Multiple Reaction Monitoring Cubed for Protein Quantification at the

Anal. Chem. 2009, 81, 9343–9352

Multiple Reaction Monitoring Cubed for Protein Quantification at the Low Nanogram/Milliliter Level in Nondepleted Human Serum T. Fortin,†,‡ A. Salvador,‡ J. P. Charrier,† C. Lenz,§ F. Bettsworth,† X. Lacoux,† G. Choquet-Kastylevsky,† and J. Lemoine*,‡ R&D Proteomique, bioMe´rieux SA, Marcy l’Etoile, France, UMR 5180 Sciences Analytiques, Universite´ de Lyon, Lyon1, France, and PSM Support, Applied Biosystems, Darmstadt, Germany Mass spectrometry-based strategies for the quantification of low-abundance putative protein biomarkers in human blood currently require extensive sample fractionation steps which hamper their implementation in a routine and robust way across clinical laboratories. We demonstrate that a technique using MS3 reconstructed chromatograms on a signature of secondary ions issued from a trapped primary product ion, termed multiple reaction monitoring cubed (MRM3), enables targeting protein biomarkers in the low nanogram/milliliter range in nondepleted human serum. The simple two-step workflow is based on a trypsin proteolysis of whole serum (100 µL) followed by enrichment of targeted proteotypic peptides on a solid phase extraction column using mixed-cation exchange resin. MRM3’s fidelity of peak detection extends the dynamic range and limit of quantitation (LOQ) of protein biomarkers to the low nanogram/milliliter range, corresponding to a concentration that is 106-fold lower than the concentration of the most abundant proteins in serum. The power of the MRM3 method is illustrated by the assay of prostate specific antigen in nondepleted human sera of patients. The results correlate well with the established method for determining PSA levels in serum, i.e., enzymelinked immunosorbent assay (ELISA) tests. In comparison to the huge efforts engaged for years to discover new protein biomarkers by proteomic strategies, the very few recently approved new biomarkers might appear dramatically disappointing. As recently suggested, this state arises in part from the high false-discovery rate of proteomic strategies, not only due to individual variability across the human samples but also simply to analytical bias. The second and probably major reason lies in the lack until recently of a consensual method for verification across large cohorts that is able to replace the gold standard immuno enzymatic enzyme-linked immunosorbent assay (ELISA) * To whom correspondence should be addressed. Je´roˆme Lemoine, UMR 5180, Baˆt. CPE, Universite´ de Lyon, Lyon 1, 69622 Villeurbanne cedex. E-mail: [email protected]. † bioMe´rieux SA. ‡ UMR 5180 Sciences Analytiques, Universite´ de Lyon. § Applied Biosystems. 10.1021/ac901447h CCC: $40.75  2009 American Chemical Society Published on Web 10/19/2009

tests in cases where specific antibodies are not available.1,2 Multiple reaction monitoring mass spectrometry (MRM-MS) coupled to stable isotope dilution (SID) carried out in triple quadrupole instruments has emerged as a possible core technology for targeted validation of individual, or sets of, biomarker proteins.3 Some essential pilot studies have been designed and thoroughly validated regarding the way to introduce a stable isotope-labeled internal standard4-6 as well as concerning the critical choice of the best responding proteotypic peptides7,8 according to validated SRM databases or predictive algorithms of electrospray ionization yield. Nonetheless, SID-MRM-based assays of protein biomarkers in a clinical environment according to the exacting Food and Drug Administration (FDA) quality standards still remain challenging. Though single or multiplexed protein assays in patient blood samples were demonstrated a few years ago for high to medium abundant proteins,9,10 the targeted clinically relevant limits of quantitation in the low nanogram/ milliliter range have only recently been achieved.11 This level of sensitivity was only accessible through cumbersome sample fractionation, combining immuno-depletion of albumin, mixedcation exchange solid phase extraction, conventional microbore reverse phase chromatography, and mass spectrometry detection. Alternative workflows combine immuno-depletion of the most abundant plasma proteins (with the effective risk of losing proteins of interest11), multidimensional liquid separations, and ultimately a mass spectrometry coupling with microbore, nanoflow chromato(1) Anderson, N. L.; Anderson, N. G.; Pearson, T. W.; Borchers, C. H.; Paulovich, A. G.; Patterson, S. D.; Gillette, M.; Aebersold, R.; Carr, S. A. Mol. Cell. Proteomics 2009, 8, 883–886. (2) Pan, S.; Aebersold, R.; Chen, R.; Rush, J.; Goodlett, D. R.; McIntosh, M. W.; Zhang, J.; Brentnall, T. A. J. Proteome Res. 2009, 8, 787–797. (3) 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. (4) Gerber, S. A.; Rush, J.; Stemman, O.; Kirschner, M. W.; Gygi, S. P. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6940–6945. (5) Brun, V.; Dupuis, A.; Adrait, A.; Marcellin, M.; Thomas, D.; Court, M.; Vandenesch, F.; Garin, J. Mol. Cell. Proteomics 2007, 6, 2139–2149. (6) Pratt, J. M.; Simpson, D. M.; Doherty, M. K.; Rivers, J.; Gaskell, S. J.; Beynon, R. J. Nat. Protoc. 2006, 1, 1029–1043. (7) Deutsch, E. W.; Lam, H.; Aebersold, R. EMBO Rep. 2008, 9, 429–434. (8) Fusaro, V. A.; Mani, D. R.; Mesirov, J. P.; Carr, S. A. Nat. Biotechnol. 2009, 27, 190–198. (9) Anderson, L.; Hunter, C. L. Mol. Cell. Proteomics 2006, 5, 573–588. (10) Lin, S.; Shaler, T. A.; Becker, C. H. Anal. Chem. 2006, 78, 5762–5767. (11) Fortin, T.; Salvador, A.; Charrier, J. P.; Lenz, C.; Lacoux, X.; Morla, A.; Choquet-Kastylevsky, G.; Lemoine, J. Mol. Cell. Proteomics 2009, 8, 1006– 1015.

