Chromatographic Separation and Sample Preparation in One Step for

Anal. Chem. 1998, 70, 3046-3052

Chromatographic Separation and Sample Preparation in One Step for MALDI Mass Spectrometric Analysis of Subpicomole Amounts of Heterogeneous Protein Samples Filippo Rusconi,*,† Jean-Marie Schmitter,‡ Jean Rossier,† and Marc le Maire§

Laboratoire de Neurobiologie, Ecole Supe´ rieure de Physique et de Chimie Industrielles de la Ville de Paris, CNRS UMR 7637, 10 rue Vauquelin, F-75231 Paris Cedex 05, France, Laboratoire de Physico- et Toxico-Chimie, UPRES A5472 CNRS, Universite´ Bordeaux I, F-33405 Talence Cedex, France, and Section de Biophysique des Prote´ ines et des Membranes, De´ partement de Biologie Cellulaire et Mole´ culaire, CEA et CNRS URA 2096, Centre d’Etudes de Saclay, F-91191 Gif-sur-Yvette Cedex, France

A rapid and efficient sample preparation method is described that allows analysis of biopolymer mixtures by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Very low amounts of complex biopolymer mixtures can be analyzed by MALDI MS without any apparent loss of material and with a substantial gain in time by combining chromatographic separation and sample preparation in a single operation. This micropurification method is based on both the formation of a submicroliter reversed-phase chromatographic bed and the use of matrix-containing eluent solutions. In this way, desalting, matrix mixing, and micropurification of a biological sample could be performed in one step. A proteolytic mixture containing Ca2+ ATPase fragments ranging from ∼1000 to ∼30 000 Da, obtained after endoproteinase AspN cleavage of sarcoplasmic reticulum vesicles, was analyzed by MALDI MS. Direct analysis by MALDI MS of this peptidic mixture did not provide any signal for the higher molecular mass species. When our micropurification technique was applied to this sample, successful mass data acquisitions for as low as 150 fmol of the ∼30 000-Da fragment were performed. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has proven to be a very powerful technique for providing biochemists with highly informational mass data of biopolymer samples1 (for a recent review, see Fenselau2). Sample preparation for analysis by MALDI MS is a crucial part of the overall procedure leading to high-quality spectra, as underlined by several authors.3-5 The first step consists of the isolation of the molecular species of interest as a purified sample or as a

mixture. This step precedes a desalting step that is needed to free the sample of contaminants such as salts, buffers, and detergents. Devices have been described that allow desalting of biological samples for analysis by mass spectrometry on a microgram scale.3,6 The third step involves the selection of a matrix that is chosen in accordance with the nature of the biopolymers to be analyzed (peptides, polypeptides, oligonucleotides, etc.)7-9 and the actual preparation of the sample for analysis (on-target cocrystallization of the sample with the matrix). As described by Kussman et al.,3 matrixes are chosen for their ability to selectively favor desorption/ionization of certain biopolymers. 2,5-Dihydrobenzoic acid (DHB) is generally used for samples containing both large and small peptides, while R-cyano-4hydroxycinnamic acid and sinapinic acid are used for analysis of respectively low- and high-molecular-mass species. Depending on the matrix used, the sample spots can be uniform or nonuniform. For example, DHB cocrystallizes with the analyte starting from the edge of the deposit, so that crystals are formed radially from the edge of the spot to its central part. The crystals formed at the edge of the sample spot will yield [M + H]+ ions, while, in the central part of the spot, the crystals will generate metal-complexed analyte ions.3,10 One characteristic of MALDI is its capability to allow analysis of complex peptide/polypeptide mixtures.11-13 This technique (for an in-depth review of biological mass spectrometry, see, e.g.,

* To whom correspondence should be addressed. Present address: IECBEcole Polytechnique, ENSCPB, Avenue Pey Berland, BP 108, F-33402 Talence Cedex, France. Tel.: +33-5.57.96.22.13. FAX: +33-5.57.96.22.15. E-mail: [email protected]. † Ecole Supe ´rieure de Physique et de Chimie Industrielles de la Ville de Paris. ‡ Universite ´ Bordeaux I. § Centre d’Etudes de Saclay. (1) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (2) Fenselau, C. Anal. Chem. 1997, 69, 661A-665A.

