Ultra Performance Liquid Chromatography Isotope Dilution Tandem

Similar HPLC methods have been applied to the absolute quantification of ... The PepSeq program of Water's BioLynx software package was used for de no...
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Anal. Chem. 2008, 80, 2688-2693

Ultra Performance Liquid Chromatography Isotope Dilution Tandem Mass Spectrometry for the Absolute Quantification of Proteins and Peptides Leah G. Luna, Tracie. L. Williams, James L. Pirkle, and John R. Barr*

Division of Laboratory Science, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Highway, MS F-50, Atlanta, Georgia 30341

A selective, rapid, and sensitive 12.7-min ultra performance liquid chromatography-isotope dilution tandem mass spectrometry (UPLC-ID/MS/MS) method was developed and compared to conventional high-performance liquid chromatography-isotope dilution tandem mass spectrometry (HPLC-ID/MS/MS) for the absolute quantitative determination of multiple proteins from complex matrixes. The UPLC analysis was carried out on an Acquity UPLC ethylene-bridged hybrid (BEH) C18 reversedphase column (50 × 2.1 mm i.d., 1.7-µm particle size) with gradient elution at a flow rate of 300 µL/min. For the HPLC separation, a similar gradient profile on a reversed-phase C18 column with dimensions of 150 × 1.0 mm at a flow rate of 30 µL/min was utilized. The aqueous and organic mobile phases were 0.1% formic acid in water and acetonitrile, respectively. Detection was performed on a triple-quadrupole mass spectrometer operated in the multiple reaction monitoring mode. Linear calibration curves were obtained in the concentration range of 1090 fmol/µL. Relative standard deviation values equal to or less than 6.5% were obtained by the UPLC-ID/MS/ MS method, thus demonstrating performance equivalent to conventional HPLC-ID/MS/MS for isotope dilution quantification of peptides and proteins. UPLC provides additional dimensions of rapid analysis time and highsample throughput, which expands laboratory emergency response capabilities over conventional HPLC. High-performance liquid chromatography coupled to a tandem quadrupole mass spectrometer (HPLC-MS/MS) utilizing multiple reaction monitoring (MRM) and isotope dilution (ID) is one of the most widely used sensitive and selective quantification techniques for small molecules1-3 and metabolites4-6 in clinical laboratory environments. Similar HPLC methods have been * To whom correspondence should be addressed. E-mail: [email protected]. Phone: 770-488-7848. Fax: 770-488-0509. (1) Holm, S. S.; Hansen, S. H.; Faber, J.; Staun-Olsen, P. Clin. Biochem. 2004, 37, 85-93. (2) DiMarco, T.; Giulivi, C. Mass Spectrom. Rev. 2007, 26, 108-120. (3) Herna´ndez, F.; Sancho, J. V.; Pozo, O. J. Anal. Bioanal. Chem. 2005, 382, 934-946. (4) Johnson, R. C.; Lemire, S. W.; Woolfitt, A. R.; Ospina, M.; Preston, K. P.; Olson, C. T.; Barr, J. R. J. Anal. Toxicol. 2005, 29, 149-155. (5) Blair, I. A.; Tilve, A. Curr. Drug Metab. 2002, 3, 463-480. (6) Dooley, K. C. Clin. Biochem. 2003, 36, 471-481.

