Protein Quantification by MALDI-Selected Reaction Monitoring Mass

Anal. Chem. 2010, 82, 5227–5237

Protein Quantification by MALDI-Selected Reaction Monitoring Mass Spectrometry Using Sulfonate Derivatized Peptides Antoine Lesur, Emmanuel Varesio, and Ge´rard Hopfgartner* Life Sciences Mass Spectrometry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland The feasibility of protein absolute quantification with matrix-assisted laser desorption/ionization (MALDI) using the selected reaction monitoring (SRM) acquisition mode on a triple quadrupole linear ion trap mass spectrometer (QqQLIT) equipped with a high-frequency laser is demonstrated. A therapeutic human monoclonal antibody (mAb) was used as a model protein, and four tryptic peptides generated by fast tryptic digestion were selected as quantification surrogates. MALDI produces mostly singly charged peptides which hardly fragment under low-energy collision-induced dissociation (CID), and therefore the benefits of using 4-sulfophenyl isothiocyanate (SPITC) as a fragmentation enhancer derivatization agent were evaluated. Despite a moderate impact on the sensitivity, the N-terminus sulfonated peptides generate nearly complete y-ion ladders when native peptides produce few fragments. This aspect provides an alternative SRM transition set for each peptide. As a consequence, SRM transitions selectivity can be tuned more easily for peptide quantitation in complex matrices when monitoring several SRM transitions. From a quantitative point of view, the signal response depending on mAb concentration was found to be linear over 2.5 orders of magnitude for the most sensitive peptide, allowing precise and accurate measurement by MALDI-SRM/MS. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was introduced in the late 1980s.1 MALDI is usually coupled with a time-of-flight (TOF) mass spectrometer and is well adapted for the analysis of organic polymers, DNA, proteins, peptides, and low molecular weight compounds (LMWC).2-4 Recently, the combination of MALDI sources with tandem mass spectrometers (ion trap, quadrupole-TOF, TOF/TOF, Fourier transform mass spectrometer, and triple quadrupole) has extended the applicability of MALDI-MS analysis, leading in complement to peptides analysis to the recent promotion of quantitative * Corresponding author. E-mail: [email protected]. Phone: +41 (0)22 379 63 44. Fax: +41 (0)22 379 68 08. (1) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Process. 1987, 78, 53–68. (2) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151–153. (3) Kaufmann, R. J. Biotechnol. 1995, 41, 155–175. (4) Jungblut, P.; Thiede, B. Mass Spectrom. Rev. 1997, 16, 145–162. 10.1021/ac100602d  2010 American Chemical Society Published on Web 05/19/2010

applications5-9 and mass spectrometry imaging10 of low molecular weight compounds. The MALDI produces mainly singly charged peptides, and their fragmentation was historically performed by postsource decay (PSD).11 However, PSD of singly charged peptides, besides a fussy use and a poor fragmentation efficiency, generates significantly more unassigned fragments than the CID of multiply charged peptides from electrospray ionization (ESI).12 Nowadays, high-quality MS/MS spectra of peptides are generated by highenergy collision induced dissociation (CID) on TOF/TOF instruments.13 The high resolution and the accuracy of these systems have promoted liquid chromatography-MALDI-TOF/TOF as an adapted platform for large-scale proteomic analyses.14,15 Other combinations of a MALDI source with tandem mass spectrometers such as MALDI-QqTOF16,17 or MALDI-IT18,19 are restricted to lowenergy CID under which singly charged peptide fragmentations do not consistently provide informative spectra.20 As suggested (5) Zabet-Moghaddam, M.; Heinzle, E.; Tholey, A. Rapid Commun. Mass Spectrom. 2004, 18, 141–148. (6) van Kampen, J. J.; Burgers, P. C.; de Groot, R.; Luider, T. M. Anal. Chem. 2006, 78, 5403–5411. (7) van Kampen, J. J.; Burgers, P. C.; Gruters, R. A.; Osterhaus, A. D.; de Groot, R.; Luider, T. M.; Volmer, D. A. Anal. Chem. 2008, 80, 4969–4975. (8) van Kampen, J. J.; Burgers, P. C.; de Groot, R.; Osterhaus, A. D.; Reedijk, M. L.; Verschuren, E. J.; Gruters, R. A.; Luider, T. M. Anal. Chem. 2008, 80, 3751–3756. (9) Wagner, M.; Varesio, E.; Hopfgartner, G. J. Chromatogr. B 2008, 872, 68– 76. (10) Stoeckli, M.; Staab, D.; Schweitzer, A. Int. J. Mass Spectrom. 2007, 260, 195–202. (11) Spengler, B.; Kirsch, D.; Kaufmann, R.; Jaeger, E. Rapid Commun. Mass Spectrom. 1992, 6, 105–108. (12) Gevaert, K.; Demol, H.; Martens, L.; Hoorelbeke, B.; Puype, M.; Goethals, M.; Damme, J. V.; Boeck, S. D.; Vandekerckhove, J. Electrophoresis 2001, 22, 1645–1651. (13) Pittenauer, E.; Allmaier, G. Comb. Chem. High Throughput Screening 2009, 12, 137–155. (14) Pan, S.; Shi, M.; Jin, J.; Albin, R. L.; Lieberman, A.; Gearing, M.; Lin, B.; Pan, C.; Yan, X.; Kashima, D. T.; Zhang, J. Mol. Cell. Proteomics 2007, 6, 1818–1823. (15) Hattan, S. J.; Marchese, J.; Khainovski, N.; Martin, S.; Juhasz, P. J. Proteome Res. 2005, 4, 1931–1941. (16) Loboda, A.; Krutchinsky, A.; Spicer, V.; Ens, W.; Standing, K. Proc. 47th ASMS Conf. Mass Spectrom. Allied Top., Dallas, TX, 1999; pp 1956-1957. (17) Shevchenko, A.; Loboda, A.; Ens, W.; Standing, K. G. Anal. Chem. 2000, 72, 2132–2141. (18) Qin, J.; Ruud, J.; Chait, B. T. Anal. Chem. 1996, 68, 1784–1791. (19) Qin, J.; Chait, B. T. Anal. Chem. 1996, 68, 2108–2112. (20) Cramer, R.; Corless, S. Rapid Commun. Mass Spectrom. 2001, 15, 2058– 2066.