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graphy.12-14 Other groups have proposed and clinically validated biomarker assays through the integration of antibody enrichment of the targeted protein15 or peptides in the so-called stable isotope standards capture by antipeptide antibodies (SISCAPA).16-18 Verification of protein biomarkers in a clinical context across hundreds of patients, however, requires a high degree of robustness and the potential for medium to high throughput (tens to one hundred samples per week). As a consequence, workflows with limited and straightforward automated sample handling should ideally be favored. We describe here a new method termed multiple reaction monitoring cubed (MRM3) aimed at the quantitation of minor proteins in complex biological matrixes like plasma or serum. Developed on the latest generation of hybrid triple quadrupole/linear ion trap mass spectrometers, MRM3 relies on the extraction of a specific pattern of second generation MS3 fragment ions issued from a trapped primary ion in order to reconstruct an ion chromatogram for subsequent quantification. EXPERIMENTAL SECTION Reagents and Chemicals. Acetonitrile and water (LC-MS grade) were obtained from Fisher Scientific (Strasbourg, France). Dithiothreitol (DTT), iodoacetamide (IAA), urea and TRIS were purchased from Sigma-Aldrich (St. Quentin-Fallavier, France). Healthy female serum was obtained from the Etablissement Franc¸ais du Sang (EFS) and patient sera diagnosed with prostate cancer were from The Hospital of Lyon-Sud. Protein Sources. Human prostate specific antigen (PSA) ultrapure was from Scipac (Sittingbourne, U.K.). TP171, TP435, and TP574 are recombinant proteins derived from Treponema pallidum (Nichols strain) DNA, cloned using pMR78 vector, and expressed in Escherichia coli BL21 or BL21 RIL strains. Core NS4 is a recombinant protein from a fusion of partial sequences of core and NS4 hepatitis C virus genes. The core NS4 gene was cloned using pMR78 vector and expressed in Escherichia coli BL21 strain. PSA ELISA Assay. PSA quantitation using ELISA was performed on VIDAS, an automated analyzer, using the VIDAS TPSA kit (bioMe´rieux, Marcy l’Etoile, France). The VIDAS TPSA assay is an equimolar test, which detects PSA bound to R-1antichymotrypsin (PSA-ACT) and free PSA (fPSA) in the same manner. PSA bound to R-2-macroglobulin (PSA-R2M) is not accessible to anti-PSA antibodies and cannot be detected using classical PSA ELISA tests such as VIDAS TPSA. VIDAS TPSA was calibrated according to the First International Standard.19 Heavy Peptide Synthesis and Qualification. Heavy isotopelabeled peptide standard LSEPAELTDAVK was synthesized in(12) Wu, S. L.; Amato, H.; Biringer, R.; Choudhary, G.; Shieh, P.; Hancock, W. S. J. Proteome Res. 2002, 1, 459–465. (13) Keshishian, H.; Addona, T.; Burgess, M.; Kuhn, E.; Carr, S. A. Mol. Cell. Proteomics 2007, 6, 2212–2229. (14) Kuhn, E.; Wu, J.; Karl, J.; Liao, H.; Zolg, W.; Guild, B. Proteomics 2004, 4, 1175–1186. (15) Kulasingam, V.; Smith, C. R.; Batruch, I.; Buckler, A.; Jeffery, D. A.; Diamandis, E. P. J. Proteome Res. 2008, 7, 640–647. (16) Anderson, N. L.; Anderson, N. G.; Haines, L. R.; Hardie, D. B.; Olafson, R. W.; Pearson, T. W. J. Proteome Res. 2004, 3, 235–244. (17) Kuhn, E.; Addona, T.; Keshishian, H.; Burgess, M.; Mani, D. R.; Lee, R. T.; Sabatine, M. S.; Gerszten, R. E.; Carr, S. A. Clin. Chem. 2009, 55, 1108– 1117. (18) Hoofnagle, A. N.; Becker, J. O.; Wener, M. H.; Heinecke, J. W. Clin. Chem. 2008, 54, 1796–1804. (19) Rafferty, B.; Rigsby, P.; Rose, M.; Stamey, T.; Gaines Das, R. Clin. Chem. 2000, 46, 1310–1317.

