Amino Acid Analysis of Peptides Using Isobaric-Tagged Isotope

Apr 13, 2009 - Prevention, 4770 Buford Highway, Atlanta, Georgia 30341. Protein quantification using stable isotope dilution mass spectrometry require...
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Anal. Chem. 2009, 81, 3979–3985

Amino Acid Analysis of Peptides Using Isobaric-Tagged Isotope Dilution LC-MS/MS Adrian R. Woolfitt, Maria I. Solano, Tracie L. Williams, James L. Pirkle, and John R. Barr* Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Highway, Atlanta, Georgia 30341 Protein quantification using stable isotope dilution mass spectrometry requires the quantification of specific peptides unique to the protein of interest. Since these peptides are used as calibration standards, accurate and precise measurement of these target peptides is critical. This peptide measurement has typically been made by amino acid analysis (AAA) using absorbance or fluorescence detection methods. This approach can be limited to only a few amino acids, is often not traceable to highquality reference standards, and not uncommonly has coefficients of variation (CVs) that exceed 10%. We report here an isobaric-tagged isotope dilution mass spectrometry method for AAA that provides excellent sensitivity, specificity, and precision; utilizes a broad range of amino acids; and uses U.S. National Institute of Standards and Technology (NIST) amino acid standards for an accuracy base. The average CV for the method applied to three different peptides with measurements on 7 different days was 3.57% (range 2.72-4.20%). We applied this method to the quantification of three NIST standard peptides and hemagglutinin, an influenza virus surface protein. Quantification of proteins is an analytical challenge. Many proteins of clinical interest are hard to purify to homogeneity, so it is often difficult to determine with high accuracy and precision the absolute amount and purity of a target protein in a standard or a complex mixture, such as a clinical sample. In the past, methods such as UV absorbance,1 the Bradford2 and Lowry3 assays, or nitrogen analysis such as the Kjeldahl total nitrogen4 method were used, with varying degrees of success. In 1996, Barr et al. first reported using stable isotope dilution mass spectrometry (IDMS) to quantify a protein by accurate measurement of one or more peptides released by enzymatic digestion.5 There has since been much interest in both relative and absolute MS-based protein quantification, and a wide range of isotope-dilution strategies have * To whom correspondence should be addressed. John R. Barr, 4770 Buford Highway, MS F-50, Atlanta, GA 30341. E-mail: [email protected]. Phone: 770-4887848. Fax: 770-488-0509. (1) Layne, E. Methods Enzymol. 1957, 10, 447–454. (2) Bradford, M. M. Anal. Biochem. 1976, 72, 248–254. (3) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265–275. (4) McKenzie, H. A.; Wallace, H. S. Aust. J. Chem. 1954, 7, 55–70. (5) Barr, J. R.; Maggio, V. L.; Patterson, D. G., Jr.; Cooper, G. R.; Henderson, L. O.; Turner, W. E.; Smith, S. J.; Hannon, W. H.; Needham, L. L.; Sampson, E. J. Clin. Chem. 1996, 42, 1676–1682. 10.1021/ac900367q Not subject to U.S. Copyright. Publ. 2009 Am. Chem. Soc. Published on Web 04/13/2009