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by Huang et al.,21 the fragmentation of singly charged peptides is sensitive to the proton sequestration by the basic amino acids. The arginine-ending peptide fragmentation is found to be less informative than for lysine-ending peptides because arginine has a greater proton affinity and thereby impedes the backbone ionization. This phenomenon is less observed for doubly charged peptides, where one of the protons is localized on the basic amino acid residue and the other is randomly located on the peptide chain. Thus, the fragmentation patterns are similar to a random cleavage along the peptide backbone.22,23 The fragmentation efficiency can be greatly improved on lowenergy CID instruments by a chemical modification of the peptides. It was observed that a protonated basic amino acid residue or a positively charged derivative located at the N-terminus of peptides promotes the formation of b-ion fragments (the reverse is true for a localized charge at the C-terminus and y-ion fragments). To control these phenomena, modifications via derivatization were investigated. Quaternary ammonium and phosphonium groups were evaluated to simplify and enhance peptide fragmentation.24,25 Alternatively, the addition of a fixed negative charge via a strong acid group (sulfonic acid) on the N-terminus of tryptic peptides enhances their fragmentation.26 This approach dramatically simplifies de novo spectra interpretation because only y-ions are detected. Keough et al. proposed that the fixed negative charge at the N-terminus counterbalances the C-terminus arginine or lysine positive charge leading to the ionization of sulfonated tryptic peptides by an additional labile proton. Because this proton is not sequestered by the C-terminus basic amino acid, it may randomly protonate the peptide backbone and enhance the fragmentation along the peptide chain.26,27 The sulfonation of peptides was investigated with several reagents: the chlorosulfonylacetyl chloride which must be used in nonaqueous solution, the sulfobenzoic acid cyclic anhydride,26 and the 3-sulfopropionic acid N-hydroxysuccinimide ester.28 The 4-sulfophenyl isothiocyanate (SPITC) was introduced in 2001 by Gevaert12 as an alternative reagent, and the method was improved by Marekov in 200329 and simplified by elimination of the organic coreagent by Wang.30 The SPTIC derivatization was successfully applied for protein identification on MALDI-TOF and MALDI-TOF/TOF instruments,31,32 and new sulfonation protocols were recently developed on the C18 solid phase28 or on-spot using the Anchor (21) Huang, Y.; Triscari, J. M.; Tseng, G. C.; Pasa-Tolic, L.; Lipton, M. S.; Smith, R. D.; Wysocki, V. H. Anal. Chem. 2005, 77, 5800–5813. (22) Hunt, D. F.; Yates, J. R., III; Shabanowitz, J.; Winston, S.; Hauer, C. R. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 6233–6237. (23) Paizs, B.; Suhai, S. Mass Spectrom. Rev. 2005, 24, 508–548. (24) Spengler, B.; Luetzenkirchen, F.; Metzger, S.; Chaurand, P.; Kaufmann, R.; Jeffery, W.; Bartlet-Jones, M.; Pappin, D. J. C. Int. J. Mass Spectrom. Ion Process. 1997, 169-170, 127–140. (25) Huang, Z.-H.; Wu, J.; Roth, K. D. W.; Yang, Y.; Gage, D. A.; Watson, J. T. Anal. Chem. 1997, 69, 137–144. (26) Keough, T.; Youngquist, R. S.; Lacey, M. P. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7131–7136. (27) Keough, T.; Lacey, M. P.; Fieno, A. M.; Grant, R. A.; Sun, Y.; Bauer, M. D.; Begley, K. B. Electrophoresis 2000, 21, 2252–2265. (28) Keough, T.; Lacey, M. P.; Youngquist, R. S. Rapid Commun. Mass Spectrom. 2002, 16, 1003–1015. (29) Marekov, L. N.; Steinert, P. M. J. Mass Spectrom. 2003, 38, 373–377. (30) Wang, D.; Kalb, S. R.; Cotter, R. J. Rapid Commun. Mass Spectrom. 2004, 18, 96–102. (31) Leon, I. R.; Neves-Ferreira, A. G.; Valente, R. H.; Mota, E. M.; Lenzi, H. L.; Perales, J. J. Mass Spectrom. 2007, 42, 1363–1374. (32) Oehlers, L. P.; Perez, A. N.; Walter, R. B. Comp. Biochem. Physiol., Part C: Toxicol. Pharmacol. 2007, 145, 120–133.