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house at bioMe´rieux (Lyon, France) using Fmoc chemistry on an ABI433A peptide synthesizer (Applied Biosystems, Foster City, CA). Two L-(13C3)alanine-N-FMOC building blocks (Euriso-Top, Saint-Aubin, France) were incorporated into the heavy peptide. Peptide purity was established using HPLC and mass spectrometry (Ion trap LCQ, ThermoFisher Scientific, Waltham, MA) to be better than 95%. Peptide concentrations were measured in 1 mg vials using an amino acid analyzer series 1100 with fluorescence detector (Agilent Technologies, Massy, France). Enzymatic Digestion. Female serum samples and standard proteins (PSA, TP171, TP435, Tp574, and core NS4) were denatured in 6 M urea, 10 mM Tris pH 8.0, 30 mM dithiothreitol at 40 °C for 40 min and then alkylated with 50 mM iodoacetamide at room temperature in the dark for 40 min. To reduce urea concentration, the samples were diluted 6-fold with water prior to overnight digestion at 37 °C with trypsin (sequencing grade modified, Promega) using a 1:30 (w/w) enzyme to substrate ratio. Tryptic digestion was stopped with formic acid to a final concentration of 0.5%, and the samples were desalted using Oasis HLB 3 cm3 (60 mg) reversed phase cartridges (Waters, Milford, MA). The Oasis cartridges were conditioned with 1 mL of methanol, then with 1 mL of water containing 0.1% formic acid prior to loading of the tryptic digest. Cartridges were washed with 1 mL of 0.1% formic acid and eluted with methanol/water (80:20, v/v) containing 0.1% formic acid. The samples were finally dried by vacuum centrifugation and resuspended in 200 µL of water/acetonitrile (95/5, v/v) containing 0.5% of formic acid. Infusion of all standards protein trypsin digests was used to optimize MRM transitions and MRM3 parameters. Liquid Chromatography-Mass Spectrometry. LC-MS analyses were performed on a system consisting of an Ultimate 3000 series HPLC instrument comprising a binary pump and autosampler (Dionex, Sunnyvale, CA), coupled to a QTRAP 5500 LC/MS/MS system hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems/MDS Analytical Technologies, Foster City, CA) equipped with a Turbo V ion source. Instrument control, data acquisition, and processing were performed using the associated Analyst 1.5 software. The LC separation was carried out on a Symmetry C18 column (100 mm × 2.1 mm, particle size 3.5 µm) from Waters (Milford, MA). Elution was performed at a flow rate of 300 µL/min with water containing 0.1% (v/v) formic acid as eluent A and acetonitrile containing 0.1% (v/v) acid formic as eluent B, employing a linear gradient from 5% B to 40% B in 25 min. The injection duty cycle was 45 min taking into account the column equilibration time. The mass spectrometer was initially tuned and calibrated using polypropylene glycol, reserpine, and Agilent Tuning Mix (all Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Q1 resolution was adjusted to 0.7 ± 0.1 amu fwhm for both MRM and MRM3 mode, referred to as unit resolution. Q3 was also set to unit resolution in MRM mode. In MRM3 mode, ion trap fill time and excitation time was set to 200 and 25 ms, respectively, resulting in a total scan time of 370 ms. In all MRM3 experiments, the scan rate was set to 10 000 Da/s and a Q0 trapping was used. MS analysis was carried out in positive ionization mode using an ion spray voltage of 5500 V. The nebulizer and the curtain gas flows were set at 50 psi using