been developed.6-8 These strategies generally offer far higher specificity than traditional methods since they are based on identification of structural components of the target proteins (i.e., peptides). IDMS methods have now become standard procedures for absolute protein quantification. One approach requires complete tryptic digestion of a protein and quantification of one or more of its unique target peptides. Quantification of those peptides is performed using isotopically labeled peptides as internal standards in liquid chromatography-MS or liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses and withstandardcurvescreatedusingthenativesyntheticpeptides.5,9-11 With complete protein digestion and availability of stable, unique peptides, this approach can offer excellent sensitivity, specificity, accuracy, and precision. The accuracy base for such IDMS methods is the peptide standard.5,9 Although the peptide materials can be highly purified, they will still contain residual salts and water, and in many cases they are hygroscopic. Therefore, gravimetric measurement alone is not acceptable to determine the amount of peptide in a standard.9 Amino acid analysis (AAA) has been widely used for this purpose.5,11 AAA is traditionally performed using acid hydrolysis followed by LC separations with either pre- or postcolumn derivatization and detection by absorbance or fluorescence. Our laboratory has used two or more commercial laboratories for AAA of peptide standards. However, in our experience, the discrepancies between different laboratories have been as much as 17%, and often as few as two or three amino acids (AAs) have been used for quantification. In addition, traceability to high-quality reference standards to ensure accuracy has been lacking. In a 1996 comparative study involving 71 facilities, interlaboratory coefficients of variation (CVs) were estimated at around 10% after excluding outliers.12 This level of variability has been considered acceptable for analysis of AAs in food.13 An AAA method has recently been presented that utilizes IDMS of 9 AAs, with U.S. National Institute of Standards and (6) Yan, W.; Chen, S. S. Briefings Funct. Genomics Proteomics 2005, 4, 27–38. (7) Bantscheff, M.; Schirle, M.; Sweetman, G.; Rick, J.; Kuster, B. Anal. Bioanal. Chem. 2007, 389, 1017–1031. (8) Bro ¨nstrup, M. Expert Rev. Proteomics 2004, 1, 503–512. (9) Burkitt, W. I.; Pritchard, C.; Arsene, C.; Henrion, A.; Bunk, D.; O’Connor, G. Anal. Biochem. 2008, 376, 242–251. (10) Williams, T. L.; Luna, L.; Guo, Z.; Cox, N. J.; Pirkle, J. L.; Donis, R. O.; Barr, J. R. Vaccine 2008, 26, 2510–2520. (11) Barnidge, D. R.; Dratz, E. A.; Martin, T.; Bonilla, L. E.; Moran, L. B.; Lindall, A. Anal. Chem. 2003, 75, 445–451. (12) Schegg, K. M.; Denslow, N. D.; Andersen, T. T.; Bao, Y.; Cohen, S. A.; Mahrenholz, A. M.; Mann, K. Tech. Protein Chem. 1997, 8, 207–216. (13) Sarwar, G.; Botting, H. G. J. Chromatogr. 1993, 615, 1–22.

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Figure 1. Schematic representation of the isobaric tag isotope dilution mass spectrometry method for amino acid analysis.

Technology (NIST) AA standards for calibration.9 Although this method offers exceptional accuracy and precision, it does not provide broad AA coverage, is labor intensive, and is not suitable for peptides that contain an amino acid more than once, since the method adjusts all AA concentrations in the standards so that the signal levels for each AA are similar. Another recent LC-MS/ MS method is based on derivatization of AAs with isobaric tags called iTRAQ reagents, which are isotopically enriched methylpiperazine acetic acid N-hydroxy succinimide esters.14 The method provides broad AA coverage, can handle more than one copy of an AA in a peptide, and has excellent sensitivity and specificity. The iTRAQ AAA reagents14,15 introduce one of two isobaric mass tags that improve reverse-phase chromatographic sensitivity and specificity and that preferentially fragment to give ions at m/z 114 or m/z 117 under MS/MS conditions. This approach permits MS detection parameters to be the same across amino acids. By contrast, other LC-MS/MS methods using underivatized AAs require that MS parameters be optimized for each individual AA.16,17 The focus of this report is the development of an isobarictagged isotope dilution mass spectrometry (IT-IDMS) method for AAA of peptides that (1) improves precision, (2) uses NIST amino acid standards for accuracy, (3) utilizes a broad array of amino acids (a total of 15), (4) has excellent sensitivity and specificity, and (5) utilizes straightforward LC-MS/MS techniques. The development of the IT-IDMS method was based on altering and expanding the iTRAQ method including changing the calibration (14) Daniels, S.; Stanick, W.; Ingle, E.; White, W.; Cox, D.; Nimkar, S.; Purkayastha, B. 10th Annual Meeting of the Association of Biomolecular Resource Facilities, Long Beach, CA, Feb 11-14, 2006. (15) Ross, P. L.; Huang, Y. N.; Marchese, J. N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.; Jacobson, A.; Pappin, D. J. Mol. Cell. Proteomics 2004, 3, 1154–1169. (16) Nagy, K.; Taka´ts, Z.; Pollreisz, F.; Szabo´, T.; Ve´key, K. Rapid Commun. Mass Spectrom. 2003, 17, 983–990. (17) Piraud, M.; Vianey-Saban, C.; Petritis, K.; Elfakir, C.; Steghens, J.-P.; Morla, A.; Bouchu, D. Rapid Commun. Mass Spectrom. 2003, 17, 1297–1311.