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applied to the absolute quantification of peptides and proteins.7-10 These quantification methods are often run on large 2.1- or even 4.6-mm-i.d. columns, which easily accommodate high flow rates to keep analysis times short. In recent years, the use of 1 mm or smaller capillary HPLC columns with low flow rates has become a common way of enhancing the sensitivity of HPLC-MS/MS methods.8-12 However, on many conventional HPLC systems, the use of small-diameter columns with low flow rates results in longer analysis and re-equilibration times.11 The main factor that impedes rapid quantitative analysis on capillary HPLC columns at higher flow rates is the inability of most HPLC systems to perform at pressures exceeding 6000 psi. The use of sub-2-µm particle sizes to achieve higher separation efficiency is not realistic because of these pressure constraints.12 To accommodate pressure limitations at higher flow rates in conventional HPLC, increased column lengths and larger particle sizes are generally utilized.13 Recent advances and commercialization of ultra performance liquid chromatography (UPLC) have permitted system pressures to reach as high as 15 000 psi thereby enabling the use of sub2-µm particle sizes and flow rates as high as 2 mL/min.14 Increased flow rate, however, produces narrow UPLC peaks requiring rapid transitional switching capabilities from the mass spectrometer’s data acquisition system to provide an adequate number of data points necessary to properly define the peak for reproducible quantification.13,15 Until recently, ultra-high-pressure liquid chromatography systems have typically been paired with fast data acquisition systems such as time-of-flight mass spectrometers.16 However, in recent years, tandem mass spectrometers that are capable of handling fast transitional switching, decreased analysis (7) Kirkpatrick, D. S.; Gerber, S. A.; Gygi, S. P. Methods 2005, 35, 265-273. (8) Mayya, V.; Rezual, K.; Wu, L.; Fong, M. B.; Han, D. K. Mol. Cell. Proteomics 2006, 5, 1146-1157. (9) Bro ¨nstrup, M. Expert Rev. Proteomics 2004, 1, 503-512. (10) Gerber, S. A.; Rush, J.; Stemman, O.; Kirschner, M. W.; Gygi, S. P. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6940-6945. (11) 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. (12) Wu, N.; Clausen, A. M. J. Sep. Sci. 2007, 30, 1167-1182. (13) Churchwell, M. I.; Twaddle, N. C.; Meeker, L. R.; Doerge, D. R. J. Chromatogr., B 2005, 825, 134-143. (14) Nova´kova´, L.; Matysova´, L.; Solich, P. Talanta 2006, 68, 908-918. (15) Li, R.; Dong, L.; Huang, J. Anal. Chim Acta 2005, 546, 167-173. (16) Loboda, A. V.; Krutchinsky, A. N.; Bromirski, M.; Ens, W.; Standing, K. G. Rapid Commun. Mass Spectrom. 2000, 14, 1047-1057. 10.1021/ac701945h CCC: $40.75