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Chip MALDI plate technology.33,34 Moreover, relative quantification of proteins and fragmentation enhancement can be performed simultaneously with a stable isotopically labeled version of SPITC35 used as an internal standard. However, the SPITC derivatization strategy suffers from the unspecific sulfonation on the ε-amino group of lysine which considerably reduces the ionization and fragmentation of lysine-ending peptides under PSD. This limitation can be overtaken by protecting the lysine residues with a guanidination step. The O-methylisourea was investigated because it converts arginine into lysine without reacting with N-terminus amine.36,37 Nowadays, the ability of MALDI-TOF/TOF instruments for peptide fragmentation has lowered the need of chemically assisted fragmentation for qualitative proteomics studies. From a quantitative point of view, MALDI-MS remains seldomly used because quantitative proteomics requires the convergence of three criteria: (i) the use of high-frequency/velocity MALDI sources to increase the analytical throughput, (ii) an efficient fragmentation of the peptides into identifiable fragments, and (iii) a proper selection of the precursor and product ions for performing selected reaction monitoring experiments (SRM). The coupling of a high repetition rate laser with a triple quadrupole linear ion trap mass spectrometer is recent.38,39 The high-speed capability of this MALDI source allows the analysis of 100 samples per day with the advantage of convenient reacquisition capability. The high selectivity of the SRM acquisition mode provided by triple quadrupole-based analyzers (QqQ or QqQLIT) reduces significantly the background noise and MALDI matrix interferences. In analogy to Wagner et al., who demonstrated ultrafast quantitation of saquinavir in human plasma by MALDI-SRM/MS,9 we investigated the potential of this technique for peptide quantitation. We also show the implementation of the chemically assisted fragmentation by using the SPITC derivatization in combination with the SRM mode from a MALDI-QqQLIT mass spectrometer to quantify a therapeutic monoclonal antibody (mAb). EXPERIMENTAL SECTION Reagents. Solutions (20 mM Tris-HCl pH ) 5.5) of recombinant human monoclonal antibody (MW ) 145 157 kDa, 53.8 µg/ µL) and its version isotopically labeled on threonines (13C4, 15N1, ∆m ) 5 u - 5.54 µg/µL), used as an internal standard (IS), were obtained from Novartis Pharma AG (Basel, Switzerland). Ammonium bicarbonate was provided by Fluka (Fluka Chemie GmbH, Buchs, Switzerland). DL-Dithiothreitol, iodoacetamide, sodium 4-sulfophenylisothiocyanate (SPITC), sodium bicarbonate, acetonitrile (HPLC grade), and Trypsin I-XS were purchased from Sigma-Aldrich (Sigma-Aldrich chemie GmbH, (33) Oehlers, L. P.; Perez, A. N.; Walter, R. B. Rapid Commun. Mass Spectrom. 2005, 19, 752–758. (34) Zhang, X.; Rogowska-Wrzesinska, A.; Roepstorff, P. J. Mass Spectrom. 2008, 43, 346–359. (35) Lee, Y. H.; Han, H.; Chang, S. B.; Lee, S. W. Rapid Commun. Mass Spectrom. 2004, 18, 3019–3027. (36) Keough, T.; Lacey, M. P.; Youngquist, R. S. Rapid Commun. Mass Spectrom. 2000, 14, 2348–2356. (37) Beardsley, R. L.; Reilly, J. P. Anal. Chem. 2002, 74, 1884–1890. (38) Corr, J. J.; Kovarik, P.; Schneider, B. B.; Hendrikse, J.; Loboda, A.; Covey, T. R. J. Am. Soc. Mass Spectrom. 2006, 17, 1129–1141. (39) Gobey, J.; Cole, M.; Janiszewski, J.; Covey, T.; Chau, T.; Kovarik, P.; Corr, J. Anal. Chem. 2005, 77, 5643–5654.

Steinheim, Germany), and formic acid and trifluoroacetic acid were obtained from Merck (Merck KGaA, Damstadt, Germany). Sequencing grade modified trypsin and chymotrypsin were provided by Promega (Promega AG Du ¨ bendorf, Switzerland). The recrystallized R-cyano-4-hydroxycinnamic acid matrix (CHCA) was provided by LaserBio Laboratories (SophiaAntipolis, France). Preparation of Standards and Quality Controls. A stock solution of native mAb (unlabeled) was diluted in ammonium bicarbonate (50 mM) to obtain a final concentration ranging from 37.5 to 15 000 ng/µL. Those solutions were split into three independent sets of nine calibration standards (37.5, 75, 125, 375, 750, 1500, 3000, 6000, and 15 000 ng/µL) and three quality controls (125, 750, and 6000 ng/µL). An amount of 5 µL of all standards and quality controls was spiked with 5 µL of IS (labeled mAb at 1.39 µg/µL) before tryptic digestion. Tryptic Digestion. An amount of 10 µL of DL-dithiothreitol (50 mM in 50 mM ammonium bicarbonate) was mixed with the standards/quality control IS-containing solutions. These samples were then reduced under agitation at 60 °C for 45 min (Thermomixer Comfort, Vaudaux-Eppendorf, Scho¨nenbuch, Switzerland). After cooling at room temperature, 10 µL of iodoacetamide (100 mM in 50 mM ammonium bicarbonate) was added for the alkylation of the cysteines, and the incubation was performed for 30 min in the dark at room temperature. Afterward, all samples were diluted with 70 µL of 50 mM ammonium bicarbonate before adding 5 µL of proteomic grade trypsin (0.27 µg/µL in 1 mM HCl). The tryptic digestions were performed in 15 min at 60 °C under agitation. Directly after the incubation, the tryptin activity was inhibited by neat formic acid addition (1 µL). SPITC Derivatization. The SPITC derivatization was adapted from a previously described method.30 In brief, 10 µL of the digest was vacuum-dried and further reconstituted in SPITC solution (4sulfophenyl isothiocyanate sodium salt monohydrate (10 µg/µL) in sodium bicarbonate buffer (20 mM), pH ) 9). To ensure a good solubilization of the pellet, the tubes were alternately sonicated (3 min) and vortexed. The solutions were incubated for 45 min at 55 °C. The reaction was subsequently quenched by addition of 4.5 µL of 1% (v/v) aqueous TFA. Solid Phase Extraction (SPE). SPITC derivatized and native digests were desalted on a C18, 96-well, microplate (ZipPlate, Millipore) according to the manufacturer protocol. Native samples consisted in 10 µL of underivatized digest mixed with 20 µL of 0.2% (v/v) aqueous TFA. SPITC derivatized samples were diluted in 10 µL of 0.1% (v/v) aqueous TFA before loading onto the ZipPlate. Peptide elution was directly performed using 15 µL of a CHCA solution (10 µg/µL in 60/40 (v/v) acetonitrile/TFA aq. 0.1%). Each sample was manually spotted five times on a stainless steel plate (1 µL by spot) leading to a 32-fold dilution factor from the initial stock solution concentrations. Sample Preparation for Qualitative Analysis. Tryptic digestion, SPITC derivatization, and SPE were performed in a similar way as described above. Briefly, all qualitative data were obtained from the same native mAb digest (unlabeled peptides). The digestion was performed in 15 min at 60 °C using a trypsin/mAb ratio (w/w) of 1/20. The digested mAb (752 ng/µL) was aliquoted in 30 µL. Two aliquots were kept native, and the others were derivatized with SPITC (30 µL of the previously described SPITC