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curve for each amino acid so that it is based on NIST AA standards, selectively using the iTRAQ-117 reagent, and adding 15 isotopically labeled AAs as internal standards in order to improve accuracy and precision. We have evaluated the performance of the method with emphasis on accuracy, precision, sensitivity, and specificity. We apply this method to the quantification of three NIST standard peptides and two peptides from hemagglutinin, an influenza virus surface protein.10

EXPERIMENTAL SECTION Method. An overview of the IT-IDMS method is presented in Figure 1. The left column describes the peptide analysis, which begins with a peptide of unknown concentration being mixed with 15 isotopically labeled amino acids. The mixture undergoes acid hydrolysis, and the resulting amino acids are then derivatized with the iTRAQ-117 reagent. This derivatization improves the chromatographic and mass spectrometric properties of the AA, thus improving the sensitivity and specificity of the method. Finally, peak-area isotopic ratios are calculated for each derivatized amino acid released from the peptide. The right column follows a similar process for generating a calibration curve for each of the 15 amino acids based on NIST AA standards. First, NIST AA standards are mixed with 15 isotopically labeled amino acids (the same concentrations of 15 amino acids used in the peptide analysis). They are then derivatized with iTRAQ-117 reagent, and peak-area isotopic ratios are calculated for each NIST amino acid. The concentration of the derivatized NIST AA standards and their peakarea isotopic ratios are then used to develop a calibration curve for each AA. From the peptide analysis, the “peptide AA/labeled AA ratio” is applied to the appropriate NIST AA calibration curve to calculate the concentration of that AA in the starting peptide. So, for example, peptide Ser-117/labeled Ser-117 would be applied to the NIST Ser-117/labeled Ser-117 calibration curve to calculate the serine concentration of the original peptide.

Materials. An amino acid mix18 (SRM 2389; around 2.5 mM for each of 17 AAs, with a range of 2.27 mM (Glu) to 2.94 mM (Arg)) and peptide standards19 (RM 8327; 1 mg/vial) were obtained from the National Institute of Standards and Technology (Gaithersberg, MD). Fifteen individual 13C- and 15N-labeled (heavy) L-amino acids were obtained from Cambridge Isotope Laboratories, Inc. (Andover, MA). L(+)-Norleucine (NorLeu; 99+%) was obtained from Acros Organics (ThermoFisher Scientific, Rockford, IL). Amino Acid 20/20 Analyzer iTRAQ kits and C18 AAA columns were from Applied Biosystems (Foster City, CA). Burdick & Jackson HPLC-grade water and acetonitrile were from Honeywell (Morristown, NJ), 6 M HCl from Pierce Biotechnology (ThermoFisher), and heptafluorobutyric acid (HFBA) and formic acid were from SigmaAldrich (St. Louis, MO). Influenza peptides were synthesized at a 1-5 mg scale by MidWest Bio-Tech (Fishers, IN). These lyophilized peptides were redissolved in-house, aliquoted in 200 µL volumes into 1.5 mL vials using a Biomex NXP Laboratory Automation Workstation (Beckman Coulter, Fullerton, CA) and then lyophilized again and stored at -70 °C until use. All AAA materials and equipment were kept and handled using clean working practices designed to avoid possible AA and protein contamination;20 most of the sample preparation steps were carried out in a clean biological safety cabinet dedicated to this purpose only. Preparation of Standards. A mixture of 15 heavy AAs was prepared in 0.1 M HCl, such that each AA was present at 15 pmol/ µL. NorLeu was prepared gravimetrically at 15 pmol/µL in 0.1 M HCl. The NIST AA standards mix was diluted by 1:166 in 0.1 M HCl to give an average of 15 pmol/µL as calculated for all AAs present except Cys.18 Seven-point calibration curves were prepared using NIST AAs as standards at average levels of 75, 112.5, 150, 225, 300, 450, and 600 pmol per AA (5-40 µL of NIST AA standards mix), each with 150 pmol of each labeled AA (10 µL of mix) and 150 pmol (10 µL) of NorLeu14 in 0.5 mL polypropylene tubes. The standards were taken to complete dryness in a rotary evaporator in preparation for derivatization. Peptide Reconstitution. Lyophilized influenza peptides (typically 2-5 nmol per vial) were suspended in 40 µL of 10% (v/v) formic acid in water and were agitated on a vortex mixer to aid reconstitution. Then, 360 µL of 0.1% (v/v) formic acid was added. NIST standard peptides (1 mg per vial) were reconstituted using a similar procedure but with 100 µL of 10% and then 900 µL of 0.1% formic acid in the original containers.19 Peptides were further diluted to a nominal concentration of 15 pmol/µL using 0.1% formic acid. Hydrolysis Procedure. To optimize quantitative performance, samples were prepared for hydrolysis so that nominal peptide loadings would produce AA levels that fall within the 75-600 pmol range of the calibration curves. For example, 150 pmol of a peptide containing 1, 2, and 3 copies of different AAs would be expected to yield 150, 300, and 450 pmol of those AAs, respectively. The totals for any Asp/Asn or Glu/Gln pairs were combined for these (18) National Institute of Standards and Technology. Certificate of Analysis, Standard Reference Material 2389, January 6, 2006. (19) National Institute of Standards and Technology. Report of Investigation, Reference Material 8327, March 19, 2007. (20) Cohen, S. A.; Strydom, D. J. Anal. Biochem. 1988, 174, 1–16.