(3) Kussmann, M.; Nordhoff, E.; Rahbek-Nielsen, H.; Haebel, S.; Rossel-Larsen, M.; Jakobsen, L.; Gobom, J.; Mirgorodskaya, E.; Kroll-Kristenson, A.; Palm, L.; Roepstorff, P. J. Mass Spectrom. 1997, 32, 593-601. (4) Xiang, F.; Beavis, R. C. Rapid Commun. Mass Spectrom. 1994, 8, 199-204. (5) Vorm, O.; Roepstorff, P.; Mann, M. Anal. Chem. 1994, 66, 3281-3287. (6) Brockman, A. H.; Dodd, B. S.; Orlando, R. Anal. Chem. 1997, 69, 47164720. (7) Strupat, K.; Karas, M.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1991, 111, 89-102. (8) Beavis, R. C.; Chait, B. T. Rapid Commun. Mass Spectrom. 1989, 3, 432435. (9) Beavis, R. C.; Chaudhary, T.; Chait, B. T. Org. Mass Spectrom. 1992, 27, 156-158. (10) Dai, Y.; Whittal, R. M.; Li, L. Anal. Chem. 1996, 68, 2494-2500. (11) Mann, M.; Talbo, G. Curr. Opin. Biotechnol. 1996, 7, 11-19. (12) Andersen, J. S.; Svensson, B.; Roepstorff, P. Nat. Biotechnol. 1996, 14, 449457.

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Burlingame et al.14 and Cotter15) is thus particularly well suited for peptide mapping studies,16 as a means to protein primary structure elucidation, as well as for protein posttranslational modification studies where the modification is extensive.17,18 However, it was found with different mass spectrometric techniques, including fast atom bombardment19,20 (FAB) and plasma desorption21,22 (PD), that the analysis of peptide mixtures rarely led to complete peptide maps. This differential desorption/ ionization of the single components of a biopolymer mixture could be, in the case of FAB and PD, correlated either to their relative hydrophobicity and/or to their net charges. Authors have counteracted this phenomenon, called spectral suppression, by either further purifying the components of the peptide mixture, taking into account the relative hydrophobicity of the different peptidic species (continuous-flow FAB mass spectrometry is one example of a purification strategy23), or derivatizing the chargebearing functional groups of the peptide mixture components.22 In MALDI MS, spectral suppression is also encountered,24 though it is not yet fully documented. This phenomenon is partly related to the fact that a proteolytic mixture can contain both lowand high-molecular-mass peptides, making it necessary to use both peptide- and polypeptide-specific matrixes for the same sample in order to obtain informational mass spectrometric data for the entire protein sequence. Experiments aimed at identifying an unknown protein or its potential posttranslational modifications very often include a proteolytic cleavage of the protein with a protease of known specificity. The MALDI MS analysis of the resultant peptidic mixture should bring the primary structure information needed to eventually identify the protein or its modification site. As happens in proteome-type experiments, if large (or extensively modified) or unperfectly purified proteins are proteolyzed, the proteolytically generated peptide mixture might be complex enough to preclude any identification due to the spectral suppression phenomenon. Thus, the peptidic mixture might need to be at least partially purified in order to minimize the spectral suppression. However, proteome-type experiments are characterized by the very low amounts of biopolymers available, which makes either desalting or partial purification on a conventional HPLC system counterproductive because of the unavoidable loss of material. (13) Beavis, R. C.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 68736877. (14) Burlingame, A. L.; Boyd, R. K.; Gaskell, S. J. Anal. Chem. 1996, 68, 599R651R. (15) Cotter, R. J. Time-of-Flight Mass Spectrometry: Instrumentation and Applications in Biological Research; ACS Professional Reference Books; American Chemical Society: Washington, DC, 1997. (16) Henzel, W. J.; Billeci, T. M.; Stults, J. T.; Wong, S. C.; Grimley, C.; Watanabe, C. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5011-5015. (17) Redeker, V.; Levilliers, N.; Schmitter, J.-M.; Le Caer, J.-P.; Rossier, J.; Adoutte, A.; Bre, M.-H. Science 1994, 266, 1688-1691. (18) Harvey, D. J.; Rudd, P. M.; Bateman, R. H.; Bordoli, R. S.; Howes, K.; Hoyes, J. B.; Vickers, R. G. Org. Mass Spectrom. 1994, 29, 753-758. (19) Naylor, S.; Findeis, A. F.; Gibson, B. W.; Williams, D. H. J. Am. Chem. Soc. 1986, 108, 6359-6363. (20) Caprioli, R. M.; Moore, W. T.; Fan, T. Rapid Commun. Mass Spectrom. 1987, 1, 15-18. (21) Nielsen, P. F.; Roepstorff, P. Biomed. Environm. Mass Spectrom. 1988, 18, 131-137. (22) Schmitter, J-.M. J. Chromatogr. 1991, 557, 359-368. (23) Caprioli, R. M.; Moore W. T. Methods Enzymol. 1990, 193; 214-237. (24) Mock, K. K.; Sutton, C. W.; Cottrell, J. S. Rapid Commun. Mass Spectrom. 1992, 6, 233-238.

