Combination of Peptide Profiling by Matrix-Assisted Laser Desorption

cell. Direct peptide profiling by MALDI-TOF MS on a single cHH-producing cell previously identified by immu- ..... presence of nitrocellulose.20 Good ...
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Anal. Chem. 1998, 70, 1805-1811

Combination of Peptide Profiling by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and Immunodetection on Single Glands or Cells Virginie Redeker,*,† Jean-Yves Toullec,‡ Joe 1 lle Vinh,† Jean Rossier,† and Daniel Soyez‡

Laboratoire de Neurobiologie, Ecole Supe´ rieure de Physique et de Chimie Industrielles de la Ville de Paris, CNRS UMR 7637, 10 rue Vauquelin, 75231 Paris Cedex 05, France, and Equipe Signaux et Re´ gulations Endocrines, Ecole Normale Supe´ rieure, CNRS EP 119, 46 rue d’Ulm, 75230 Paris Cedex 05, France

The combination of two sensitive and powerful analytical techniques on the same biological sample was examined: (i) matrix-assisted laser desorption/ionization timeof-flight mass spectrometry (MALDI-TOF MS),which gives informative peptide profiling on complex samples such as organs or cells; (ii) immunological tools such as enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry to probe for specific peptides in biological extracts or cells. The cellular expression of the two precursors of the hyperglycemic hormone (cHH) was analyzed in neurosecretory cells (30-µm diameter) from the crayfish Orconectes limosus. Neurohemal organs were used to optimize the sample preparation and to demonstrate that, after peptide fingerprinting by MALDITOF MS, the sample can be recovered from the MALDI plate for further immunological analysis by ELISA. It was also established that, after immunocytochemistry following 4% paraformaldehyde fixation of the organ, the stained tissue could be recovered for further MALDI-TOF MS analysis. This dual characterization was successfully scaled down to the level of a single crayfish neurosecretory cell. Direct peptide profiling by MALDI-TOF MS on a single cHH-producing cell previously identified by immunocytochemistry demonstrated that both procHH isoforms were expressed in each cell analyzed. During the past decade, matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been developed into a very sensitive technique for the determination of protein molecular mass and for the analysis of complex peptide mixtures within biological matrixes.1-4 MALDI-TOF MS offers a number of advantages over other mass spectrometric * Corresponding author: (Tel) (33) (0) 1.40.79.47.65; (fax) (33) (0) 1.40.79.47.57; (e-mail) [email protected]. † Ecole Supe ´ rieure de Physique et de Chimie Industrielles. ‡ Ecole Normale Supe ´ rieure. (1) Caroll, J. A.; Beavis, R. C. In Laser Desorption and Ablation; Miller, J. C., Haglund R. F., Jr., Eds; Academic Press: San Diego, 1996; Chapter 7. (2) Mann, M.; Talbo, G. Curr. Opin. Biotechnol. 1996, 7, 11-19. (3) Andersen, J. S.; Svensson, B.; Roepstorff, P. Nat. Biotechnol. 1996, 14, 449457. (4) Stults, J. T. Curr. Opin. Struct. Biol. 1995, 5, 691-698. S0003-2700(97)01309-7 CCC: $15.00 Published on Web 03/20/1998

© 1998 American Chemical Society

techniques, including the ability to analyze mixtures of peptides and proteins over a very broad mass range, a high sensitivity (from the picomole to the high-attomole range), and a high tolerance for contaminants (salts, buffers, and even some detergents). This ionization mode also leads to clear spectra with major singlecharged ions. Consequently, MALDI has been proven to be a powerful tool for the direct analysis of complex biological mixtures such as bacteria cells,5,6 small pieces of dissected tissues, and single giant neurons from the freshwater snail Lymnaea stagnalis which have cell bodies of 100-µm diameter.7-11 The ability to directly obtain a peptide fingerprint at the cellular level demonstrates the high performance of the MALDI-MS approach for the study of bioactive peptide synthesis and processing. Other sensitive tools developed for peptide studies include enzyme-linked immunosorbent assay (ELISA) and immunocytochemical studies, which are frequently employed to probe for polypeptides, for example, in the analysis of cell-specific hormone expression.12 Combination of both immunodetection and peptide profiling on the same cellular extract or on the same single cell provides a valuable approach to characterize polypeptide expression and processing in order to understand cellular diversity. In crustaceans, a number of neurohormones are synthesized by a cluster of 20-50-µm-diameter neurosecretory cells (the X-organ) located within the eyestalks.13 The axon of these cells (5) Claydon, M. A.; Davey, S. N.; Edwards-Jones, V.; Gordon, D. B. Nat. Biotechnol. 1996, 14, 1584-1986. (6) Holland, R. D.; Wilkes, J. G.; Rafii, F.; Sutherland, J. B.; Persons, C. C.; Voorhees, K. J.; Lay, J. O. Rapid Commun. Mass Spectrom. 1996, 10, 12271232. (7) van Veelen, P. A.; Jimenez, C. R.; Li, K. W.; Wildering, W. C.; Geraerts, W. P. M.; Tjaden, U. R.; van der Greef, J. Org. Mass Spectrom. 1993, 28, 15421546. (8) Jimenez, C. R.; van Veelen, P. A.; Li, K. W.; Wildering, W. C.; Geraerts, W. P.; Tjaden, U. R.; van der Greef, J. J Neurochem. 1994, 62, 404-407. (9) Li, K. W.; Hoek, R. M.; Smith, F.; Jimenez, C. R.; van de Schors, R. C.; van veelen, P. A.; Chen, S.; van der Greef, J.; Parish, D. C.; Benjamin, P. R.; Geraerts, W. P. M. J. Biol. Chem. 1994, 269, 30288-30292. (10) Van Strien, F. J.; Jespersen, S.; van der Greef, J.; Jenks, B. G.; Roubos E. W. FEBS Lett. 1996, 379, 165-170. (11) Jimenez, C. R.; Li, K. W.; Smit, A. B.; van Minnen, J.; Janse, C.; van Veelen, P.; Dreisewerd, K.; Zeng, J.; van der Greef, J.; Hillenkamp, F.; Karas, M.; Geraerts, W. P. M. In Mass Spectrometry in Biological Sciences; Burlingame, A. L., Carr, S. A., Eds; Humana Press. Totowa, NJ, 1996; pp 227-243. (12) Gilon, P.; Mallefet, J.; De Vriendt, C.; Pauwels, S.; Geffard, M.; Campistron, G.; Remacle, C. Histochemistry 1990, 93, 645-654.