© 2008 American Chemical Society Published on Web 03/19/2008

time and increased resolution have become commercially available.17,18 Coupling these new tandem mass spectrometers with UPLC, 10-fold improvements in speed as well as increased resolution due to narrower peak widths is not uncommon.13,14 Additionally, the compressed peaks tend to have higher intensities since the material is eluting in a more concentrated band, thus compensating for a reduced dwell time for the mass spectrometer to record each data point. To date, UPLC-ID/MS/MS has been applied to quantify small molecules,17 pesticides,19-21 and a variety of pharmaceuticals.14,15,18,22-27 We now demonstrate the application of UPLCID/MS/MS to the simultaneous quantification of target proteins from vaccines. This UPLC method was developed for measurement of hemagglutinin of several different influenza subtypes, most notably, those that are currently included in annual seasonal vaccines. Since the vaccine manufacturing process is currently dependent upon a quantitative method that requires antibody production,28,29 a process that is extremely time-consuming,30 alternate paths are sought to shorten vaccine production time. The UPLC method could be applied to numerous vaccines so that the target proteins can be rapidly, accurately, and precisely monitored throughout multiple stages in the production cycle.31 EXPERIMENTAL SECTION Target Selection. The viral protein mixture consisted of a split trivalent vaccine that contained proteins from influenza subtypes H1N1, H3N2, and HB (i.e., A/New Caledonia/20/99 (H1N1)-like virus, A/California/7/2003 (H3N2)-like virus, and B/Shanghai/ 361/2002-like virus) to identify conserved target peptides for each hemagglutinin subtype. Tryptic digestion of the viral protein mixture was conducted by adding 90 µL of a 0.1% solution of Rapigest (Waters, Bedford, MA) to 10 µL of the viral protein solution. Total protein concentration was not measured, but it was estimated that 2-10 µg of hemagglutinin was digested in each sample. For discovery of potential peptide targets, the proteins were incubated overnight at 37 °C with 1 µmol of modified trypsin (Promega, Madison, WI) for complete protein digestion. The protein digest (5 µL) was injected onto a Symmetry300 (Waters) (17) Barcelo-Barrachina, E.; Moyano, E.; Galceran, M. T.; Lliberia, J. L.; Bago, B.; Cortes, M. A. J. Chromatogr., A 2006, 1125, 195-203. (18) New, L.-S.; Saha, S.; Ong, M. K.; Boelsterli, U. A.; Chan, E. Rapid Commun. Mass Spectrom. 2007, 21, 982-988. (19) Mezcua, M.; Aguera, A.; Lliberia, J. L.; Cortes, M. A.; Bago, B.; FernandezAlba, A. R. J. Chromatogr., A 2006, 1109, 222-227. (20) Wang, X.; Zhao, T.; Gao, X.; Dan, M.; Zhou, M.; Jia, W. Anal. Chim. Acta 2007, 594, 265-273. (21) Zhang, Y.; Jiao, J.; Cai, Z.; Zhang, Y.; Ren, Y. J. Chromatogr., A 2007, 1142, 194-198. (22) Al-Dirbashi, O.; Aboul-Enein, H.; Jacob, M.; Al-Qahtani, K.; Rashed, M. Anal. Bioanal. Chem. 2006, 385, 1439-1443. (23) Leandro, C. C.; Hancock, P.; Fussell, R. J.; Keely, B. J. J. Chromatogr., A 2007, 1144, 161-169. (24) Ma, Y.; Qin, F.; Sun, X.; Lu, X.; Li, F. J. Pharm. Biomed. Anal. 2007, 43, 1540-1545. (25) Mensch, J.; Noppe, M.; Adriaensen, J.; Melis, A.; Mackie, C.; Augustijns, P.; Brewster, M. E. J. Chromatogr., B 2007, 847, 182-187. (26) Salque`bre, G.; Bresson, M.; Villain, M.; Cirimele, V.; Kintz, P. J. Anal. Toxicol. 2007, 31, 114-118. (27) Yu, K.; Di, L.; Kerns, E.; Li, S. Q.; Alden, P.; Plumb, R. S. Rapid Commun. Mass Spectrom. 2007, 21, 893-902. (28) Williams, M. S. Vet. Microbiol. 1993, 37, 253-262. (29) Adams, T.; Osborn, S.; Rijpkema, S. Vaccine 2005, 23. (30) Cox, N. J.; Tamblyn, S. E.; Tam, T. Vaccine 2003, 21, 1801-1803. (31) Kaslow, D. C. Hum. Vaccines 2007, 3, 1-7.