nitrogen. The Turbo V ion source was operated at 500 °C with the auxiliary gas flow (nitrogen) set at 40 psi. An MRM-Initiated Detection and Sequencing (MIDAS Workflow) experiment was used to scout for suitable MRM transitions using purified tryptic digests of recombinant proteins and PSA. Q1 and Q3 masses as well as collision energy values were predicted from the PSA primary sequence using MRM Pilot 1.0 software (Applied Biosystems, Foster City, CA). Preparation of Standard and Quality Control Samples. A total of 80 µg of each protein TP171, TP574, TP435, core NS4, and PSA were digested according to the procedure above. Digested proteins were pooled to give a stock solution at a concentration of 20 µg/mL. Serum standards and quality control samples were prepared by diluting the 20 µg/mL stock solution into digested blank female serum to reach the desired concentrations of 10, 20, 50, 100, 500, and 1000 ng/mL per protein, respectively. To build the calibration curve for quantification of PSA in patient sera, PSA solution at 1.14 mg/mL was diluted into human female serum to give a 200 µg/mL stock solution and the concentration was verified using the VIDAS TPSA ELISA kit. The human female serum sample used was also previously tested by the VIDAS TPSA ELISA kit to make sure that levels of endogenous PSA were below the detection limits. Serum standards and quality control samples were prepared by diluting the 200 µg/ mL stock solution with an ELISA tested blank female serum before to reach the desired concentrations of 1, 5, 10, 50, 100, 500, and 1000 ng/mL of PSA. Digested serum spiked with PSA and patient serum were spiked with 50 µL of an internal standard ((13C6) LSEPAELTDAVK) solution at a concentration of 3.1 fmol/µL and desalted using the procedure outlined above. Digested serum was reconstituted in 1 mL of acetate buffer (200 mM, pH 3.0) and fractionated on a MCX cartridge (60 mg; Waters, Milford, MA). The cartridge was first conditioned with 1 mL of methanol and 1 mL of acetate buffer, pH 3.0. The digested serum in acetate buffer was loaded on the MCX cartridge and then was washed successively with 1 mL of acetate buffer, pH 3.0, and 1 mL of methanol. The peptide fraction containing the peptide of interest, LSEPAELTDAVK from PSA, was eluted with 1 mL of solvent mixture methanol/acetate buffer, pH 5.5 (50:50, v/v). The eluate was then dried using vacuum centrifugation and resuspended in 200 µL of a mixture of acetonitrile/ water (5:95, v/v) containing 0.5% of formic acid. RESULTS AND DISCUSSION We recently documented for the first time the clinical assay by mass spectrometry of a protein biomarker in the low nanogram/milliliter concentration of serum without immuno-enrichment of the targeted compound.11 The protocol combined albumin immuno-depletion, trypsin digestion, and solid phase extraction of the proteotypic peptide. In most of the similar studies published so far, immuno-depletion of the most abundant plasma or serum proteins was systematically adopted to limit the biological interferences across the targeted MRM channels. The aim of the present work was to use a signature of multiple second/generation product ions produced by two-stage collision induced decay (CID) fragmentation, or CID-MS/MS/MS, to increase the specificity of mass spectrometric detection to a degree where the less easily automatable and time-consuming immuno-depletion is no longer needed to remove MRM interferences.

As illustrated by Figure 1, MRM3 has been developed on a state-of-the-art hybrid triple quadrupole linear ion trap instrument. Since the introduction of hybrid triple quadrupole linear ion trap instruments in 2002,20 significant improvements have been made in the ability to perform fragmentation in the lowpressure linear ion trap21 implemented in these instruments. These improvements include an increase in the rf drive frequency for increased fragment trapping efficiency,22 the introduction of auxiliary axial electrodes to manipulate ions inside the linear ion trap for increased sensitivity,23,24 and the use of a pulsed gas valve to locally increase the pressure in the trapping/fragmenting region for reduced excitation time.24 These improvements have been implemented in the hybrid triple quadrupole linear ion trap instrument used in this study. An MRM3 ion chromatogram is reconstructed from selected specific fragment ions produced from a primary product ion trapped in the Q3 linear ion trap and subsequently activated by resonant excitation. This primary product ion is selected among the most intense MRM transitions observed for a proteotypic peptide. To assess first the intrinsic gain in specificity of detection of the MRM3 method compared to the conventional MRM operating mode, a trypsin digest of a human female serum was spiked with trypsin hydrolyzed bacterial protein models TP171, TP574, TP435, core NS4, and the prostate specific antigen (PSA) over three decades of concentration, ranging from 0 to 1000 ng/ mL. The proteotypic peptides were experimentally selected from a list of theoretical MRM transitions generated by the MRM Pilot software. All transitions were calculated for the doubly protonated precursor ions of predicted tryptic peptides. Only peptides free of methionine and cysteine amino acid residues were kept over the m/z range 400 to 1000 Th. Finally, only the most likely observed y product ions were simultaneously evaluated in MRM mode analyzing the protein standard digests to definitely confirm the two most sensitive transitions per peptide. This study led to retaining [M + 2H]2+ ions at m/z 501.3, 793.6, 719.4, 476.3, and 636.8 for peptide sequences ELADALLEK, FVPVAVPHELK, ELYELIDSNSVR, ALESFWAK, and LSEPAELTDAVK representing proteins TP171, TP574, TP435, core NS4 and PSA, respectively. As revealed by parts A and D of Figure 2, the MRM chromatograms of the targeted transitions for the two proteotypic peptides of TP453 and core NS4 proteins models spiked at 100 ng/mL exhibit numerous interfering peaks. This redundancy across the selected m/z values within a given transition channel is one of the major drawbacks encountered during the development of MRM-based assays.25,26 One attempt proposed to enhance the reliability of detection is to follow multiple transitions for a (20) Hager, J. W.; Le Blanc, J. C. Y. Rapid Commun. Mass Spectrom. 2003, 17, 1056–1064. (21) Collings, B. A.; Stott, W. R.; Londry, F. A. J. Am. Soc. Mass Spectrom. 2003, 14, 622–634. (22) Collings, B. A. J. Am. Soc. Mass Spectrom. 2007, 18, 1459–1466. (23) Loboda, A.; Krutchinsky, A.; Loboda, O.; McNabb, J.; Spicer, V.; Ens, W.; Standing, K. Eur. J. Mass Spectrom. 2000, 6, 531–536. (24) Collings, B. A.; Romaschin, M. A. J. Am. Soc. Mass Spectrom. 2009, 9, 1714–1719. (25) Sherman, J.; McKay, M. J.; Ashman, K.; Molloy, M. P. Proteomics 2009, 9, 1120–1123. (26) Duncan, M. W.; Yergey, A. L.; Patterson, S. D. Proteomics 2009, 9, 1124– 1127.