estimates. We based these calculations either on pre-existing AAA measurements for a sample or on the results of survey hydrolyses for that sample. Three types of samples underwent hydrolysis: peptide samples of unknown concentration, NIST peptide standards, and a solvent blank for quality assurance. To monitor recoveries, samples of the NIST AA standards mix were also subjected to hydrolysis. Peptide samples consisting of a nominal 180 pmol of peptide in 12 µL of 0.1% formic acid were mixed with 180 pmol of the labeled 15 AA mix and 180 pmol of NorLeu in polypropylene tubes. Similarly, an average of 180 pmol of NIST AA mix in 12 µL of 0.1 M HCl was also mixed with 180 pmol of the labeled 15 AA mix and 180 pmol of NorLeu in polypropylene tubes. A 50 µL solvent blank was mixed the same way. For the peptide samples, NIST AA samples, and the solvent blank, the remaining hydrolysis was done the same way. Volumes containing 150 pmol of each sample (i.e., peptide, NIST standard, or solvent blank) were transferred directly to the bottom of 6 × 50 mm disposable glass tubes from Kimble glass, Inc. (Sylvania, OH) previously cleaned with concentrated sulfuric acid and stored in an oven at 165 °C until use. Samples were taken to complete dryness in a rotary evaporator. Tubes were then placed in clean hydrolysis chambers, and 200 µL of 6 M HCl-containing 1% phenol was added to the bottom of each chamber. The chambers were then evacuated and flushed with dry nitrogen three times, before being sealed under vacuum using the Eldex H/D Workstation (Eldex Laboratories, Inc., Napa, CA). Hydrolysis was carried out at 150 °C for 120 ± 10 min. Hydrolysates were resuspended in 50 µL of 0.1 M HCl, immediately transferred to 0.5 mL polypropylene tubes, and taken to complete dryness in a rotary evaporator. Since the hydrolysis procedure is an important component to the overall variance of AAA,21 we developed a peptide analysis protocol which tracked each individual hydrolysis sample and distributed replicates evenly across the four hydrolysis chambers that could be processed concurrently using the Eldex H/D workstation. Each hydrolysis chamber contained a set of samples that constituted an analytical run. An analytical run (one hydrolysis chamber) included two QC samples consisting of a NIST standard peptide, one blank, one NIST AA mix sample as a recovery marker, and up to seven unknown peptide samples to be measured. In general, we obtained repeat peptide samples from multiple randomly selected vials to incorporate variance due to aliquoting. Each hydrolysis sample was analyzed by LC-MS/MS in duplicate. We investigated the effect of hydrolysis times (0.5, 1, 2, 3, 6, and 18 h) using three NIST standard peptides, A, B, and C, with the Eldex H/D workstation and vapor-phase acid hydrolysis at 150 °C. The optimum hydrolysis time was 2 h. Samples in each hydrolysis experiment were quantified using individual NIST AA standard calibration curves prepared and analyzed simultaneously. Furthermore, to evaluate day-to-day reproducibility, at least three separate hydrolysis experiments were carried out on different days. Derivatization with iTRAQ Reagents. Dry hydrolysates and calibration standards were derivatized according to the Amino Acid 20/20 Analyzer protocol.14 Briefly, AA samples were mixed with ¨ .; Andersen, T. T.; Apostol, I.; Fox, J. W.; Paxton, R. J.; Strydom, (21) Yu ¨ ksel, K. U D. J. Tech. Protein Chem. 1994, 6, 185–192.