The technique described in this report permits the simultaneous achievement of the three sample preparation steps, described in detail earlier in this section, that are required for efficient analysis by MALDI MS of biological samples. This method involves both desalting of a biopolymer mixture and stepgradient microseparation of the components of this mixture on a submicroliter C18 reversed-phase chromatographic bed. Furthermore, direct on-target elution of the adsorbed biopolymer molecules is achieved by successive use of different matrix-containing eluent solutions of increasing acetonitrile content. Thus, this method permits simultaneously a reversed-phase-like chromatographic separation of the biopolymer mixture and a thorough mixing of the eluted molecular species with different peptide- or polypeptide-specific matrixes in the same single experiment. We demonstrate the validity of this method on a complex peptide/polypeptide mixture of Ca2+ ATPase fragments ranging from ∼1000 to ∼30 000 Da, obtained after endoproteinase AspN proteolysis of sarcoplasmic reticulum vesicles. Indeed, when MALDI MS analyses were performed directly on this sample, no mass data could be obtained for the ∼30 000-Da molecular mass species. When our micropurification technique was applied to this sample, spectral suppression was diminished, leading to successful mass data acquisitions for as low as 150 fmol of the ∼30 000 Da fragment. In addition, the technique allows us to use both a peptidespecific matrix for small peptides and a protein-specific matrix for large polypeptides in a single experiment, thus achieving optimized experimental conditions for mass spectrometric analysis of very low amounts of biopolymer mixtures. EXPERIMENTAL SECTION Preparation of Ca2+ ATPase Fragments. Proteolytic fragments of Ca2+ ATPase were obtained by a modification of the method described by le Maire et al.,25 Juul et al.26 and Champeil et al.27 Sarcoplasmic reticulum vesicles (SRV, 75 µL of a 40 mg total protein/ml solution) in 3 mL of 100 mM Bis-Tris, pH 6.5 (Sigma, St. Louis, MO) were treated with 1 µg of endoproteinase AspN (sequencing grade, Boehringer Mannheim) at 20 °C for 20 h. The reaction was stopped by adding of 10 µL of 100 mM EDTA and by placing the reaction tubes on ice for 10 min. The reaction mixture was then centrifuged for 60 min at 217000g at 4 °C (tabletop ultracentrifuge TL 100, Beckman, Palo Alto, CA). The supernatant (referred to in the next sections of this report as the AspN supernatant) contained soluble Ca2+ ATPase fragments and was used for subsequent steps of purification and mass spectrometric analyses. To purify the soluble Ca2+ ATPase fragments that might interact with nucleotides, the AspN supernatant was further affinity-chromatographed on a reactive red-120 resin (Sigma).28,29 For this, a column was packed in a P1000 Gilson pipet tip with 300 µL of the reactive red-120 resin. The resin was washed with (25) le Maire, M.; Deschamps, S.; Møller, J. V.; Le Caer, J.-P., Rossier, J. Anal. Biochem. 1993, 214, 50-57. (26) Juul, B.; Turc, H.; Durand, M. L.; Gomez de Gracia, A.; Denoroy, L.; Møller, J. V.; Champeil, P.; le Maire, M. J. Biol. Chem. 1995, 270, 20123-20134. (27) Champeil., P.; Menguy, T.; Soulie´, S.; Juul, B.; Gomez de Gracia, A.; Rusconi, F.; Falson, P.; Denoroy, L.; Henao, F.; le Maire, M.; Møller, J. V. J. Biol. Chem. 1998, 273, 6619-6631. (28) Coll, R. J.; Murphy, A. J. J. Biol. Chem. 1984, 259, 14249-14254. (29) Stokes, D. L.; Green, N. M. Biophys. J. 1990, 57, 1-14.

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Figure 1. SDS-PAGE analysis of the Ca2+ ATPase soluble fragments. The soluble Ca2+ ATPase fragments were released upon treatment of sarcoplasmic reticulum vesicles with endoproteinase AspN. Lane 1, soluble fragments contained in the supernatant (AspN supernatant, see Experimental Section); lane 2, reactive red-120 column eluate (two major fragments were retained on the column with apparent molecular masses of ∼30 (p29/30) and ∼3 kDa (p3/ 4)); lane 3, RP-HPLC purified p29/30 fragment; lane 4, RP-HPLC purified p3/4 fragment. Note that, because of the silver-staining procedure of the gel,33 some of the low-molecular-mass peptides of lane 1 were lost.