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Figure 1. Schematic representation of MRM3 developed on a hybrid triple quadrupole linear ion trap mass spectrometer.

given proteotypic peptide. Figure 2C demonstrates that summing the most intense MRM signals may indeed result in some case in higher counts per second (cps) for the target peptide. However, the shape of the target peaks of parts C and F of Figure 2, unambiguously, shows that such a sum may in turn dramatically damage the specificity of detection. As underlined by Molloy and co-workers,25 this redundancy within the respective transition channel of both 12C target peptide and 13C standard becomes more and more critical as the concentration of the targeted analyte decreases. The nonzero intercepts of the calibration curves drawn either from unique (Figure 3A,D) or summed (Figure 3D,F,C) transitions perfectly illustrate this feature. In contrast, the MRM3 ion chromatograms (parts B and E of Figure 2) display a nearly complete removal of the predominant interferences detected in the MRM mode. These MRM3 chromatograms were reconstructed from the extraction of selected signatures of two and three intense secondary MS3 product ions, respectively. Since this additional MS3 filtering stage cannot fully preclude from interfering secondary product ions, the degree of “purity” of the MS3 spectrum was compared to one carried out on the proteotypic peptide issued from the pure protein standard digest, taking into account their conserved relative intensities. Alternatively, this comparison could be made directly on the AQUA peptide standard. In this preliminary report, only two and three MS3 major product ions were retained to build the MRM3 signature. The optimal number of fragment ions required to reduce the interferences will, 9346


however, be dependent on the peptide sequence as well as the peptide concentration. Indeed, as the concentration decreases, the greater the number of product ions selected, the higher is the likelihood of detecting contaminating interferences from nontarget peptides. To further evaluate the impact of the additional MRM3 filtering stage toward the limits of detection and quantification, calibration curves were drawn by establishing a linear regression function of the peak area of ELYELIDSNSVR peptide versus concentration of the nominal standard TP435 protein in MRM mode and in MRM3 mode (Figure 3A,B). Linearity was observed in the MRM mode between 100 and 1000 ng/mL for the transition 719.4/677.4 and between 50 and 1000 ng/mL for the transition 719.4/677.4/562 + 544.5 in MRM3 mode. A linear regression function was similarly established with the peak area of ALESFWAK tryptic peptide versus concentration of the nominal standard Core NS4 protein in SRM and MRM3 mode (Figure 3C,D). Linearity was observed in the MRM mode between 50 and 1000 ng/mL for transition 476.3/767.4 and between 10 and 1000 ng/mL for transition 476.3/767.4/603.2 + 404.3 + 532.2 in MRM3 mode. Determination (r2) was greater than 0.99, and differences between nominal and calculated concentrations were lower than 15% in all cases. Thus, the relationship between the chromatographic peak areas reconstructed from MRM3 experiments and protein concentrations showed an extended range of linearity down to the lowest concentration. In contrast, the intercept values of the MRM curves for the five protein models studied