solution was used). Native and SPITC derivatized digests were desalted with a ZipPlate and either eluted in 30 µL of CHCA matrix solution (10 µg/µL in 60/40 (v/v) acetonitrile/TFA aq. 0.1%) for MALDI analysis or eluted in 60/40 (v/v) acetonitrile/TFA aq. 0.1%, vacuum-dried, and reconstituted in TFA aq. 0.1% for HPLC analysis. HPLC-QqQLIT Analysis. The HPLC-MS system was employed for qualitative purposes. The system consisted of a Shimadzu (Duisburg, Germany) HPLC system configured in a binary high-pressure gradient mode (SIL-HTc autosampler, two LC-ADvp pumps), and 6 µL of digest was injected onto a 2.1 × 100 mm-3.5 µm XTerra MS C18 reversed-phase column (Waters, Milford, MA, USA) operated at the flow rate of 250 µL/min. The mobile phase A consisted of 0.1% formic acid in water and mobile phase B of 0.1% formic acid in acetonitrile. Peptides were eluted by a linear gradient starting from 5% B to 60% B in 15 min. Electrospray ionization (Turbo V ion source) was operated in positive mode. The MS data were collected using a 4000 QTRAP hybrid triple quadrupole linear ion trap mass spectrometer in EPI (Enhanced Product Ion) mode (AB/MDS Sciex, Concord, ON). EPI is a product ion mode where Q3 is used as a linear ion trap (trapping time ) 100 ms) for collecting fragment ions. The Q0 trapping mode was activated, and the nominal CEs (i.e., taking into consideration the charge state of the precursors) applied to DTLMISR, AEDTAVYYCAR, NTLYLQMNGLR, and SGTASVVCLLNNFYPR were, respectively, 60 eV for the first three peptides and 70 eV for the last one (unless otherwise stated). MALDI-QqQLIT and MALDI-TOF/TOF Analyses. MALDIMS/MS experiments were performed on a MALDI-4000 QTRAP equipped with a prototype MALDI source (AB/MDS Sciex, Concord, ON). The Nd:YAG laser possesses a wavelength of 355 nm and a high frequency up to 1000 Hz. For the qualitative study, samples were analyzed in Enhanced MS (EMS, this mode uses the Q3 as a LIT for scanning ions, trapping time 10 ms) and EPI mode (trapping time ) 100 ms). The same samples were also analyzed on a 5800 MALDI-TOF/TOF (AB/MDS Sciex, Concord, ON). The quantitative study was performed using the MALDI-4000 QTRAP in SRM mode (dwell time ) 20 ms). The laser frequency and energy were set according to the mass spectrometer acquisition mode: 200 Hz/2.0 µJ with EMS, 500 Hz/2.2 µJ with EPI, and 1000 Hz/2.3 µJ with SRM. All acquisitions were done in rastering mode. Quantitative data processing was performed with MultiQuant software v.1.2.0.6 (AB/MDS Sciex, Concord, ON) using the summation algorithm. The numerical analysis was performed using a linear regression (y = ax + b) and a 1/x2 weighting factor. RESULTS AND DISCUSSION The aim of the present work was (i) to investigate the chemical derivatization of the N-terminal of peptides to enhance the fragmentation of singly charged peptides and (ii) to demonstrate the feasibility of the quantitation of peptides using MALDI-SRM/ MS on a triple quadrupole linear ion trap. The MALDI 4000 QTRAP instrument is equipped with a rapid moving target plate (from 0.1 to 30 mm/s) and a high repetition rate laser (up to 1000 Hz). Its quadrupoles (Q1 and Q3) operated in RF/DC mode have a mass range from m/z 5 to m/z 2800, and this is less than a reflectron TOF but remains adequate for most peptides. As a matter of fact, an in silico tryptic digestion of the yeast proteome has shown a majority of peptide masses distributed Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

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Figure 1. MALDI-QqQLIT MS full scan spectra of the mAb’s tryptic digest under its native form and after SPITC derivatization. Native and derivatized peptides are symbolized as 2, AEDTAVYYCAR; *, DTLMISR; 9, NTLYLQMNGLR; and b, SGTASVVCLLNNFYPR. Data were acquired on the MALDI-QqQLIT setup using Q3 as a LIT.

between 800 and 1200 Da with a mean around 1200 Da.40 The originality of this system is to combine the selectivity of the triple quadrupole MS for performing SRM experiments with the high throughput capability of the MALDI source (i.e., a single spot is analyzed within a few seconds). Moreover, a single analysis requires minimal sample consumption (e.g., depending on the spot size, 4-8% of a 1 µL spot surface is ablated by the laser in rastering mode). Therefore, it allows an elegant way of reacquiring the samples at any time and a convenient storage of the samples on MALDI plates in a crystalline form. In addition, thanks to the acquisition speed, a multiple determination (i.e., five spots) of the same sample can be performed for accuracy improvement. We have investigated from a bioanalytical perspective the quantification of a therapeutic mAb. A general approach for the absolute quantification of proteins by LC-MS/MS consists of monitoring protein-specific peptides by single reaction monitoring (SRM) experiments.41 The uniqueness of these peptide sequences becomes critical when the analyte is extracted from complex biological matrices such as plasma. In our study, tryptic peptides are generated from a purified protein (mAb), and this requirement is minored. In a previous work,42 the peptide generation rates under different heating conditions (heating plate, microwave oven) were determined. In most cases, 30 min trypsin digestion generates peptide concentrations equivalent to an overnight digestion. This is important in a bioanalytical setup where large sets of samples are expected to be analyzed on a daily basis. Therefore, this strategy was applied for the present work using a 15 min digestion under conduction/convection thermal transfer. Peptide Mass Fingerprinting. Figure 1 shows the MS spectra acquired on the MALDI-QqQLIT of the mAb tryptic digest (15 min at 60 °C) before and after a SPITC derivatization. Table (40) Cagney, G.; Amiri, S.; Premawaradena, T.; Lindo, M.; Emili, A. Proteome Sci. 2003, 1, 5. (41) Lange, V.; Picotti, P.; Domon, B.; Aebersold, R. Mol. Syst. Biol. 2008, 4, 222. (42) Lesur, A.; Varesio, E.; Hopfgartner, G. J. Chromatogr. A 2010, 1217, 57– 64.