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Table 1. Precursor and Product Ions of Derivatized Native and Isotopically Labeled Amino Acids native derivatized amino acids amino acid Ser Gly His Asp (Asn)b Met-Sulfoxide Thr Ala Glu (Gln)b Arg Pro Cys Lysc Val NorVal Met Tyr Ile Leu NorLeu Phe

precursor ion m/z 250.2 220.1 300.2 278.2 310.2 264.2 234.2 292.2 319.2 260.2 266.1 435.3 262.2 262.2 294.2 326.2 276.2 276.2 276.2 310.2

isotopically labeled derivatized amino acids product ion m/za 117.0 117.0 117.0 117.0 117.0 117.0 117.0 117.0 117.0 117.0 117.0 117.0 117.0 117.0 117.0 117.0 117.0 117.0 117.0 117.0

amino acid

precursor ion m/z

product ion m/z

Ser C3, N1 Gly 13C2, 15N1 His 13C6 Asp 13C4, 15N1

254.1 223.1 306.2 283.2

117.0 117.0 117.0 117.0

Thr 13C4 Ala 13C3, 15N1 Glu 13C5, 15N1 Arg 13C6 Pro 13C5, 15N1

268.2 238.2 298.2 325.2 266.2

117.0 117.0 117.0 117.0 117.0

Lysc 13C6, 15N2 Val 13C5, 15N1

443.3 268.2

117.0 117.0

Tyr 13C9, 15N1 Ile 13C6, 15N1 Leu 13C6

336.3 283.2 282.2

117.0 117.0 117.0

Phe 13C9, 15N1

320.2

117.0

13

15

a Native derivatized amino acids listed here are tagged with the iTRAQ-117 isobaric reagent. A set of native derivatized amino acids having iTRAQ-114 isobaric tags (not shown) are similar except that all product ions are m/z 114. b Acid hydrolysis quantitatively converts Asn to Asp and Gln to Glu. c Lys is derivatized twice with the iTRAQ-117 isobaric reagent.

5 µL of the “AA labeling buffer” included in the iTRAQ kits (containing borate buffer and 150 pmol of norvaline (NorVal)) and 5 µL of iTRAQ reagent-117 in isopropanol and then were incubated for at least 30 min at room temperature. Next, 1 µL of hydroxylamine solution was added to each sample, and after at least 5 min of incubation, the samples were taken to dryness. The samples were then reconstituted in 25 µL of iTRAQ standard-114 solution (containing 150 pmol of each iTRAQ-114 tagged AA) and diluted 20-fold with 0.1% formic acid for LC-MS/MS analysis. The Amino Acid 20/20 Analyzer protocol includes the use of iTRAQ-114 tagged AA internal standards and NorVal and NorLeu. The IT-IDMS method does not require or use the iTRAQ-114 tagged materials. Calibration is provided solely by NIST AA standards, so NorLeu is not needed. Isotopically labeled internal standards are used, so iTRAQ-114 tagged AA internal standards are not needed. However, the presence of these compounds does not affect any other aspect of the IT-IDMS method performance, and the compounds can be included for an additional quality assurance check, if desired. For example, various problems with the iTRAQ reagent system can be diagnosed based on the NorVal and NorLeu ratios. LC System. An Acquity UPLC system (Waters Corporation, Milford, MA) was used for all chromatographic separations. The LC column (C18 AAA, 4.6 mm × 150 mm), solvent system, and analytical gradient were as described in the Amino Acid 20/20 Analyzer protocol. Briefly, solvents A and B were water and acetonitrile respectively, both with 0.005% (v/v) HFBA and 0.1% (v/v) formic acid. The LC autosampler temperature was kept at 5 °C throughout all analyses. The injection volume was 5 µL, and iTRAQ labeled AAs were eluted with a linear gradient of 2% solvent B to 28% solvent B in 14.3 min at 0.7 mL/min, with the column held at 50 °C. The column was washed with a 2 min pulse of 100% solvent B then equilibrated with 2% solvent B. Retention times ranged between 4 and 14 min, and the LC cycle time was 24.8 min. 3982

