, 1:5 trypsin: protein mass ratio, and a 4 h incubation at 37 °C.


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LC-MS/MS Analysis. Peptide separation and MRM analysis were performed on an Agilent Technologies 1200 series coupled to an AB Sciex API 5000 triple quadrupole mass spectrometer with a QJet ion guide. Separation of peptides was performed with an Agilent C18 HPLC column (2.1 mm x 150 mm, 3.5 µm) at a flow rate of 0.2 mL/min over a 20 min gradient from 5 % to 45 % acetonitrile containing 0.1 % formic acid. Acquisition methods used the following parameters: ion spray voltage of 3000 V, curtain gas of 69 kPa (10 psi), ion source gas of 379 kPa (55 psi), and source temperature of 600 °C. Scheduled MRM was performed with a 60 s MRM detection window and 1 s target scan time. An initial list of MRM transitions were experimentally screened for the three most intense transitions per peptide. A single run consisting of a series of MRM transitions at differing collision energies (CE) and declustering potentials (DP) was created by making incremental adjustments of the product ion m/z value at the hundredth decimal place.17 The CE and DP values were varied by ±15 in 5 steps of 3 relative to the default equations CE = 0.036(Q1) + 8.8 for doubly and CE = 0.0544(Q1) - 2.4 for triply charged precursors and DP = 0.0729(Q1) + 31.117. MRM data was also collected on an Agilent 6460 QQQ LC/MS system with an Agilent Technologies 1200 series HPLC (Santa Clara, CA). Acquisition methods were as follows: fragmentor 135 V, electron multiplier 500 V, and capillary voltage 3500 V. Dynamic MRM scan type was used with a delta retention time of 60 s. Collision energies were optimized for each peptide from the default Agilent equations: CE = 0.031(Q1) + 1 for doubly and CE = 0.036(Q1) - 4.8 for triply charged precursors as described for the API 5000. Optimize instrument parameters are shown in Supplemental Information Table S1. Data Analysis. MRM peak area integration was performed using Skyline 2.5, AB SCIEX Analyst 1.6, or Agilent MassHunter Qualitative Analysis B.06 and an Excel spreadsheet was used to calculate peak area ratios. Peak integration was manually inspected and adjusted if necessary. The peak ratios from three transitions were averaged to yield the peptide ratios. Representative peptide ratios were plotted versus digestion time to determine optimal digestion



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incubation. Calibration curves were constructed by linear regression analysis of the peak area ratios plotted versus the molar ratios of the calibrant to the standard. All experiments were performed in duplicate with three replicate injections to assess error and reproducibility. Data is represented as the mean ± SD.

RESULTS AND DISCUSSION Calibrant and Standard Characterization. Proteotypic peptides for quantification of each cytokine protein were experimentally determined. A list of peptides was generated from an in silico tryptic digestion and corresponding light and heavy transitions were calculated using the OrgMassSpecR program (http://orgmassspecr.r-forge.r-project.org). Peptides were selected based on signal intensity, lack of cysteine and methionine residues, uniqueness, and molecular weight and screened for the three most intensive transitions per peptide. (Supplemental Information Table S-2). These transitions were used to access the level of stable isotope incorporation into the 15N-labeled calibrant proteins calibrants. The isotopic incorporation was calculated from the percentile of the area of the labeled peak to the sum of the labeled and unlabeled peaks based on three Q-peptides per protein. IL1B, IFNG, and TNFA were found to have 99.9 % ± 0.1 %, 99.3 % ± 0.2 %, and 99.9 % ± 0.1 % isotopic incorporation, respectively. These values were accepted as complete labeling and no correction for labeling efficiency was made during data analysis (Supplemental Information Figure S-1).