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1 lists the peptides assigned by Peptide Mass Fingerprint and further confirmed by MS/MS sequencing with the MALDI-QqQLIT and with a MALDI-TOF/TOF. At first glance, the response factor between native peptides and their sulfonated analogues as well as their signal-to-noise ratio do not change significantly (within a factor of 2). Under the conditions investigated, the SPITC derivatization of the selected peptides was not found to be complete. Signals from native peptides were detected in SPITC derivatized samples. Practically, at least 25% of the original signal from SGTASVVCLLNNFYPR and DTLMISR (50% from NTLYLQMNGLR and AEDTAVYYCAR) was measured after the sulfonation reaction. Wang et al.30 suggest that sodium bicarbonate in aqueous solution is the most convenient buffer for SPITC derivatization, even if the reaction can be incomplete. Indeed, they have observed that triethylamine (TEA) may increase the reaction yield, but the elimination of this base before MS analysis is challenging, which induces a significant signal suppression of the derivatized peptides. Three lysine-ending peptides (FNWYVDGVEVHNAK, EPQVYTLPPSREEMTK, and EIVLTQSPDFQSVTPK) are identified after SPITC derivatization; they are sulfonated at the N-terminus. Such derivatization products were also described by Lee et al.,43 and they suggest that a moderate SPTIC excess relative to the peptide concentration and a reaction pH around 8 hamper the derivatization of the side chain primary amino group of lysine (pKa around 11). The lysine ε-ammonium group remains mostly protonated, preventing its derivatization at such pH on the contrary of the N-terminal R-amino group (pKa ) 7.8). An atypical disulfonated version (on the N-terminus and lysine side chain) of DNSKNTLYLQMNGLR was also clearly identified by MS/MS. Selection of Monitored Peptides. The effect of SPITC derivatization on peptides fragmentation and quantitation on a QqQLIT was investigated for four peptides. The peptides studied (43) Lee, Y. H.; Kim, M. S.; Choie, W. S.; Min, H. K.; Lee, S. W. Proteomics 2004, 4, 1684–1694.

Table 1. Comparative Peptide Mass Fingerprint Results of the mAb Tryptic Digest with and Without SPITC Derivatizationa Native

SPITC

heavy chain experimental m/z

missed cleavage

heavy chain sequence

--835.5

0

K.DTLMISR.T

--838.5 1186.6 1318.5 1322.6

0 0 0 0

K.ALPAPIEK.T K.GPSVFPLAPSSK.S R.AEDTAVYYCAR.D K.NTLYLQMNGLR.A

1677.6 1807.8 1873.7 ---

0 0 0

K.FNWYVDGVEVHNAK.T R.VVSVLTVLHQDWLNGK.E K.TTPPVLDSDGSFFLYSK.L

1904.8

1

R.EPQVYTLPPSREEMTK.N

2138.8

0

R.TPEVTCVVVDVSHEDPEVK.F

experimental m/z

missed cleavage

732.5 835.7 1050.6 838.5 -----1533.5 1322.7 1537.6 1892.6 ----1981.7 2196.6

1 0 0 0

K.VDKR.V K.DTLMISR.T K.DTLMISR.T R.FTISR.D

SPITC (N-term) SPITC (N-term) SPITC (N-term)

0 0 0 0

R.AEDTAVYYCAR.D K.NTLYLQMNGLR.A K.NTLYLQMNGLR.A K.FNWYVDGVEVHNAK.T

SPITC (N-term) SPITC (N-term) SPITC (N-term)

1 1

R.DNSKNTLYLQMNGLR.A R.DNSKNTLYLQMNGLR.A

1904.8 2119.7 ---

1 1

R.EPQVYTLPPSREEMTK.N R.EPQVYTLPPSREEMTK.N

SPITC (N-term) SPITC (N-term) and SPITC (K) SPITC (N-term)

light chain experimental m/z

missed cleavage

sequence

derivatization position

light chain sequence

----1285.6

0

K.YASQSFSGVPSR.F

--1797.7

0

K.SGTASVVCLLNNFYPR.E

experimental m/z

missed cleavage

sequence

derivatization position

845.6 1084.5 1285.7 1500.6 2003.7 2012.7

1 1 0 0 0 0

K.VDIKR.T K.SFNRGEC.K.YASQSFSGVPSR.F K.YASQSFSGVPSR.F -.EIVLTQSPDFQSVTPK.E K.SGTASVVCLLNNFYPR.E

SPITC (N-term) SPITC (N-term) SPITC (N-term) SPITC (N-term) SPITC (N-term)

a

All peptides were confirmed by MS/MS sequencing (MALDI-QqQLIT and MALDI-TOF/TOF). Bold entries are peptides found in the two samples (native and its respective SPITC version).

(ranging from 800 to 1800 Da) were selected according to the three following criteria: (i) arginine-ending peptides were favored to prevent the addition of two sulfonate groups since SPITC derivatization may react with free primary amines; (ii) peptides must at least include one threonine residue because the internal standard (IS) used for the protein quantitation is a fully isotopically labeled version of the mAb (all threonines, 13 C4, 15N1, ∆m ) 5 u); (iii) peptide MS response should be sufficient. Table 2 summarizes the selected SRM transitions for the quantitative analysis of both native and SPITC derivatized peptides, as well as for the IS peptides. Qualitative Aspects of MS/MS Fragmentation. The SPITC derivatization produces N-terminal sulfonic acid peptides (Scheme 1) with an increased mass of 215 u. As described by Keough and co-workers in 1999,26 the sequestered positive charge at the C-terminal arginine residue is counterbalanced by the negative charge of the sulfonic acid group. Because arginine is already protonated on its side chain, the additional proton required for peptide ionization is supposed to be randomly delocalized on the peptide backbone. Since N-terminal fragments (b-ions) hold a negatively charged group (sulfonic acid), they should remain undetectable when the precursor ion is singly charged, thus promoting the detection

C-terminal fragments (y-ions). In Figure 2, the MS/MS spectra of the four SPITC derivatized peptides exhibit rich y-ion ladders, whereas their native homologues produce few informative product ion spectra, except for the peptide DTLMISR (Figure 2b). For the longest peptide, it should be noted that increasing the collision energy does not lead to further informative peptide fragmentation (Figure 2d, native). The same behavior was observed for the native NTLYLQMNGLR peptide; however, at least one b-ion could be assigned (Figure 2c, native). MS/MS spectra of the four selected native peptides were compared to their respective SPITC derivatized versions. The comparison was done by taking into account only b- and y-ions, on the basis of the influence of high- versus low-energy CID fragmentation. Figure 3 presents the summary of the assigned product ions for the different peptides fragmented with MALDIQqQLIT and MALDI-TOF/TOF instruments. This study focuses on the investigation of the SPITC derivation potential for MALDI-SRM/MS peptide quantitation, rather than on a comparison between both types of mass spectrometers. At a first glance, low-energy and high-energy CID of SPITC derivatized peptides produce rich y-ion ladders with the exception of some b°-ions (i.e., b-ions carrying the SPITC moiety). The major benefit of SPITC derivatization is definitely observed for the singly charged peptides when using low-energy CID fragmentation (i.e., MALDI-QqQLIT instrument). Indeed, the CID of three native Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