C18 column with dimensions of 150 mm × 0.320 mm i.d. Mobile phase A was 0.1% formic acid (Fluka Biochemica, Buchs, Switzerland) in water (Sigma Aldrich, St. Louis, MO) while mobile phase B was 0.1% formic acid in acetonitrile (ACN; Burdick & Jackson, Muskegon, MI). Chromatography was completed with a Waters NanoAcquity at a flow rate of 3 µL/min and a gradient beginning at 5% B and continuing to 50% B in 60 min. Full-scan MS/MS experiments were performed on a Waters QTOF Premier. The PepSeq program of Water’s BioLynx software package was used for de novo analysis of the sequence (MS/MS) data. MSPattern of Protein Prospector32 used the sequence tag determined from PepSeq to search the nonredundant database of the National Center for Biotechnology Information (NCBI) for protein identification. Preparation of Stocks, Working Stocks, and Calibration Solutions. Based on the three conserved native peptide sequences, chosen as targets of the hemagglutinin species of HA1, HA3, and HB subtypes, both unlabeled and isotopically labeled peptides were synthesized (Sigma Genosys, The Woodlands, TX). Isotopically labeled peptides contained one amino acid residue labeled with 13C and 15N to provide mass differences of +7 or +10 Da (Figure 1). All synthetic peptides were delivered lyophilized (each in a 1-nmol quantity). Upon reconstitution, each peptide was diluted in 20 µL of 10% formic acid and an additional 180 µL of 0.1% formic acid to provide 5 pmol/µL stock solutions (per manufacturer instructions). Working stock solutions of each peptide were prepared at a concentration of 0.5 pmol/µL by taking 40 µL of the stock solution and diluting into 360 µL of 0.1% formic acid. Each of the three unlabeled peptide working stock solutions were used to prepare five calibration standards containing 10, 30, 50, 70, and 90 fmol/µL of each peptide. Each calibration standard solution was spiked with 50 fmol/µL of each of the three labeled peptides. Preparation of Sample Digests for Protein Quantification. Ten microliters of a 2006-2007 commercially available trivalent split vaccine was removed from a single-dose vial, which contained 550 ( 10 µL. Ten microliters of Rapigest (Waters Corp., Bedford, MA) was added to the sample aliquot. The mixture was vortexed, briefly centrifuged, heated at 100 °C for 5 min, and subsequently allowed to cool to room temperature. A 10-µL aliquot of sequencing grade modified trypsin (Promega, Madison, WI) at 17.2 pmol/µL was added to the cooled mixture, vortexed, briefly centrifuged, and incubated at 37 °C for 1 h. To the resulting solution, 10 µL of each labeled peptide and 40 µL of HPLC grade water was added to the digest for a total digest volume of 100 µL, which was a 10-fold dilution from the original sample. UPLC Conditions. The analytical column utilized was a UPLC BEH 50 × 2.1-mm-i.d. reversed-phase C18 (1.7-µm particle size, Waters Corp.). The aqueous mobile phase (A) consisted of HPLC grade water with 0.1% formic acid, while the organic phase (B) was ACN with 0.1% formic acid. The sample manager was set to a temperature of 4 °C and programmed to deliver a 2-µL full loop injection, and a 5.6-time loop overfill was utilized for injections. The needle draw rate was set to 10 µL/min. Both pre- and postinjection, the injection needle was washed with 200 µL of mobile phase B followed by 600 µL of weak wash solution of 98% (32) Clauser, K. R.; Baker, P.; Burlingame, A. L. Anal. Chem. 1999, 71, 28712882.

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Figure 1. HA1 native and its corresponding isotopically labeled peptide analogue (a) The top spectrum shows the native parent m/z of 634.8(+2), and the top right spectrum shows the labeled (+10 Da) parent m/z of 639.8(+2). (b) The overlaid spectra of the labeled and native peptides. The phenylalanine (F) amino acid residue was isotopically labeled to provide a 10-Da shift in mass in the y3-y10 product ions from that of the native product ions.