The amino acid sequence of QconCAT Q1 includes ten Q-peptides, three from IL1B, three from IFNG, and four from TNFA. Expressed QconCAT Q2 encodes for the same ten Q-peptides with natural flanking sequences of six amino acids on both the C-terminal and N-terminal sides of the peptides (Supplemental Information Figure S-2). The expression and the molecular weights of


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the full length recombinant and QconCAT proteins were assess by SDS-PAGE and found at their expected molecular weights with high purity (Supplemental Information Figure S-3).

Digestion Condition Optimization. It has been well established that digestion conditions significantly influence the number of measureable peptides and individual peptide concentrations, highlighting the need to identify reaction conditions that ensure complete and reproducible digestion prior to accurate quantification.18–21 Several conditions for protein digestion were evaluated for optimal peptide release including denaturant, pH, temperature, enzyme, and enzyme concentration (Figure 1). Unfortunately, but not unexpectedly, a single set of digestion conditions optimal for all peptides was not found. Instead, various digestion parameters were found to yield the highest signal for the peptides analyzed. This nonstoichiometry of protein digestions has been previously seen18 and is attributed to denatured protein structure and the identify of amino acid residues surrounding the cleavage sites. For example, the highest peak area ratio for peptide IPVALGLK was achieved using a 1:5 trypsin: protein mass ratio while the trypsin/Lys-C mix yield the highest signal ratio for peptide SLVMSGPYELK. Interestingly, these peptides are from the same protein, ILB1. There was also variation between peptide digestion efficiency depending on the denaturant used. Either urea or RapiGest as denaturant was optimal for most peptides, but one peptide showed enhanced signal with SDS.

A pH change from 8.0 to 7.0 had the least affect on peak area ratios with an average ratio of 1.17 and less than 30 % variability among the peptides evaluated. None of the peptides showed enhanced digestion in the presence of acetonitrile or incubation at 48 °C. Digestion with both trypsin at a ratio of 1:5 and a trypsin/Lys-C mix at a ratio of 1:25 outperformed trypsin at a protein ratio of 1:50. Overall, digestion with trypsin at a protein ratio of 1:5 lead to the maximal number of peptides with peak area ratios greater than one and was used for calibration curve



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generation. Denaturation with 0.1 % RapiGest was selected for calibration curve sample preparation since the use of urea resulted in high variability in the extent of peptide digestion.

Peptide Release and Stability. In addition to digestion conditions affecting peptide quantities, the dynamic production and decay characteristics of peptides during digestion has been shown to impact quantification results.18,19,22 Time course digestion profiles for peptide release and stability from IL1B, IFNG, and TNFA reveal various peptides from each protein to have different formation and stability characteristics. The peptides DDKPTLQLESVDPK from IL1B, FFNSNK from IFNG, and VNLLSAIK and IAVSYQTK from TNFA display rapid release, reaching maximum signal by 4 h, and maintain steady signals with slow decay through 24 h of digestion (Figure 2A). Peptides that are released rapidly and show stability over time are optimal candidates for quantification by standard peptides as quantitative accuracy will be maintained.

The peptides SLVMSGPYELK from IL1B, LTNYSVTDLNVQR from IFNG, and ANALLANGVELR from TNFA show rapid formation with continual signal decrease over the remainder of the digestion (Figure 2B). Peptides that are released rapidly, but also exhibit rapid signal decay will lead to variability in quantitative accuracy. Discrepancies between the synthetic standard and sample peptides will arise, especially if longer digestion times are used or if the standard peptide is added post-digestion.22

The peptides IPVALGLK from IL1B and DDQSIQK from IFNG exhibit slow production without reaching a signal plateau after 24 h (Figure 2C). The sequences surrounding the cleavage sites of these peptides have been previously shown to promote missed cleavages and are classified as slow trypsin cleavage sites, explaining the slow formation.3, 21, 22 The amino acids flanking IPVALGLK are rich in negatively charged, acidic residues (Asp and Glu) while peptide DDQSIQK has a Phe residue at position P2, relative to the trypsin cleavage site. Peptides that


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are slowly released from the target protein are not optimal for use as surrogate standard peptides for quantification. The relative amounts of these peptides in the sample may be less than the amount of synthetic standard peptide added. This will result in an over estimation of target protein present in the sample.