Figure 2. Comparison of specificity of detection between conventional LC-MRM and LC-MRM3 experiments for two protein models spiked at 50 ng/mL in serum. TP 435 protein: (A) MRM extracted ion chromatogram of transition 719.4/677.4, (B) MRM3 extracted ion chromatogram of transition 719.4/677.4/562 + 544.5, (C) MRM extracted ion chromatogram of summed transitions 719.4/677.4 + 719.4/790.4 + 719.4/903.5. Core NS4 protein: (D) MRM extracted ion chromatogram of transition 476.3/767.4, (E) MRM3 extracted ion chromatogram of transition 476.3/ 767.4/603.2 + 404.3 + 532.2, (F) MRM extracted ion chromatogram of summed transitions 476.3/767.4 + 476.3/638.3 + 476.3/551.3. Analytical Chemistry, Vol. 81, No. 22, November 15, 2009

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Figure 3. MRM and MRM3 calibration curves for ELYELIDSNSVR peptide tracking TP435 protein (A, B, C) and for ALESFWAK peptide tracking core NS4 protein (D, E, F). Proteins were digested by trypsin and spiked into a trypsin digest of female serum at final concentrations of 5, 10, 50, 100, 500, and 1000 ng/mL (three replicates per concentration point).

were consistent with systematic peak contaminations by predominant interferences below 50 ng/mL. On average, moving from MRM to MRM3 resulted in a 3- to 5-fold improvement of the limits of detection and quantification for 9348


the five protein models (Table 1). Regarding the statistical analyses of the triplicate experiments recorded for each protein, moving from MRM to MRM3 results only on a slight reduction of accuracy and precision. This might be attributed to the

Table 1 concentration (ng/mL) TP171

MRM3 MRM

core NS4

MRM3 MRM

TP574

MRM3 MRM

TP435

MRM3 MRM

PSA

MRM3 MRM

PSA clinical sample (with MCX)

LOD (S/N ) 3)

LOQ (S/N ) 10)

3

10

12

40

3

10

17

>50

1008.3 1.9 0.8 1004.3 1.9 0.4

17

50

463 2.2 -7.4 448.3 3.7 -10.3

1016.7 0.6 1.7 1043.3 1.5 4.3

20

80

160

500

90.5 4.6 -9.5 91.6 7.0 -8.4

534.7 3.9 6.9 491.3 2.6 -1.7

1093.3 1.9 9.3 1003.3 0.6 0.3

3

10

45.9

93.3

479.0

1018.5

1.5

4.5 -8.1 53.2 2.4 6.3

3.3 -6.7 103.7 2.4 3.7

23.6 -0.6 479.3 2.8 -4.1

7.2 1.8 1009.7 2.0 0.9

10

50

100

500

1000

mean precisiona accuracyb mean precisiona accuracyb

10.9 31.8 14.9

46.0 12.4 -7.9 56.8 5.4 13.7

93.5 2.5 -6.4 93.9 4.4 -6.1

516.0 3.9 3.2 498 2.4 -0.4

991.7 0.8 -0.8 1002.3 0.7 0.2


11.3 16.0 13.2

47.0 6.7 -5.9 54.7 8.17 9.4

92.4 6.4 -7.6 101.1 19.3 1.1

516.7 3.2 3.3 511.3 1.3 2.3

992.7 0.8 -0.7 994 0.4 -0.6


56.3 23.1 12.5

106.4 10.7 6.4 104.7 21.8 4.7

476.3 9.2 -4.7 491 8.4 -1.8


63.3 15 26.5

106.6 14.6 6.6 105.7 1.9 5.7

45.2 8.7 -9.6 49.2 3.75 -1.7


MRM3

mean

MRM

precisiona accuracyb mean precisiona accuracyb

10.3 1.2 2.7

9.45 3.9 -5.7

a Expressed as relative standard deviation (RSD): (standard deviation/mean) × 100. concentration added)/concentration added] × 100.

relatively long duty cycle of the MRM3 compared to the MRM mode leading to fewer sampling points per chromatographic peak. With regard to both conventional MRM experiments carried out in triple quadrupole instruments and to product ion monitoring (PIM) experiments performed in ion trap instruments, MRM3 combines the benefits of conventional MRM assays with the increased selectivity of ion trap MS/MS/MS experiments. In the implementation of MS/MS/MS on hybrid triple quadrupole/linear ion trap mass spectrometer, the first fragmentation step is performed in the Q2 quadrupole collision cell of the mass spectrometer.21 In this process, efficient prefiltering of the ion current in Q1 greatly reduces the introduction of background ions into the ion trap mass analyzer, which in turn increases the loading capacity for the peptide of interest. Second, significant increases in the efficiency of the consecutive resonant excitation CID process performed in the linear ion trap have recently been realized,22 leading to increased MS/ MS/MS sensitivity.