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Mass Spectrometer. Multiple reaction monitoring (MRM) data were obtained using an Applied Biosystems 4000 QTrap mass spectrometer operated in positive ion electrospray mode at unit resolution with a standard Turbo-V ion source. Most settings were as specified in the Amino Acid 20/20 Analyzer protocol (IS 1500, TEM 600, CUR 20, GS1 60, GS2 60, CAD medium, DP 30, EP 10, CE 30, and CXP 5). A total of 49 MRM transitions in three time windows were monitored, as shown in Table 1. Dwell times for Ser, Gly, His, Asp, Met-Sulfoxide, Thr, Ala, and Glu were 35 ms. Dwell times for Arg, Pro, and Cys were 75 ms, and the dwell times for Lys, Val, NorVal, Met, Tyr, Ile, Leu, NorLeu, and Phe were 35 ms. Results reported are the average of duplicate LC-MS/ MS measurements. Data Analysis. The Applied Biosystems Analyst 1.4.2 software was used for peak detection, and all peaks were manually reviewed. All further data processing including quantification and visualization was performed using custom Microsoft Excel 2003 spreadsheets and macros. NIST AA standard curves were fitted using a quadratic least-squares method with 1/x weighting, using the exact NIST levels of each AA.18 The Excel macros compared observed and expected AA levels for each sample to compute AA purity values and then averaged the AA purity for each AA expected to be present in the sample to obtain a peptide purity value.21 In addition, data were analyzed for the presence of outliers, external contamination, and acceptable accuracy and precision. RESULTS AND DISCUSSION We have developed an isobaric-tagged isotope dilution mass spectrometry method for amino acid analysis to quantify peptides. Accurate peptide concentrations are the basis for protein quantification that uses unique protein peptides from enzymatic digestion. Compared to traditional AAA methods, the IT-IDMS method shows improved accuracy, precision, and sensitivity. The IT-IDMS method incorporates isotopically labeled amino acids as internal standards, NIST-certified reference materials as AA

Figure 2. Representative LC-MS/MS reconstructed ion chromatograms of isobaric tagging reagent (iTRAQ) labeled AAs, derivatized after acid hydrolysis for 2 h at 150 °C. Each AA was present in the hydrolysis at a nominal 150 pmol level. (a) NIST AA reference material SRM 2389 comprising 17 AAs, plus NorValine and NorLeucine, with iTRAQ-117 tags. (b) 15 isotopically labeled AAs with iTRAQ-117 tags, used as internal standards. Except for NorValine and NorLeucine, AAs are annotated using single-letter AA codes. One unknown on the MRM channel for Ala is annotated with “?”. Table 2. Results of Amino Acid Analysis Quality Control Validation Using NIST Peptides and NIST Amino Acid Recovery Standards NIST values

IT-IDMS method

iTRAQ methoda

NIST material subjected to hydrolysis

NIST purityb

NIST expanded uncertainty value (U, %)d

purity (mean)

coefficient of variation (CV, %)

purity (mean)

coefficient of variation (CV, %)

number of hydrolysesc

peptide A peptide B peptide C NIST amino acid recovery standard

0.69 0.73 0.67 1.000

15.9 19.2 23.9 3.69e

0.647 0.697 0.743 0.999

3.80 2.72 4.20 2.08

0.640 0.681 0.728 0.985

6.21 6.27 5.76 4.22

43 46 47 26

a The iTRAQ method was modified in these analyses to include NIST amino acid calibration standards. b For the peptides, NIST purity is the fractional peptide mass purity. For the NIST AA mix, the NIST purity is the average AA purity and is given as 1.000 because each AA concentration is known. c Each hydrolysate sample was analyzed by LC-MS/MS in duplicate. d NIST purity ±U defines an interval with a confidence level of 95% containing the true purity. e This is the average of the U values for all AAs except Cys, expressed as a percentage of the NIST purity. Uncertainties are shown as percentages to facilitate comparisons with the CV values.

standards to anchor the accuracy of results to NIST materials, and derivatization using the iTRAQ-117 isobaric tagging reagent to provide excellent chromatographic separation and sensitivity for each individual amino acid. Calibration of the IT-IDMS Method Using NIST Reference Materials and Labeled Internal Standards. The NIST AA standards included 17 out of 20 AAs (Table 1). Figure 2a shows a LC-MS/MS reconstructed ion chromatogram for the NIST AA standards after derivatization with the iTRAQ-117 reagent. Each AA was present at a nominal level of 150 pmol in the hydrolysis, and it can be seen that the relative responses for most derivatized AAs are similar (within a factor of about 4), with the exception of Cys, Lys, and Arg. In addition, there is baseline chromatographic separation between the Val, NorVal isobaric doublet and the Ile, Leu, NorLeu isobaric triplet. The same MS/MS fragmentation was employed for each AA, enabling uniform ion source parameters to be used, and the m/z 117 iTRAQ tag was the only fragment ion present at above 15% relative intensity in product ion scans from each parent m/z to m/z 50. Furthermore, the m/z 114-117 region of the spectra is relatively free of interferences.15 In contrast, the underivatized NIST AA mixture yielded widely differing relative responses (in excess of 20-fold) using a water/ acetonitrile solvent system with 0.1% pentadecafluorooctanoic acid and 0.1% formic acid modifiers (data not shown). As shown in Figure 1, 15 isotopically labeled AAs were added to the peptide samples prior to hydrolysis and to the NIST AA