Analytical Strategy. Calibration curves were constructed from the LC-MRM analysis of a series of tryptic digests where 6 different concentrations of heavy isotope labeled proteins (IL1B, IFNG, and TNFA) were spiked with a constant concentration of standard full length protein, QconCAT, or synthetic peptide. Each sample was injected in triplicate and three transitions were monitored for each peptide resulting in 9 ratio determinations per concentration. Linear regression analysis was performed on the peak area ratios (heavy/light) versus concentration ratios for all target peptides. Inferences regarding quantitative accuracy, precision, digestion efficiency, and linearity, were used to comparatively evaluate each quantitative scheme. Calibration curves were generated from experimental data sets collected on both an Agilent 6460 QQQ and an AB Sciex API 5000 triple quadrupole mass spectrometer. Supplemental Information Table S-3 summarizes the calibration curve accuracy and linearity for all standards and target peptides across both instrument platforms. Regression analysis results showed high reproducibility across instrument platforms, but results from the 6460 QQQ are highlighted. Furthermore, regression analysis based on individual peptide transitions showed high correlation (r2 ≥ 0.998) (Supplemental Figure S-4).

Method Quantification Comparison. Full length protein standards resulted in slopes very close to unity (slope accuracy of 0.58 to 1.15 accepted as unity) within the set confidence interval and precise mean peak area ratios with a maximum CV of 13.3 % and median CV of 3.2 %. A slope of unity reveals accurate peptide quantification and complete digestion. The



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calibration curves also had strong linearity with r2 ≥ 0.993 except for peptide IPVALGLK (Figure 3).

The QconCAT construct without natural flanking sequences (Q1) resulted in variable performance depending on the peptide. Three peptide calibration curves for ANALLANGVELR, FFNSNK, and DDQSIQK had slopes near unity (1.04 ± 0.01, 1.03 ± 0.03, 0.87 ± 0.06) and strong linearity (r2 = 0.999, 0.997, 0.982) suggesting accurate quantification (Figure 3). However, the calibration curves for several peptides within Q1 had slope values above or below unity indicating reduced or enhanced digestion efficiency and inaccurate quantification (Figure 4). Peptides VNLLSAIK, IAVSYQTK, and LTNYSVTDLNVQR had slopes of 28.0 ± 0.33, 2.02 ± 0.09, and 2.03 ± 0.04, respectively, indicating inhibited release from the QconCAT compared to the full length protein standard. Peptide IPVALGLK had a slope of 0.38 ± 0.02 signifying enhanced release from the QconCAT compared to the full length protein.

Analysis of the amino acid sequences surrounding the tryptic cleavage sites proved insufficient to reliably predict peptide release from the QconCAT.. The release of some peptides from the QconCat, in comparison to the full length proteins, could be explained by primary sequence. For example, peptide DDQSIQK has an adjacent Phe residue, known to promote missed cleavages, in the native protein as well as the QconCAT resulting in similar digestion efficiencies and equal peptide release.24 In the QconCAT, IAVSYQTK has an acidic Glu residue proximal to a tryptic cleavage site which is known to promote missed cleavages.25 The cleavage sites in the full length protein are surrounded by neutral residues, not known to influence trypsin digestion efficiency, explaining the decreased yield observed for this peptide from the QconCAT. However, sequence based explanations are not as clear for other peptides. LTNYSVTDLNVQR has missed cleavage promoting Glu and dibasic residues near its cleavage site in the full length protein and neutral amino acids in the QconCAT. Yet, release of this peptide is decreased in the


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QconCAT compared to the calibrant protein. FFNSNK has a missed cleavage promoting dibasic site in the native protein which is replaced by a neutral residue in the QconCAT leading to incorrect predictions of enhanced peptide release from the QconCAT. Finally, release of VNLLSAIK was reduced in the QconCAT even though the cleavage sites for VNLLSAIK are surrounded by neutral residues in both the QconCAT and full length protein.