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Table 2. Sequences, SRM Transitions, Collision Energy, and Monitored Product Ions of the Four Selected Tryptic Peptides and Their Respective IS Peptidesa peptide sequence

precursor ion (m/z for Q1)

product ion (m/z for Q3)

DTLMISR D(13C4,15N1Thr)LMISR DTLMISR D(13C4,15N1Thr)LMISR AEDTAVYYCAR AED(13C4,15N1Thr)AVYYCAR AEDTAVYYCAR AED(13C4,15N1Thr)AVYYCAR NTLYLQMNGLR N(13C4,15N1Thr)LYLQMNGLR NTLYLQMNGLR N(13C4,15N1Thr)LYLQMNGLR SGTASVVCLLNNFYPR SG(13C4,15N1Thr)ASVVCLLNNFYPR SGTASVVCLLNNFYPR SG(13C4,15N1Thr)ASVVCLLNNFYPR

835.4 840.4 1050.4 1055.4 1318.7 1323.7 1533.6 1538.6 1322.7 1327.7 1537.7 1542.7 1797.9 1802.9 2012.8 2017.8

312.2 317.4 720.3 725.3 1003.4 1008.4 732.3 732.3 329.2 334.2 1208.6 1213.6 810.4 810.4

CE (eV)

fragment

70

b3-H2O

60

y6

80

y8

80

y5

110

b3

85

y10

105

y6

a Values in italics are related to the SPITC derivatized peptides. Collision energy (CE) was optimized by monitoring the SRM traces intensity when ramping this parameter by steps of 5 eV.

Scheme 1. Sulfonation of N-Terminus Amine by SPITC Derivatizing Reagent

peptides on the MALDI-QqQLIT instrument did not give sufficient fragments for sequence identification. As a matter of fact, high-energy CID on MALDI-TOF/TOF instrument produces a comprehensive fragmentation (combination of b- and y-ions) for all native peptides. The product ion spectra of the derivatized singly charged peptides were compared with that of the doubly and triply charged native peptides generated by ESI as illustrated in Figure 4. The multicharged peptides were generated with an ESI source coupled with a QqQLIT mass spectrometer. As expected for both native and SPITC derivatized peptides, doubly charged peptide fragmentation is the most informative and comparable to MALDI-TOF/TOF spectra. These observations advocate the benefit mostly for singly charged peptides under low-energy CID fragmentation. This allows us to perform peptide sequencing on the MALDI-QqQLIT instrument. The success of TOF/ TOF instruments has probably lessened, from a “classical proteomics” point of view, the needs of chemically assisted derivatization originally developed for MALDI-PSD/MS. Nevertheless, if TOF/TOF instruments are characterized by highenergy fragmentation capability, their selectivity remains inferior to triple quadrupole MS operated in SRM mode. Trypsin-Grade Selection. Direct plasma digestion requires a significant amount of trypsin due to the high protein concentration in this biological matrix. Therefore, a clinical study for biomarkers or biopharmaceuticals generating large numbers of samples is requiring a large quantity of trypsin. As a consequence, the cost of such analyses is largely dependent on the trypsin quality, and a nonproteomic grade trypsin is generally recommended.44 This grade of trypsin is much cheaper than the proteomic grade (e.g., 12 000 times less in our instance) but is

neither protected against autolysis nor treated with L-1-tosylamide2-phenylethyl chloromethyl ketone (TCPK) to inhibit chymotrypsin activity. In our case, the nonproteomic grade trypsin was able to generate three from the four selected peptides: indeed the AEDTAVYYCAR peptide was not detected based on its respective SRM traces (data not shown). The stability of this peptide might be impeded by a pseudotrypsin45 and/or a chymotryptic activity because a digestion with a TCPK-treated trypsin (i.e., proteomic grade) did produce this missing peptide. As a comparison, a digestion performed with the nonproteomic grade trypsin and a codigestion performed with a mix of proteomic grade trypsin and chymotrypsin showed very similar peptide SRM traces. In addition, peptide NTLYLQMNGLR signal response is also slightly lowered when the mAb is digested with low-cost trypsin. These results probably indicate remaining chymotryptic activity which interferes with some tryptic peptides. Despite this drawback, low-cost trypsin digestion remains as efficient as the proteomic grade for the two other peptides. Protein quantitation is mainly achieved on the basis of only one peptide (with several SRM transitions) used as a quantitative surrogate; therefore, a low cost trypsin is probably the most reasonable choice for large scale studies. The aim of our work was to investigate the effects of SPITC derivatization on a maximum number of peptides from a single protein, and therefore we decided to select a proteomic grade trypsin. Quantitative Analysis by MALDI-SRM/MS. For the analysis of low molecular weight pharmaceutical compounds, accurate and precise quantitation was demonstrated in biological matrices over 3 orders of magnitude by MALDI-SRM/MS without any chromatographic step prior detection.9 In MALDI, the crystallization process engenders a spatial inhomogeneous distribution of the

(44) Hagman, C.; Ricke, D.; Ewert, S.; Bek, S.; Falchetto, R.; Bitsch, F. Anal. Chem. 2008, 80, 1290–1296.

(45) Keil-Dlouha, V. V.; Zylber, N.; Imhoff, J.; Tong, N.; Keil, B. FEBS Lett. 1971, 16, 291–295.

5232


Figure 2. MS/MS spectra of four peptides acquired on the MALDI-QqQLIT at low-energy CID in enhanced product mode. Both the native and the SPITC derivatized peptide are compared: (a) AEDTAVYYCAR, (b) DTLMISR, (c) NTLYLQMNGLR, and (d) SGTASVVCLLNNFYPR.