2 M NaCl and equilibrated (equilibration buffer, 1 mM CaCl2, 1 mM MgCl2, 20 mM Mops pH 8.0, 20% glycerol, 1 mM dithiothreitol). After loading of the sample (approximately 0.2 mg of soluble Ca2+ ATPase fragments), the column was rinsed with 100 mM Bis-Tris, pH 6.5, 1 mM EDTA. The column was further washed with the equilibration buffer plus increasing concentrations of NaCl (25 and 50 mM). Elution of the bound molecular species was performed by applying the elution buffer to the column (equilibration buffer supplemented with 10 mM ADP and 150 mM NaCl). All the fractions were collected for analysis by sodium dodecyl sulfate-polyacrylamide tricine gel electrophoresis30 (SDSPAGE, see Figure 1; these tricine gels are made in-house), mass spectrometry, and further purification by RP-HPLC of the bound Ca2+ ATPase fragments. RP-HPLC Separation of Endoproteinase AspN Cleavage Products. The experiments aimed at purifying the ∼3-kDa peptide retained on the reactive red-120 resin were conducted as follows: in order to minimize the amount of the ∼30-kDa polypeptide loaded onto the RP-HPLC column, the sample was first acidified (0.1% TFA) and loaded on a C18 reversed-phase resin (Sep-Pak, Waters Corp., Milford, MA), and elution was performed with 45% acetonitrile in deionized water, 0.1% TFA (v/v). The eluate (containing low-molecular-mass peptides and low amounts of the ∼30-kDa polypeptide, see Results and Discussion) was vacuum-dried, recovered in 200 µL of deionized water, 0.1% TFA (v/v), and further subjected to HPLC on a RP column (C18-RP300, Brownlee Labs). The elution was performed at room temperature at a flow rate of 200 µL/min. Solvent A was deionized water, 0.1% TFA (v/v), and solvent B was 80% acetonitrile in deionized water, (30) Scha¨gger, H.; von Jagow, G. Anal. Biochem. 1987, 166, 368-379.

3048 Analytical Chemistry, Vol. 70, No. 14, July 15, 1998

0.09% TFA (v/v). The sample was injected at time 0, and buffer B was 1%. The gradient was from 1% B to 10% B in 1 min, then from 10% B to 60% B in 50 min, and finally from 60% B to 100% B in 10 min. Fractions were collected for further analysis by mass spectrometry and SDS-PAGE (see Figure 1). The experiments aimed at defining the molar ratio between the ∼3-kDa peptide and the ∼30-kDa polypeptide were conducted as follows: the sample (reactive red-120 column eluate) was acidified (0.1% TFA, v/v) and subjected to HPLC on a RP column. The sample was injected at time 0, and buffer B was 15%. The gradient was from 15% B to 45% B in 30 min, then from 45% B to 70% B in 50 min, and finally from 70% B to 100% B in 10 min. Fractions were collected for Edman degradation chemistry sequencing. Matrix Solutions Preparation. The preparation of the different 2,5-dihydrobenzoic acid (DHB, Sigma, St. Louis, MO) solutions was as follows: an initial solution of the DHB matrix was prepared in 40% acetonitrile/water, 0.1% TFA (v/v) at a concentration of 0.4 M. Four different matrix solutions of varying acetonitrile content (15%, 30%, 40%, and 60% acetonitrile) were prepared by diluting 4 times this initial solution with variable amounts of acetonitrile and water. These four matrix-containing eluent solutions enabled us to achieve a step-gradient microchromatography (see below) of peptides and polypeptides while simultaneously mixing the eluted molecular species with matrix. R-Cyano-4-hydroxycinnamic acid (ACHCA, Sigma) and 3,5dimethoxy-4-hydroxycinnamic acid (sinapinic acid, Aldrich, St. Louis, MO) were also used in this work and were prepared according to the same procedure as for DHB, except that the initial solution was a saturated solution at 40% acetonitrile. The unsolubilized matrix was pelleted, and 50 µL of the supernatant was used for subsequent dilutions. Sample Desalting for MALDI MS Analysis. Desalting of the sample was performed on the same micropurification device as described in the text. The sample was acidified to 0.1% TFA (v/v) and loaded onto the 0.3-µL microchromatography bed. However, no chromatographic separation was performed. After desalting of the sample, all the molecular species were eluted into an Eppendorf tube with a matrix-free eluent solution (8 µL of 80% acetonitrile-0.1% TFA (v/v) in water). The eluate was concentrated/evaporated, and the sample was then resolubilized and deposited according to the classical dried droplet procedure. Mass Spectrometry. MALDI time-of-flight (MALDI-TOF) spectra of the peptides and polypeptides were obtained with a Voyager-Elite Biospectrometry Workstation mass spectrometer (PerSeptive Biosystems Inc., Framingham, MA; laser pulse width ) 3 ns, laser wavelength ) 337 nm). All the analyses were performed with the DHB matrix, unless otherwise specified. Each spot was analyzed twice, by setting the parameters of the spectrometer such as to detect both low-molecular-mass peptides (LMM spectrometer settings, reflector and positive modes, accelerating voltage 20 kV, grid voltage 72%, extraction delay 100 ns, low-mass gate 500 Da) and high-molecular-mass polypeptides (HMM spectrometer settings, linear and positive modes, accelerating voltage 25 kV, grid voltage 95%, extraction delay 250 ns, low-mass gate 1000 Da). Spectra were calibrated externally using the [M + H]+ ions from different peptidessACTH (clip 7-38), average m/z 3660.17; ACTH (clip 18-39), monoisotopic