Furthermore, peptides found to have slopes not equal to unity were not limited to a specific peptide class. Peptide IPVALGLK showed slow release, LTNYSVTDLNVQR decayed rapidly after being released, and IAVSYQTK was a rapidly released, stable peptide. Based on these results, the selection of peptides and peptide order in QconCAT construct design is not completely predictable. Some peptides predicted to perform quantitatively well based on primary sequence analysis near the cleavage sites showed poor quantitative accuracy and vice versa.

Quantification Using QconCATs with Flanking Sequences. Calibration curves and regression analysis for a QconCAT construct with natural flanking sequences of six amino acids (Q2) showed improved quantitative performance compared to Q1 (Figure 4). Peptides VNLLSAIK, IAVSYQTK, and LTNYSVTDLNVQR, and IPVALGLK had slopes of 1.06 ± .02, 1.03 ± 0.01, 1.09 ± 0.0, and 0.93 ± 0.03, respectively and r2 values ≥ 0.995. The mean peak area ratios for Q2 had a maximum CV of 33.8 % and median CV of 3.5 %, slightly lower precision than observed with the full length proteins. The inclusion of natural flanking sequences of six amino acids resulted in slopes near unity for peptides that showed inaccurate quantification when non-native residues appeared in close proximity to trypsin cleavage sites. These results suggest that accurate quantification using QconCAT technology can be achieved with a few caveats. Peptide release from the native proteins and QconCAT proteins must be equimolar as peptides with enhanced or inhibited proteolytic release will have negative effects on quantitative accuracy leading to overestimation or underestimation of protein concentration. This makes



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QconCAT design and optimization of peptide order very important. However, it may not be readily evident which amino acids will influence digestion efficiency or the number of residues surrounding the cleavage site that should be considered. Inclusion of natural flanking sequences of six residues within the QconCAT design enabled peptide release most similar to the full length protein and allowed for accurate quantification of more peptides.

Quantification Using Standard Peptides with Cleavage Sites. Calibration curves were constructed from a series of labeled calibrant proteins spiked with unlabeled synthetic peptides. The synthetic peptides consisted of the native peptide or included natural flanking sequences of variable length (Supplemental Information Table S-4). The natural flanking sequences provided cleavable sites within the synthetic peptide to better mimic the cleavage characteristics of the native peptide. Quantification by amino acid analysis was performed post resolubilization to address two main concerns regarding the use of synthetic peptides, accurate concentration determination and quantitative resolubilization. Two peptides, DDKPTLQLESVDPK and IPVALGLK, with flanking sequences of 0 (tryptic peptide alone), 2, 4, or 6 amino acids were added to the calibrants prior to digestion to account for peptide degradation during this process. Regression analysis showed slopes equal to or near unity for peptide DDKPTLQLESVDPK regardless of presence or length of flanking sequence. This peptide was an optimal candidate for quantification, especially by synthetic peptides, because it displayed rapid release and slow decay during the time course digestion experiments. The cleavage sites of peptide DDKPTLQLESVDPK are also bordered by amino acids not known to promote missed cleavages or influence tryptic digestion accounting for why similar levels of standard peptide with and without flanking sequences were detected.