standards calibration curves. Figure 2b shows a reconstructed ion chromatogram for these 15 labeled AAs used as internal standards. For the NIST AA standards, we constructed 7-point calibration curves which spanned the range of 75-600 pmol for each AA. The isotopically labeled AAs were present at 150 pmol throughout. The addition of these labeled AAs did not produce any consequential interferences. The accuracy of the AA measurements was then directly based on NIST AA standards. Improvements in accuracy and/or precision were expected since losses of individual AAs during hydrolysis were accounted for and corrected using the corresponding labeled AAs as internal standards. As noted in the methods section, hydrolysis is an important step and care must be taken to ensure complete hydrolysis of the peptide being analyzed. To compare the iTRAQ method with the IT-IDMS method for AA analysis, we analyzed 150 pmol NIST AA hydrolysis recovery samples in a total of 26 separate analytical runs obtained on seven different days by both methods. The original iTRAQ method was enhanced by using NIST AA standards for calibration so that the same NIST accuracy base was used for the purpose of the method comparison. Results of the comparison are presented in the last row of Table 2. Results are expressed as average purity to be consistent with how the results are reported for the NIST peptides. Purity is the number of moles of each AA measured divided by the expected number of moles present, while average purity is the average for all AAs quantified (Cys, Met, Met sulfoxide, NorVal, and NorLeu are Analytical Chemistry, Vol. 81, No. 10, May 15, 2009

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excluded). A value of 1.000 would indicate an average 100.0% recovery for each quantified NIST AA. The estimate by the original iTRAQ method was 0.985 with a CV of 4.22%. The estimate by the IT-IDMS method was 0.999 with a CV of 2.08% (a 51% improvement in precision) and a slight improvement in the accuracy. Measurement on three NIST peptides (discussed later in the text) and additional measurements in our laboratory (not shown) consistently showed about a 50% improvement in precision by the IT-IDMS method compared to the iTRAQ method. In addition to established benefits of isotopically labeled internal standards, the use of these standards instead of NorLeu provides the ability to analyze NorLeu in peptides of interest. For example, in several projects in our laboratory, we have substituted Met with NorLeu in peptides used as toxin substrates, so quantification of NorLeu is important for these analyses. IT-IDMS Analysis of NIST Peptides. We evaluated the accuracy and precision of the IT-IDMS method for peptide quantification by analyzing three NIST standard peptides: peptide A (AA sequence DAEPDILELATGYR), peptide B (KAQYARSVLLEKDAEPDILELATGYR), and peptide C (RQAKVLLYSGR). Sample preparation and method measurements were conducted on seven different days to capture both within-day and amongday sources of variation. We also compared the iTRAQ and ITIDMS methods, utilizing the NIST AA standards for calibration of both methods as described above for the NIST AA recovery measurements. The results are summarized in the first three rows of Table 2. For each of the three NIST peptides, the ITIDMS method was able to quantify the peptide concentration using quantitative results for every AA in the peptide. The NIST peptide mass purities are 0.69 ± 0.11, 0.73 ± 0.14, and 0.67 ± 0.16 for peptides A, B, and C, respectively,19 expressed as a fraction of 1.00 ± U. The 1.00 represents 100% peptide mass purity of the 1.0 mg of lyophilized material. Similar to the AA purity described above, the peptide mass purity is the number of moles of peptide measured divided by the number of moles of peptide calculated based on gravimetric measurement of the peptide standard. Since peptide standards are expected to contain salts and water, the true amount of peptide present in a sample is expected to be less than that estimated by weight. U is the NIST expanded uncertainty value and is equal to 4.303 times the overall standard deviation of the peptide mass purity measurements obtained by three laboratories. Two laboratories used an alanine-based method of AAA with precolumn derivatization, and the third laboratory used ultraviolet (UV) absorbance measurements.19 For these peptides, the range set by ±U about the mean corresponds to a confidence level of 95% that the true peptide mass purity is within this range. We calculated nominal peptide loadings of 150 pmol based on the assumption of 1.00 peptide mass purity, but with the expectation that the true levels were around 100 pmol for each peptide. Results for the IT-IDMS method for the three peptides were close to the NIST values and were within the NIST expanded uncertainty ranges.19 The means obtained using the iTRAQ method (using the iTRAQ m/z 114 tagged internal standards, NorLeu as a recovery standard, and the same NIST AA calibration curves) were similar, but for each peptide the precision was better with the IT-IDMS method. This finding was in agreement with the approximate 50% improvement in 3984