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Figure 1. Organization of the preprocHH hormone and its related peptides. The preprohormone contains several domains flanked by proteolytical processing sites indicated by the vertical bars.Two isoforms of preprocHH have been identified. They differ by only one amino acid in the cPRP (cHH precursor-related peptide) sequence where a serine residue at position 33 is changed to asparagine. [M + H]+ average masses were calculated for each peptide released by the proteolytic cleavage. The signal peptide corresponding to the sequence MVSFRTMWSLVVVVVVASLASSGVQG has a mass of 2711.3 Da. The cPRP Ser and cPRP Asn peptides have the sequence RSVEGSSRMERLLSSGSSSSEPLSFLSQDQSVS where the underlined residue is Ser and Asn, respectively. cPRP Ser and Asn peptides have masses of 3518.8 and 3545.8 Da, respectively. The cHH peptide has a mass of 8401.7 Da corresponding to the sequence QVFDQACKGIYDRAIFKKLDRVCEDCYNLYRKPYVATTCRQNCYANSVFRQCLDDLLLIDVLDEYISGVQTV with the following posttranslational modifications: the N-terminal glutamine residue is modified to pyroglutamate, and there are three disulfide bonds and a C-terminal amidation.

terminates in a neurohemal organ (the sinus gland) where the neuropeptides are stored in large amounts (i.e., ∼50-100 pmol/ gland) before their release into the hemolymph. The major neurohormone stored in the sinus gland is the crustacean hyperglycemic hormone (cHH), a peptide of 72 amino acid residues which is involved in the control of homeostasis and the stress response via the regulation of blood glucose levels.14 Like many hormonal peptides, cHH is first synthesized as part of a preprohormone which is then processed by proteolytic cleavages during cellular transport (Figure 1). In the crayfish Orconectes limosus eyestalk, the X-organ contains ∼30 cHH-producing neurosecretory cells (30-µm diameter).15 cDNA cloning revealed the presence of two molecular variants of procHH.16 At the peptide precursor level, the two procHH variants differ by a single amino acid residue located at the C-terminus of the cHH precursor-related peptide (cPRP) flanking the cHH. The C-terminal amino acid of the cPRP peptide sequence, containing 33 amino acid residues, is either a serine or an asparagine residue. The theoretical average masses calculated for the corresponding protonated peptides are 3518.8 and 3545.8 amu, respectively. Although a number of biochemical and immunochemical studies have been devoted to the cHH-producing neurosecretory system of crustaceans,17,18 the question of the cellular specificity (13) Kleinholz, L.; Keller, R. In Hormones and Evolution; Barrington, E. J. W., Ed.; Academic Press: New York, 1979; pp 159-213. (14) Keller, R.; Sedlmeier, D. In Endocrinology of selected invertebrate types; Gilbert, L. I., Miller, T. A., Eds.; Springer-Verlag: New York, 1988; pp 315-326. (15) Gorgels-Kallen, J. L.; Van Herp, F.; Leuven, R. S. E. W. J. Morphol. 1982, 174, 161-168. (16) De Kleijn, D. P. V.; Janssen, K. P. C.; Martens, G. J. M.; Van Herp, F. Eur. J. Biochem. 1994, 224, 623-629. (17) Van Herp, F.; Kallen, J. L. In Comparative aspects of neuropeptides; Stefano, G. B., Florey, E., Eds.; Manchester University Press: Manchester, U.K., 1990; pp 361-364.