Figure 3. Effect of SPITC derivatization on the peptide fragmentation under low-energy CID (MALDI-QqQLIT) and high-energy CID (MALDITOF/TOF). Only y- and b-ion series are taken into consideration. b°-ion is a b-ion carrying the SPITC moiety.

analyte on the spot, and multiple analyses of the same sample significantly improve precision and accuracy in particular when nonlabeled internal standards are used. Due to the highthroughput capability of MALDI, the total analysis time is not seriously affected by multiple analyses. In the case of LC-MALDI setup, only a signal spot would be analyzed. In our study, each sample was spotted five times and analyzed in rastering mode using a single pass of the laser through the spots. The SRM responses of these five replicates were integrated as a “single

peak” for averaging the variability. A homologue (13C4, 15N1 threonine) of the mAb was used as an internal standard. This isotopically labeled protein was digested together with the analyte to balance digestion, sample preparation, and spotting variability. At the end, the quantification was based on analytes/ IS area ratios. The amount of protein in a single MALDI spot, from the least to the most concentrated spot, can be estimated to range from 1.2 to 470 ng (8 to 3250 fmol) considering that digestion is complete and that SPE recovery is total. Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

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Figure 4. Effect of the charge state and SPITC derivatization on the peptide fragmentation under low-energy CID (ESI-QqQLIT). Only y- and b-ion series are taken into consideration. b°-ion is a b-ion carrying the SPITC moiety.

Table 3. Precision and Accuracy Results of MALDI-SRM/MS of the mAb Using the Peptide DTLMISR With and Without SPITC Derivatization Native DTLMISR sample C01 C02 C03 C04 C05 C06 C07 C08 C09 QC high QC med QC low R slope intercept

conc. (ng/µL) 15000 6000 3000 1500 750 375 125 75 37.5 6000 750 125

n)1

n)2

n)3

14882 6528.0 3061.4 1509.5 756.65 355.17 117.37 71.390 39.240 6381.5 779.55 122.16 0.99841 0.00110 -0.00550

15907 6201.6 3154.2 1406.1 761.97 366.66 119.05 68.210 39.810 6114.2 768.31 122.85 0.99783 0.00118 -0.00563

13827 6313.3 3186.1 1550.9 748.34 397.21 116.54 64.190 40.680 5731.3 828.29 125.03 0.99591 0.00115 0.00127

mean (ng/µL)

RSD %

accuracy %

14872 6347.7 3133.9 1488.9 755.65 373.01 117.65 67.930 39.910 6075.7 792.05 123.35

7.0 2.6 2.1 5.0 0.9 5.8 1.1 5.3 1.8 5.4 4.0 1.2

99.1 105.8 104.5 99.3 100.8 99.5 94.1 90.6 106.4 101.3 105.6 98.7

SPITC DTLMISR sample

conc. (ng/µL)

n)1

n)2

n)3

mean (ng/µL)

RSD %

accuracy %

C01 C02 C03 C04 C05 C06 C07 C08 C09 QC high QC med QC low R slope intercept

15000 6000 3000 1500 750 375 125 75 37.5 6000 750 125

15051 6168.6 2979.2 1438.8 745.15 362.76 144.52 66.240 38.100 6201.5 756.01 127.50 0.99656 0.00071 -0.00597

15904 6142.8 2993.4 1366.7 765.93 379.93 125.82 70.270 38.590 6524.2 801.60 117.57 0.99855 0.00072 -0.00400

14852 6495.3 3210.9 1535.1 791.51 370.99 100.77 64.240 42.230 5751.9 687.70 118.85 0.99628 0.00081 -0.00485

15269 6268.9 3061.2 1446.8 767.53 371.23 123.70 66.917 39.640 6159.2 748.44 121.31

3.7 3.1 4.2 5.8 3.0 2.3 17.7 4.6 5.7 6.3 7.7 4.5

101.8 104.5 102.0 96.5 102.3 99.0 99.0 89.2 105.7 102.7 99.8 97.0

The DTLMISR peptide was selected to demonstrate the applicability of MALDI-SRM/MS to perform peptide quantitation in its native form. The analyte/IS area ratio was found to be linear for about 2.5 orders of magnitude with precision better than 6% 5234


and accuracy in the range of 98-106% based on QCs (Table 3). It is noteworthy to mention that the MS/MS spectrum of the native form generates an informative spectrum, and therefore good SRM transitions can be selected. For the three larger remaining

Table 4. Precision and Accuracy Results of MALDI-SRM/MS of the mAb Using the Peptide AEDTAVYYCAR With and Without SPITC Derivatizationa Native AEDTAVYYCAR sample

conc. (ng/µL)

n)1

n)2

n)3

mean (ng/µL)

RSD %

accuracy %


15000 6000 3000 1500 750 375 125 75 37.5 6000 750 125

13633 6040.0 3320.9 1452.8 768.98 368.89 5575.6 703.85 0.99566 0.00061 0.01560

14890 6475.3 3065.7 1346.7 735.60 385.37 5506.4 706.47 0.99785 0.00057 0.01113

13829 6417.0 2970.2 1523.8 761.66 370.17 5591.5 647.73 0.99872 0.00052 0.01457

14117 6310.7 3118.9 1441.1 755.41 374.81 5557.8 686.02 -

4.8 3.7 5.8 6.2 2.3 2.4 0.8 4.8 -

94.1 105.2 104.0 96.1 100.7 99.9 92.6 91.5 -

SPITC AEDTAVYYCAR sample

conc. (ng/µL)

n)1

n)2

n)3

mean (ng/µL)

RSD %

accuracy %


15000 6000 3000 1500 750 375 125 75 37.5 6000 750 125

14630 6039.8 2968.0 1399.0 771.80 341.69 167.88 60.950 5740.8 732.37 90.480 0.98431 0.00071 0.02996

16515 5924.9 3004.0 1308.8 786.90 383.36 115.48 78.170 6153.5 766.07 125.76 0.99644 0.00067 0.00618

16333 6465.9 2774.5 1365.9 739.46 402.05 108.77 80.240 7060.7 803.53 107.63 0.99477 0.00058 0.02875

15826 6143.5 2915.5 1357.9 766.05 375.70 130.71 73.120 6318.3 767.32 107.96

6.6 4.6 4.2 3.4 3.2 8.2 24.8 14.5 10.7 4.6 16.3

105.5 102.4 97.2 90.5 102.1 100.2 104.6 97.5 105.3 102.3 86.4

a

In bold ) accuracy or RSD values above 20%.