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Table 3. Amino Acid Analysis on Influenza Hemagglutinin Peptides from H5N1 Strains hemagglutinin peptidea

mean, nmol/vial

standard deviation

coefficient of variation (CV, %)

LVLATGLR LVLATGL+7R TLDFHDSNVK TLDFHDSNV+6K

5.266 1.893 3.147 3.886

0.168 0.065 0.056 0.076

3.18 3.44 1.78 1.95

a Each peptide was hydrolyzed at the optimal 150 pmol level and analyzed on 3 separate days using the IT-IDMS method. Single hydrolysis samples were taken from 12 separate vials of each peptide.

precision observed with other analyses comparing the iTRAQ and the IT-IDMS method. IT-IDMS Analysis of Hemagglutinin Peptides. Hemagglutinin is an influenza virus surface protein that is an important determinant of the virulence of the virus. Influenza vaccines are required to contain specified amounts of inactivated hemagglutinin. Digestion of hemagglutinin by trypsin releases several characteristic peptides that can be quantified. The molar concentration of these peptides equals the molar concentration of the hemagglutinin.10 Accurate and precise measurement of hemagglutinin is needed for production of effective influenza vaccine. We analyzed four peptides: two of the peptides from tryptic digestion of hemagglutinin (TLDFHDSNVK and LVLATGLR) and their isotopically labeled analogues (TLDFHDSNV+6K and LVLATGL+7R), where V+6 and L+7 represent stable isotopically labeled Val and Leu with the mass increases in daltons. The peptides had previously been aliquoted into 864 vials, and 12 vials were selected for analysis. On each of three days, samples were selected from four vials and processed through the IT-IDMS method, totaling 12 independent analyses per peptide. In this analysis design, sources of overall method error included both within-day and among-day sources. Nominal levels of each peptide were close to 150 pmol. Each analytical run included NIST peptide B or C as a quality control material, NIST AA standards as a recovery check for the hydrolysis step, and solvent blanks prepared from the same batches of solvent as had been used to aliquot the peptides. Table 3 shows the IT-IDMS results obtained over the 3 days of analysis. Measured CVs for the purities of the four peptides were 3.18%, 3.44%, 1.78%, and 1.95%. These peptide concentrations were measured using results from every AA in each peptide, with the exception of the isotopically labeled Val and Leu (because of interferences with the isotopically labeled internal standards). The CV range (1.8-3.5%) was similar to the analyses of the three NIST peptides. CONCLUSIONS Protein quantification using stable IDMS requires the quantification of specific peptides that are unique to the protein of interest. Since the concentrations of these peptides expressed as moles equates to the protein molar concentration, accurate and precise measurement of these peptides is important. NIST provides a certified standard mix of 17 amino acids that can be used as an accuracy base for the amino acid analysis of

target peptides. These peptides can, in turn, be used for mass spectrometry-based protein quantification. We have presented a new method for AAA of peptides that is based on LC-MS/MS and uses NIST AA standards for calibration, iTRAQ-117 reagents for better chromatographic sensitivity and specificity, and 15 isotopically labeled amino acids as internal standards to provide IDMS performance. This method allows for amino acid determination of nearly all amino acids that would be present in a target peptide intended for protein quantification. The method CV including within-day and among-day variance is in the 1.8-4.2% range. Measurement of the three NIST peptide standards showed good agreement with NIST values. We have also applied the IT-IDMS method to characterize a set of four peptides that are used for quantification of hemagglutinin in influenza vaccine preparations. Results showed excellent precision and provided accuracy for the hemagglutinin measurement that is traceable to NIST AA standards.

ACKNOWLEDGMENT A.R.W. and M.I.S. contributed equally to this work. We thank Dr. Scott Daniels from Applied Biosystems, whose guidance and advice made this work possible. We thank Dr. Jurgen G. Schmidt (Los Alamos National Laboratories) and Steven Fulkerson (Applied Biosystems) for useful discussions. References in this article to any specific commercial products, process, service, manufacturer, or company do not constitute an endorsement or a recommendation by the U.S. government or the Centers for Disease Control and Prevention. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of CDC.

Received for review February 17, 2009. Accepted March 31, 2009. AC900367Q

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