Figure 2. MALDI MS analysis of the reactive red-120 column eluate. Here, 0.1 µL of the reactive red-120 column eluate (∼150 fmol of the p29/30 and p3/4 fragments) was desalted and analyzed.

m/z 2465.2sand the [M + H]+ ions from horse apomyoglobin monomer and dimer (monomer, average m/z 16 952.5; dimer, average m/z 33 904). The laser energy required to desorb/ionize the calibrant molecules was used as the starting laser energy for subsequent mass spectrometry experiments. If desorption/ ionization could not be achieved using this threshold laser energy, this energy was progressively increased up to twice its initial value. All these calibrants were from Sigma. Edman Degradation Sequencing. Peptides were sequenced by automated Edman degradation using a model 794 protein sequencer (Perkin Elmer-Applied Biosystems Division, Foster City, CA). The amount of peptide/polypeptide loaded was typically 10 pmol. RESULTS AND DISCUSSION Two Ca2+ ATPase Fragments Might Bind Nucleotides. Coll and Murphy28 have demonstrated that detergent-solubilized Ca2+ ATPase can be purified on a reactive red-120 column and that the mechanism of purification is affinity chromatography, with the ATPase binding the reactive red-120 ligand in its nucleotide binding site. To purify the Ca2+ ATPase fragments that might bind nucleotides, a mixture of water-soluble proteolytic fragments of this protein was loaded onto a reactive red-120 column and eluted by ADP (see Experimental Section). Upon analysis of the eluate by SDS-PAGE, two major molecular species were observed, with apparent molecular masses of 30 (p29/30) and 3 kDa (p3/4), respectively (Figure 1, lane 2). Lane 1 of this gel shows the migration pattern obtained for the AspN supernatant. The p29/30 fragment, starting near S350 and ending near S610, corresponds to a domain which can be obtained by treatment of the sarcoplasmic reticulum vesicles with various proteases.26,27 However, p3/4 is a new proteolytic fragment that is probably retained on the reactive red-120 column because it binds to p29/30. To identify these two molecular species that were retained on the reactive red-120 column, we analyzed the eluate by MALDI-TOF mass spectrometry. Upon analysis of low amounts of this sample (after desalting, corresponding to 150 fmol of both the p29/30 and p3/4 molecular species), no signal could be obtained for the p29/30 polypeptide, while the p3/4 peptide generated reasonably intense signals (m/z 3464.14, Figure 2, HMM spectrometer settings). This finding was reminiscent of the commonly encountered spectral suppression phenomenon that is particularly evident when mass spectrometric analyses are performed on unresolved

Figure 3. Microchromatography device description. The microchromatography device was set up with use of a GelLoader tip, an Eppendorf tube, the resin, and a low-speed centrifuge. The elution is performed by use of a Pipetman, and the eluate is directly deposited onto the MALDI target (see text for details).

biopolymer samples, especially when large polypeptides are present along with low-molecular-mass peptides. Microchromatography of the Reactive Red-120 Eluate for Analysis by Mass Spectrometry. To diminish the spectral suppression phenomenon observed in the previous section of this report, we set up a microchromatography device that would allow us to perform a step-gradient chromatographic separation of very low amounts (