b

>100

50

15

4.5

50

Expressed as % difference: [(concentration found -

The nature and power of MRM3 for significantly improving the specificity and sensitivity of detection of proteotypic peptides is illustrated by the clinical assay of prostate specific antigen. In order to reach the clinically relevant targeted limit of quantitation of 4 ng/mL, we applied a previously optimized proteotypic peptide enrichment based on mixed-cation exchange solid phase extraction on a cartridge format11 just after trypsin hydrolysis of the crude sera. This extraction phase could also be easily implemented online using an additional chromatographic module. Its interest is also to reduce the amount of peptide loaded onto the analytical reversed-phase column to prolong column lifetime and reduce unnecessary nonpeptidic contamination of the analytical system by, e.g., salts or lipids. At this stage, one should mention that this two-step workflow is likely the simplest among those published today. It should be emphasized that whether a multiplexed analysis of a complex peptide panel is targeted, the SPE enrichment strategy is less suitable since it is highly probable that the peptides will be distributed over different fractions. In this case, Analytical Chemistry, Vol. 81, No. 22, November 15, 2009

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the pooling of many fractions may in turn probably nearly annihilate the benefit of SPE enrichment. A potential downside of using MRM3 experiments for accurate quantification of candidate biomarkers using internal standards is the relatively long duty cycle of one MRM3 experiment (350 ms) in relationship to the chromatographic peak widths typically observed in analytical HPLC. To test the feasibility of using internal, heavy isotope-labeled peptide standards (i.e., AQUA peptides), a spiking experiment including (13C6) LSEPAELTDAVK was performed (Figure 4A). The overlaid chromatograms monitoring the light LSEPAELTDAVK peptide (MRM3 transition 636.8/943.5/627 + 698 + 779 + 797) tracking PSA and (13C6) heavy AQUA standard (MRM3 transition 639.8/ 949.5/630 + 704 + 785 + 803) show chromatographic peaks defined by 10-12 data points for both the endogenous peptide and the internal standard. This clearly demonstrates the feasibility of using internal standards. Provided that multiple peptides in an assay can be chromatographically resolved, protein assays for 15-20 candidates including internal standards to be tested concomitantly in MRM3 can be conceived where different MRM3 experiments are performed in different retention time windows. In order to evaluate the dynamic range of a PSA assay with internal standardization by (13C6) LSEPAELTDAVK peptide, aliquots of a human female serum preliminary tested for the absence of endogenous PSA by the VIDAS TPSA ELISA kit was spiked with PSA protein between 1 and 1000 ng/mL. The AQUA internal standard was added just prior to LC-MRM3 analysis to each duplicate along the calibration curve to a constant final concentration equivalent of 50 ng/mL of PSA protein. Linearity was observed between 5 and 1000 ng/mL for the MRM3 transition 636.8/943.5/(627 + 698 + 779 + 797) tracking the PSA protein with a regression factor of r 2 > 0.99. The differences between the nominal and calculated concentrations were lower than 15% (Figure 4B). In comparison, the calibration curve drawn with the summed three most intense MRM channels displays only linearity between 10 and 50 ng/mL (Figure 4C). This study led us to define the MRM3 limits of detection (LOD) and quantitation (LOQ) for peptide LSEPAELTDAVK, calculated as a magnitude of, respectively, 3 and 10 times the standard deviation of noise in a blank sample (3σ), at, respectively, 1.5 and 4.5 ng/mL. Note that the scope of the present work was not to present a reliable validated assay of PSA but more a preliminary evaluation of the ability of MRM3 to quantitatively detect highly diluted proteins in serum or plasma with good accuracy. Nonetheless, the good correlation between the results obtained by MRM3 mass spectrometry and by the established ELISA tests for three patients diagnosed with cancer (Figure 4D) provides a definitive illustration that MRM3 should be considered a promising method among the available emerging tools dedicated to protein biomarker verification. Note that all experiments were deliberately performed using a conventional bore liquid chromatography format instead of the gold standard nanoflow chromatography owing to its higher robustness and because it is the format used in the clinical laboratories for routine analysis. Thus the question arises about 9350