HPLC grade water, 2% ACN, and 0.1% formic acid. A gradient profile was utilized at a flow rate of 300 µL/min. Initially, the mobile phase consisted of 98% A and 2% B and held for 2 min. An 8.3% change per minute was utilized over the next 3 min where the mobile phases were 73% A and 27% B, respectively. At 5.10 min, the gradient was stepped to 2% A and 98% B for 3.2 min to clean the column and then stepped to 98% A and 2% B for the next 4.5 min to re-equilibrate the column to initial conditions. The total run time was 12.7 min. HPLC Conditions. The binary solvent and sample managers on the NanoAcquity system (Waters Corp.) were configured for capillary flow rates by using 0.001-in. Peeksil tubing from the injector on the sample manager to the head of the analytical column. The analytical column was a 150 mm × 1 mm i.d. Symmetry300 reversed-phase C18 (3.5-µm particle size, Waters Corp.). The aqueous mobile phase (A) consisted of HPLC-grade water with 0.1% formic acid, while the organic phase (B) was ACN with 0.1% formic acid. A 2-µL full loop injection with 3-time loop overfill was utilized for injections. The needle draw rate was set to 5 µL/min. Both pre- and postinjection, the injection needle was washed with 200 µL of mobile phase B, followed by 600 µL of weak wash solution of 95% HPLC grade water, 5% ACN, and 0.1% formic acid. A gradient profile was employed at a flow rate of 30 µL/min. Initially, the mobile phase consisted of 95% A and 5% B. After 10 min, the gradient was stepped to 12% B. A 1.1% change per minute was utilized over the next 14 min, where the mobile phases were 73% A and 27% B, respectively. After 24 min, total the gradient was stepped to 5% A and 95% B for 20 min to clean 2690 Analytical Chemistry, Vol. 80, No. 8, April 15, 2008

the column and then stepped to 95% A and 5% B for the next 30 min to equilibrate column to initial conditions. The total run time was 74 min. Mass Spectrometry Quantification. The UPLC column eluent was introduced into a 4000-Q TRAP (Applied Biosystems, Foster City, CA) triple-quadrupole tandem mass spectrometer with a turbo ion spray interface. The instrument was operated in the positive ion mode with segmented MRM monoisotopic mass-tocharge (m/z) quantifying transition pairs of the following: 622.8/ 857.5, 634.8/1011.4, and 623.3/704.4 for HA3, HA1, and HB native peptides and 626.3/864.5, 639.4/1021.4, and 626.8/711.4 for the corresponding labeled peptides, respectively. Two additional transition pairs utilizing the same conditions were monitored for peptide confirmation for each of the three peptides. A complete list of the transitions along with the sequences of the target peptides derived from HA1, HA3, and HB is provided in Table 1 along with the optimized declustering potential and collision energies for each peptide transition pair. Instrument parameters were as follows: curtain gas 25, collision gas high, ion spray voltage 5500 V, source temperature 400 °C, source gases 40, cell entrance 5, collision cell exit potential of 13, dwell time of 110 ms per transition. Instrument control and data processing were performed with the Applied Biosystems Analyst software version 2.1.4. The automated baseline-to-baseline peak integration algorithm within the Analyst software was utilized to define peak areas for quantification. The HPLC column eluent was introduced into a TSQ Quantum triple-quadrupole tandem mass spectrometer with an electrospray

Table 1. Native and Labeled Peptide Sequences for the HA3, HA1, and HB Proteinsa

a Underlined amino acid indicates the isotopic label and the numbers in parentheses indicates the mass difference between the native and labeled peptide. Quantification, labeled, and confirmation multiple reaction monitoring ion transitions are shown as well as the declustering potential and collision energy optimization for each peptide.

interface (Thermo Scientific, Waltham, MA). The instrument was operated in the positive ion mode with MRM m/z quantifying transitions as stated above. Instrument parameters were as follows: spray voltage 4000 V, sheath gas 4, auxiliary gas 2, capillary tube temperature 300 °C, and a collision gas of 1.5 mTorr. Collision energies and tube lens were optimized for each peptide. Data processing and instrument control were performed with the Thermo Scientific Xcalibur software. RESULTS AND DISCUSSION Target and Transition Selection. Target peptides from each HA3, HA1, and HB subtypes were identified via data-dependent LC-MS/MS analysis. The target peptides are shown in Table 1 and were chosen because they are conserved throughout the subtype, have ion signals concentrated in one charge state rather than distributed across many, and contain no methionine or tryptophan, which can oxidize either in vivo or during sample processing. Since there are other peptides that could potentially have the same mass-to-charge ratio as the target peptide, a second stage of mass spectrometry (MS/MS) was utilized to add selectivity to the analysis. Figure 1a shows the precursor m/z isotopic distribution for the HA1 target peptide and its labeled analogue as acquired on the QTOF Premier. The spectrum of the precursor peptides showed that the (M + 2H)2+ ion was dominant for both the native and the labeled peptides. When the native and labeled precursors were subjected to MS/MS fragmentation, and deconvolution of the multiply charged ions was performed, the fragmentation pattern (Figure 1b) confirmed the amino acid