peptides, none or only one fragment could be selected for the SRM transitions jeopardizing their detection. On the contrary, SPICT derivatives of all four peptides generated good quality MS/ MS spectra leading to exploitable SRM transitions (Figure 2). For the DTLMISR derivatized peptide, the response was also found to be linear for 2.5 orders of magnitude with a precision better than 8% and accuracy in the range of 97-103% based on QCs (Table 3). The SPITC derivatization increases the linearity range of AEDTAVYYCAR by a factor of 5 but slightly reduces the limit of quantitation for NTLYQMNGLR (factor of 2) (Tables 4 and 5). The largest peptide (SGTASVVCLLNNFYPR) benefits from SPITC derivatization because no fragments were actually detectable from its native homologue (Table 6). For these peptides, five individual measurement points had an accuracy over 20% (three based on standards and two based on QCs), and one precision based on standard was found to be more than 20% (in bold). These results suggest that a major advantage of SPITC derivatization from a quantitative point of view is the selectivity enhancement and the possibility to monitor several SRM transitions. The SPITC derivatization helps to produce a set of alternative transitions (mainly y-ions series) for each peptide. This has

two key benefits: the first one is the confirmation of the peptide identity by using several SRM transitions, and the second one is the possibility to change the SRM transitions in the case of matrix interferences. For quantitation, the use of a labeled peptide as internal standard is almost mandatory to compensate for variations during the complete workflow. CONCLUSION The feasibility of protein quantification by MALDI-SRM/MS using tryptic peptides of a monoclonal antibody was demonstrated. Good precision and accuracy could be obtained over 2.5 orders of magnitude. Low-energy CID spectra of singly charged peptides in their native form generate only a few or no exploitable fragments depending on their size and sequence which challenges the selection of sensitive and selective SRM transitions. This limitation can be overcome by the use of a SPITC derivatizing agent acting as a peptide CID fragmentation enhancer. While the sensitivity seems to be comparable for native and derivatized peptides, the greatest benefit of SPITC derivatization is the possibility of defining a wide set of SRM transitions for each peptide. This aspect is essential if SRM transitions are subjected to interferences from the complex background of the sample. The Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

5235

Table 5. Precisions and Accuracy Results of MALDI-SRM/MS of the mAb Using the Peptide NTLYLQMNGLR With and Without SPITC Derivatizationa Native NTLYLQMNGLR sample

conc. (ng/µL)

n)1

n)2

n)3

mean (ng/µL)

RSD %

accuracy %


15000 6000 3000 1500 750 375 125 75 37.5 6000 750 125

15891 6637.0 3104.7 1374.0 773.26 346.05 98.650 85.540 6957.9 785.01 120.62 0.99105 0.00067 0.03398

15614 5743.5 2756.0 1349.7 806.84 399.73 142.20 67.800 5276.0 806.43 119.56 0.99442 0.00075 0.00440

15080 5769.2 2788.3 1472.8 747.85 428.29 127.39 72.270 4849.8 801.41 129.84 0.99725 0.00076 0.00774

15528 6049.9 2883.0 1398.8 775.98 391.36 122.75 75.203 5694.6 797.62 123.34

2.7 8.4 6.7 4.7 3.8 10.7 18.0 12.3 19.6 1.4 4.6

103.5 100.8 96.1 93.3 103.5 104.4 98.2 100.3 94.9 106.3 98.7

SPITC NTLYLQMNGLR sample

conc. (ng/µL)

n)1

n)2

n)3

mean (ng/µL)

RSD %

accuracy %


15000 6000 3000 1500 750 375 125 75 37.5 6000 750 125

14365 5835.3 2785.6 1461.6 823.33 419.62 118.76 5977.8 853.13 115.46 0.99628 0.00070 0.01812

15009 5508.1 2914.0 1373.1 839.01 423.24 118.41 5753.6 770.55 142.05 0.99483 0.00075 -0.00366

12874 5849.7 2860.6 1394.8 825.62 481.80 112.22 5511.4 648.47 134.73 0.98626 0.00081 0.00427

14083 5731.0 2853.4 1409.9 829.32 441.55 116.46 5747.6 757.38 130.75

7.8 3.4 2.3 3.3 1.0 7.9 3.2 4.1 13.6 10.5

93.9 95.5 95.1 94.0 110.6 117.7 93.2 95.8 101.0 104.6

a


Table 6. Precisions and Accuracy Results of MALDI-SRM/MS of the mAb Using the Peptide SGTASVVCLLNNFYPR with SPITC Derivatizationa SPITC SGTASVVCLLNNFYPR sample

conc. (ng/µL)

n)1

n)2

n)3

mean (ng/µL)

RSD %

accuracy %


15000 6000 3000 1500 750 375 125 75 37.5 6000 750 125

13783 6371.6 3213.2 1408.8 756.72 6334.3 741.90 0.99568 0.00063 0.12293

14894 5847.6 3270.8 1383.7 764.79 6264.1 662.28 0.99658 0.00062 0.18207

12285 6879.4 3068.4 1579.8 718.82 6145.5 925.83 0.98687 0.00064 0.01630

13654 6366.2 3184.1 1457.4 746.78 6248.0 776.67 -

9.6 8.1 3.3 7.3 3.3 1.5 17.4 -

91.0 106.1 106.1 97.2 99.6 104.1 103.6 -

a


combination of a rapid tryptic digestion (15 min) with the highspeed analysis of MALDI-SRM/MS opens new possibilities for 5236


high-throughput quantitation of proteins. Despite that in our study no chromatographic step was used, application to complex

biological samples such as plasma requires a LC separation prior to MALDI-SRM/MS analysis. One of the key advantages of LCMALDI-SRM/MS approaches compared to LC-ESI-SRM/MS is that the sample has been collected onto the MALDI target and can be reanalyzed at any time with the same or with different SRM settings. ACKNOWLEDGMENT We acknowledge Dr. Stephan Bek, Novartis Pharma AG (Basel, Switzerland), for providing the mAb and its labeled version

and fruitful collaboration. We are grateful to Dr. Patrick Pribil from AB SCIEX (Concord, Canada) for analyses performed on the 5800 TOF/TOF instrument and to Dr. J.C. Yves Le Blanc for fruitful discussions.

Received for review March 6, 2010. Accepted April 28, 2010. AC100602D


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Protein Quantification by MALDI-Selected Reaction Monitoring Mass

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