the intrinsic gap of sensitivity between nanospray and conventional electrospray flows. A previous study reported an average 2-fold improvement in absolute signal intensity moving from 200 to 0.1 µL/min27 but at the cost of reduced reproducibility as determined by relative standard deviation values. A 10- to 20-fold improvement is likely more conceivable owing to the more efficient desolvation process of droplets with smaller diameter when they are produced under nanoflow conditions. Nonetheless, the loading capacity of a conventional bore 2.1 mm inner diameter chromatography column is at least 2 orders of magnitude higher than that of a 75 µm internal diameter column (assuming the column length and particle size are similar). Hence, there is no doubt that the ability to inject greater amounts of peptides on conventional bore systems, e.g., equivalent to 1.5 mg of hydrolyzed proteins versus 10-50 µg for nanoflow chromatography, may fully offset the decrease in signal intensity moving from nanospray to electrospray conditions. Furthermore, the volumes of serum or plasma typically used during the clinical verification step are not critically a limiting factor in contrast to proteomic discovery phase experiments performed from biopsies or primary cell cultures, even though in this study we started from as little as 100 µL of patient serum for the PSA assay. Reflecting on further improving the sensitivity of the present strategy to achieve routine assays of any biomarker present in serum below the nanogram/milliliter concentration range, several aspects seem obvious. Clearly, increasing the amount of serum to digest beyond 1 mL is not reasonable considering that a reliable clinical evaluation based on replicated analyses would require excessive blood sampling. One alternative would be to replace the MCX fractionation step on an SPE cartridge by a much more resolutive strong cation exchange chromatography in order to increase the concentration of the targeted peptide and reduce the interfering peaks. However, the most striking potential increase of sensitivity would likely arise from the specific enrichment of targeted peptides using antibodies prior to LC-MS/MS analysis. The interest of this strategy called stable isotope standards and capture by antipeptide antibodies (SISCAPA) has already been illustrated by many groups that have succeeded in biomarker assays down to the nanogram/milliliter of plasma.16-18 CONCLUSIONS Compared to strategies that use antibodies either for extensive depletion of the most abundant plasma proteins or for enrichment of the targeted protein or peptide (e.g., SISCAPA), the MRM3 approach demonstrates equivalent or even improved limits of quantification. The two-step sample preparation workflow used in this study is based on whole serum trypsin proteolysis and targeted peptide enrichment through MCX solid phase extraction and is easily amenable to automation on commercially available multiwell plate devices. It is not dependent on the availability of specific antibodies for the proteins of interest and represents a markedly simplified and accelerated process compared to previously published protocols. The duty cycle of MRM3 acquisition (350 ms) is compatible with the use of isotopically labeled internal standards and, through segmentation of the retention time scale, with scheduled analysis of multiple proteotypic peptides per injection. (27) Gangl, E. T.; Annan, M. M.; Spooner, N.; Vouros, P. Anal. Chem. 2001, 73, 5635–5644.

Figure 4. (A) Overlaid LC-MRM3 extracted ion chromatograms of transition 636.8/943.5/627+ 698 + 779 + 797 and transition 639.8/949.5/630 + 704 + 785 + 803 monitored, respectively, for light LSEPAELTDAVK peptide tracking PSA protein (spiked at 50 ng/mL in serum) and for (13C6) heavy LSEPAELTDAVK internal standard (spiked at an equivalent concentration of 50 ng/mL of PSA protein in serum). (B) Calibration curve after internal standardization (IS) of MRM3 transition 636.8/943.5/627 + 698 + 779 + 797 monitored for LSEPAELTDAVK peptide tracking the PSA protein biomarker spiked in a human female serum (duplicate analysis). (C) Calibration curve after internal standardization (IS) of summed MRM transitions 636.8/943.5 + 636.8/472.3 + 636.8/312.2. (D) LC-MRM3 extracted ion chromatograms of transition 636.8/943.5/627 + 698 + 779 + 797 monitored for LSEPAELTDAVK peptide tracking PSA in serum of one blank female serum and three patients diagnosed with prostate cancer. Analytical Chemistry, Vol. 81, No. 22, November 15, 2009

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Therefore, MRM3 coupled to conventional bore chromatography should be considered a potential sensitive, specific, and cost-effective core technology for the future clinical verification of putative biomarkers across hundreds of patients. The high specificity of detection of the MRM3 approach also opens up new avenues for the reliable quantitation of highly diluted proteins in other complex matrixes such as cellular extracts or, for instance, stoichiometric assays of post-translationaly modified isoforms of regulating proteins involved in biological systems.

support. C.L. would like to thank Yves Le Blanc of MDS Analytical Technologies for fruitful discussion of the technical aspects of MRM3. J.P.C. also thanks Carolyn Roitsch for careful reading of the manuscript.

ACKNOWLEDGMENT The authors are grateful to the EZUS Lyon 1 Company and the French Institut National du Cancer (INCa) for their financial

Received for review July 1, 2009. Accepted October 9, 2009.

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SUPPORTING INFORMATION AVAILABLE MS3 spectra supporting the reconstructed MRM3 chromatograms. This material is available free of charge via the Internet at http://pubs.acs.org.

AC901447H

Multiple Reaction Monitoring Cubed for Protein Quantification at the

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