sequence of the peptide. Because the phenylalanine (F) was isotopically labeled, the y-ion series reflected a 10-Da shift in mass for each product ion from y3 through y10 compared to its native counterpart. The three most intense ions were chosen for MRM and were composed of the monoisotopic precursor ion and the three most intense monoisotopic product ions (Table 1). No other peptides in the digest demonstrated these selected MRM transitions. UPLC-ID/MS/MS Run Optimization. A 12.7-min gradient was utilized for the analysis, which included a 2-min hold at initial conditions to equilibrate peptides on column after injection by flowing ∼3.8 column volumes of solvent through the system before releasing the gradient for peptide elution. The 3-min gradient elution to 27% B eluted all peptides in at least 5.7 column volumes of solvent, while at least 6.1 and 8.5 column volumes were utilized respectively for column washing and re-equilibration to initial conditions (Figure 2). Results demonstrate that sufficient column washing was performed as no carryover was observed from run to run or between replicate runs of digests. The initial column hold and peptide elution profile was more than sufficient for peptide separation and generated highly reproducible peaks with mean retention times of 3.95 ((0.010), 4.49 ((0.004), and 5.03 ((0.003) min for HA3, HA1, and HB peptides, respectively. Figure 2 illustrates the decrease in overall run time from 74 min using HPLC to 12.7 min with UPLC while maintaining baseline separation between peptides. Differences in peak intensity for each of the peptides were expected as the analysis was Analytical Chemistry, Vol. 80, No. 8, April 15, 2008

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Figure 2. Total ion chromatogram relative comparison for HA3, HA1, and HB, respectively, by the 12.7-min UPLC method (a) and the 74-min HPLC conventional method (b). Table 2. Calculated Concentrations for HA3, HA1, and HB Viral Hemagglutinin Proteins from a Commercial Seasonal Vaccine

performed on different mass spectrometers with different ionization sources and collision cell designs. However, the area ratio of the native to labeled peptides remained unchanged between the 2692

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two different methods, and results from the quantitative analysis of the trivalent vaccine were comparable. The chromatograms further demonstrate that, with optimal conditions of 30 µL/min