The calibration curve for synthetic peptide IPVALGLK +0 yield a slope of 0.04 ± 0.00 which would result in an overestimation of protein concentration as the concentration of this standard


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peptide was not dependent on tryptic digestion for peptide release and subsequent detection (Figure 5). This peptide displayed slow peptide release in the time course study, not even reaching a signal plateau after 24 h of digestion. The tryptic cleavage sites of IPVALGLK are surrounded by acidic Asp and Glu residues which are known to promote missed cleavages in tryptic digestions. Both peptides with flanking sequences of 2 amino acids showed incomplete tryptic digestion with high signal intensity for missed cleavage of the N-terminal internal cleavage site and low signal intensity for the non-cleaved and fully tryptic peptides (Supplemental Information Figure S-5). Calibration curves were not constructed for this synthetic peptide. Peptides with 4 and 6 residue flanking sequences lead to slopes of 0.15 ± 0.02 and 0.77 ± 0.06, respectively (Figure 5). The quantitative discrepancies associated with peptides that are slowly released or rapidly decay may be compensated with the inclusion of natural flanking sequences. The use of natural flanking sequences to incorporate a cleavage site in synthetic peptides will provide more freedom in the types of peptides that can be used with quantitative accuracy, such as peptides with these non-optimal digestion kinetics. This is especially important for small proteins with inherently fewer tryptic cleavages from which to select quantitative peptides. Our results show significant improvement in quantitative performance for peptide IPVALGLK with the addition of flanking sequences 6 amino acids in length. Longer flanking sequences may provide higher quantitative accuracy, but may be more expensive and challenging to synthesize. The additional expense of synthesizing longer peptides could be offset by minimizing the need to screen peptides for optimal digestion efficiency and the increased likelihood that the peptides will provide accurate quantification, limiting the need to synthesize additional peptides.

CONCLUSIONS We are the first to report a comprehensive, side by side study of recombinant protein, QconCAT, and synthetic peptide standards including the evaluation of natural flanking



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sequences. Overall, full length proteins provided robust accuracy based on slopes of unity, linearity, and high precision for all target peptides. This method also minimizes the evaluation process of optimizing amino acid sequence patterns surrounding cleavage sites and evaluating digestion efficiencies of candidate quantitative peptides. Moreover, the MRM assays developed for this study could be utilized for the quantification of three clinically important proteins in a myriad of applications.

ASSOCIATED CONTENT

Corresponding author * Address: 9600 Gudelsky Dr., Rockville, MD 20850, E-mail: [email protected]

Notes The authors declare no competing financial interest.

Acknowledgments Certain commercial materials, instruments, and equipment are identified in this manuscript in order to specify the experimental procedure as completely as possible. In no case does such identification imply a recommendation or endorsement by the National Institute of Standards and Technology nor does it imply that the materials, instruments, or equipment identified are necessarily the best available for the purpose.

Supporting Information Available Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.


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(16) Kuhn, E.; Whiteaker, J. R.; Mani, D. R.; Jackson, A. M.; Zhao, L.; Pope, M. E.; Smith, D.; Rivera, K. D.; Anderson, N. L.; Skates, S. J.; Pearson, T. W.; Paulovich, A. G.; Carr, S. A. Mol. Cell. Proteomics MCP 2012, 11, M111.013854. (17) Sherwood, C. A.; Eastham, A.; Lee, L. W.; Risler, J.; Mirzaei, H.; Falkner, J. A.; Martin, D. B. J. Proteome Res. 2009, 8, 3746–3751. (18) Lowenthal, M. S.; Liang, Y.; Phinney, K. W.; Stein, S. E. Anal. Chem. 2014, 86, 551–558. (19) Van den Broek, I.; Smit, N. P. M.; Romijn, F. P. H. T. M.; van der Laarse, A.; Deelder, A. M.; van der Burgt, Y. E. M.; Cobbaert, C. M. J. Proteome Res. 2013, 12, 5760–5774. (20) Loziuk, P. L.; Wang, J.; Li, Q.; Sederoff, R. R.; Chiang, V. L.; Muddiman, D. C. J. Proteome Res. 2013, 12, 5820–5829. (21) Brownridge, P.; Beynon, R. J. Methods San Diego Calif 2011, 54, 351–360. (22) Shuford, C. M.; Sederoff, R. R.; Chiang, V. L.; Muddiman, D. C. Mol. Cell. Proteomics MCP 2012, 11, 814–823. (23) Ye, M.; Pan, Y.; Cheng, K.; Zou, H. Nat. Methods 2014, 11, 220–222. (24) Monigatti, F.; Berndt, P. J. Am. Soc. Mass Spectrom. 2005, 16, 13–21. (25) Siepen, J. A.; Keevil, E.-J.; Knight, D.; Hubbard, S. J. J. Proteome Res. 2007, 6, 399–408.