flow rate on a 1.0 × 150 mm, 3.5-µm particle size C18 column, maximum sensitivity and selectivity is attained by HPLC at the cost of method speed. There is no significant tradeoff between sensitivity and speed with a UPLC method. Higher flow rate (10 times higher flow rate in this case) and increased column diameter (2.1 vs 1.0 mm) is compensated for by smaller particle size (1.7 vs 3.5 µm). In addition, UPLC concentrates the same amount of peptides into very narrow peaks with higher signal-to-noise ratios than conventional HPLC. Peak asymmetry factor values were evaluated. A value of 1.0 indicates a perfectly symmetric peak. Results show asymmetry factor values ranged from 0.8 to 1.4 for all native and labeled peptides, demonstrating insignificant peak fronting and negligible tailing. Peak width values measured at half peak height ranged from 2.98 to 3.10 s for unlabeled peptides and 3.02 to 3.09 s for the labeled peptides. Peak widths were ∼25% of those found using conventional HPLC. Peak height intensity was assessed for resolution. Results show that the highest standard concentration (90 fmol/µL) produced average peak height intensity counts of 1.2 × 104 ((450), 1.6 × 104 ((714), and 1.2 × 104 ((436), while the lowest standard concentration (10 fmol/µL) resulted in average peak height counts of 1.3 × 103((32), 1.8 × 103 ((76), and 1.1 × 103 ((40) for HA3, HA1, and HB respectively. Since a smaller particle size was utilized during this separation, resolution is maintained, if not increased over conventional HPLC due to the increased number of theoretical plates even at high flow rates. Each respective set of quantification, ISTD, and confirmation ion transition pairs were further divided into periods to provide a total period scan time including pauses of 0.58 s. Average baseto-base peak widths of 13.26 ((1.89), 17.03 ((0.57), 14.02 ((2.02), 17.26 ((1.51), 17.49 ((0.73), and 17.96 ((1.10) s were obtained for HA3, HA1, and HB unlabeled and labeled peptide standards respectively. Since the dwell time for each ion was 110 ms and the total cycle time per period was 0.58 s, a minimum of 23 data points across these narrow peaks was obtained. UPLC-ID/MS/MS Analysis of Calibration Standards. Four replicate injections were performed for each calibration standard solution containing all three native peptides and their labeled counterparts. The concentration of the native peptides ranged from 10 to 90 fmol/µL with corresponding labeled peptides spiked at 50 fmol/µL in each standard solution. The mean area ratios (unlabeled/labeled) were plotted against expected concentrations for each standard. Linear regression without weighting was applied to the data sets and expected concentration versus area ratio calibration curves were generated for each peptide. Regression analysis resulted in equations of y ) 58400x + 1.290, y ) 34.800x + 0.2220, and y ) 31.200x + 0.4250 for HA3, HA1, and HB, respectively. At the 95% confidence level standard error

in the slope resulted in (1.63, (1.76, and (1.01 and corresponding R2 values of 0.9943, 0.9932, and 0.9966 for HA3, HA1, and HB, respectively. Protein Quantification Results. Four 1-h digests were each injected in quadruplicate for a total of 16 replicate runs for HA3, HA1, and HB, respectively. The area ratio of the native to the spiked labeled internal standard was computed and applied to the peptide concentration versus area ratio calibration curve to obtain the concentrations for each protein in femtomoles per microliter (Table 2). Results obtained for HA3, HA1, and HB demonstrated mean protein concentration values of 32.43 ((2.10), 36.51 ((1.32), and 70.87 ((3.69) fmol/µL, with corresponding relative standard deviation values of 6.47, 3.60, and 5.21%, respectively. To ensure that the samples had completely digested after 1 h, four 3-h digests were each injected in quadruplicate. The 3-h digest results demonstrated that the mean protein concentration for all three proteins fell within the standard deviations obtained for the 1-h digests (data not shown). A set of standards were injected along with the digested peptides and demonstrated no significant runto-run change in the calibration for all three proteins. CONCLUSIONS We have demonstrated and applied a UPLC-ID/MS/MS for the simultaneous quantification of multiple peptides and proteins. Compared to conventional HPLC methodology, it would take ∼74 min to achieve the same initial equilibration, elution, washing, and re-equilibration as obtained by the 12.7-min UPLC method. For rapid analytical emergency response capabilities, conventional HPLC imparts at most 19 runs in a 24-h period, whereas the UPLC method provides the capability of 4-5 runs/h and 113 runs/day. Thus, in the scope of emergency response situations, this allows a single laboratory the capacity to rapidly analyze and quantify 791 samples/week with the use of only one instrument. In addition, the current UPLC gradient provides more than sufficient separation between analytes and can be adapted to handle additional peptides for protein quantification. ACKNOWLEDGMENT The authors thank Dr. David Schieltz and Dr. Thomas Blake at the CDC for their advice. Reference in this article to any specific commercial products, process, service, manufacturer, or company does not constitute its endorsement or recommendation by the U.S. Government or Centers for Disease Control and Prevention (CDC). The findings and conclusions in this report are those of the author(s) and do not necessarily represent the views of the CDC. Received for review September 17, 2007. Accepted January 24, 2008. AC701945H

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