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Figure Legends

Figure 1. Effect of digestion condition on peptide release. Data points represent the peak area ratio of the unlabeled proteins (14N) to the spike-in labeled proteins (15N) digested with the condition indicated. Peak area ratios were normalized to condition (1). Conditions include: (1) 0.1 % RapiGest, (2) pH 7.0, (3) 8 mol/L urea, (4) 0.1 % SDS, (5) 80 % acetonitrile, (6) 48 °C, (7) 1:5 trypsin: protein, and (8) 1:25 trypsin/Lys-C: protein. Data and error bars display mean and standard deviation for three transitions per peptide and duplicate experiments.



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Figure 2. Time course analysis showing peptide release and stability for 9 peptides from ILB1 (peptides DDKP, SLVM, and IPVA), IFNG (peptides FFNS, LTNY, and DDQS), and TNFA (peptides VNLL, IAVS, and ANAL) during proteolytic digestion. Peptides are grouped by class: (A) rapid release and stable over time, (B) rapid release and subsequent signal decay, and (C) slow release. Normalized peak area ratios of the unlabeled proteins (14N) to the spike-in labeled proteins (15N) are plotted versus incubation time. Data and error bars represent the mean and standard deviation for three transitions per peptide and duplicate experiments.


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Figure 3. Calibration curves and regression analysis from a series of digests containing recombinant protein (Top) or QconCAT protein without flanking sequences (Bottom) spiked into samples with varying concentrations of 15N isotope labeled calibrant proteins. Measured peak areas (averaged across three transitions and three replicates for a total of nine measurements per concentration) were plotted against the expected molar ratios. The slope (m) and intercept (b) from the regression analysis are shown with standard error for a 95 % confidence interval.



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Figure 4. (A) Cartoon version of QconCAT constructs. The top QconCAT is a concatenation of the natural peptide sequences with N-terminal Met and C-terminal His6-tag (shown in blue). The bottom QconCAT consists of the same peptides with 6-amino acid long, N-terminal and Cterminal natural flanking sequences (shown in purple) concatenated into the overall sequence. (B) Calibration curves and regression analysis from a series of digests containing QconCAT protein without flanking sequences (Top) or QconCAT protein with natural flanking sequences of 6 amino acids (Bottom) spiked into samples with varying concentrations of 15N isotope labeled calibrant proteins. Measured peak areas (averaged across three transitions and three replicates for a total of nine measurements per concentration) were plotted against the expected molar ratios. The slope (m) and intercept (b) from the regression analysis are shown with standard error for a 95 % confidence interval.


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Figure 5. Calibration curves and regression analysis from a series of digests containing synthetic peptides without (+0) or with natural flanking sequences of +4 or +6 amino acids spiked into samples with varying concentrations of 15N isotope labeled calibrant protein. Measured peak areas (averaged across three transitions and three replicates for a total of nine measurements per concentration) were plotted against the expected molar ratios. The slope and standard error for a 95 % confidence interval for peptide DDKPTLQLESVDPK are 1.09 ± 0.02, 1.21 ± 0.03, 1.02 ± 0.02 and for peptide IPVALGLK are 0.04 ± 0.00, 0.15 ± 0.02, 0.77 ± 0.06 for synthetic peptides +0, +4, and +6, respectively.



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for TOC only


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Quantitative Performance of Internal Standard Platforms for Absolute

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