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of the cHH prohormone isoforms has never been addressed. Furthermore, this neurosecretory system offers a number of advantages for the development of combined MS and immunochemical methods, in situ: the same peptides exist either in large amounts in a compact tissular structure (the sinus gland) or in small amounts in discrete areas (X-organ cells). To determine whether both cHH precursors are expressed in the same neurosecretory cell, the presence of cPRPs in O. limosus neurosecretory cells was determined by direct peptide mass fingerprinting. Since the eyestalk neurosecretory system contains a number of different cell types, it was necessary to distinguish the cHH-producing cells from the others. This was achieved by immunohistochemical methods using anti-cHH antibodies after paraformaldehyde fixation. We first compared the efficiency of several matrixes and sample preparation methods for direct MALDI-TOF MS peptide profiling, of both single sinus gland and single X-organ extracts. Then we examined the possibility of performing an immunological detection by ELISA on samples that had been used for direct peptide profiling by MALDI-TOF MS. Sample recovery was performed with three different MALDI matrixes. Finally, we performed mass spectrometry analysis on single sinus glands and single cells that had been previously submitted to the immunohistochemical procedure (4% paraformaldehyde fixation and immunodetection). The combination of mass spectrometry and immunohistochemical analysis at the single-cell level revealed the coexistence of both cHH precursors in each single cHH-producing cell analyzed. EXPERIMENTAL SECTION Reagents. R-Cyano-4-hydroxycinnamic acid (R-CHCA), 2,5dihydroxybenzoic acid (DHB) from Sigma Chemicals (St. Louis, MO), and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, SA) from Aldrich were used as matrixes for MALDI experiments in the presence or absence of pure nitrocellulose (pore size 0.45 mm; Millipore Corp., HAHY, Bedford, MA). ACTH (18-39 and 7-38 clips), insulin, apomyoglobin, and angiotensin were purchased from Sigma Chemicals. AMDEX goat anti-rabbit IgG-alkaline phosphatase (ref V020302) was purchased from Amersham Life Science (Buckinghamshire, England). Normal goat serum, goat anti-rabbit peroxidase conjugate, p-nitrophenyl phosphate disodium salt (phosphatase substrate tablets), and diaminobenzidine (DAB) were from Sigma. Animals. Tissues were dissected from adult crayfish (Orconectes limosus) obtained from the Saoˆne river (France). The animals were maintained on a natural photoperiod in filtered, recirculated freshwater at 15 °C. Animals were anesthesized by cooling in ice, prior to eyestalk removal. The medulla terminalis containing the X-organ and the sinus gland were dissected for extract preparation or for immunohistochemical treatment before MALDI analysis. Mass Spectrometry. Instrumentation. The spectra were recorded in the linear mode (except in Figure 6, where the reflectron mode was used) on a MALDI-TOF mass spectrometer (Voyager Elite, Perseptive Biosystem, Inc. Framingham, MA) (18) Martin, G.; Sorokine, O.; Van Dorsselaer, A. Eur. J. Biochem. 1993, 211, 601-607.

equipped with a delayed extraction device.19 For laser desorption, a nitrogen laser beam (λ ) 337 nm, 3-ns-wide pulse at 20 Hz, laser power set just above the desorption threshold, diameter 100 µm) was focused on the target, and the ions were detected by the dual-channel plate linear detector after a flight length of 2 m. Delayed extraction time was set at 175 ns. A total of 150-256 shots were averaged for each acquired spectrum. External calibrations were performed with either a mixture of ACTH clip 18-39 and 7-38 with average m/z ratios corresponding to [M + H]+ of 2466.71 and 3660.17 Da, respectively, or a mixture of insulin and apomyoglobin with average m/z ratios corresponding to 2867.75 and 8476.75 Da, respectively, for the biprotonated ions and 5838.2 and 16952.5 Da, respectively, for the monoprotonated ions. Sample Preparation for Glands and Organ Extracts. The extracts were prepared as follows: one sinus gland (i.e., neurohemal organ) or one X-organ (containing 30 cHH-producing cells) was placed in 10 µL of acetonitrile/water 30:70 (v/v), 0.2% TFA and sonicated for 30-60 s. Different matrixes (R-CHCA, SA, DHB) were used with the dried droplet method, with the thinlayer method (with nitrocellulose), and with the sandwich method (with nitrocellulose).20 For the dried droplet method, 0.2-0.5 µL of the sample was mixed with the same volume of saturated matrix solution (R-CHCA, SA, DHB). R-CHCA and SA were saturated solutions in acetonitrile/water 30:70 (v/v), 0.1% TFA, and DHB was saturated in 0.1% TFA. For the thin-layer method, R-CHCA and SA were prepared and used as decribed previously,21 and 0.20.5 µL of the sample was spotted on the matrix layer. In the sandwich method, a saturated solution of R-CHCA or SA in acetone/water in a 99:1 (v/v) ratio was mixed with a solution of 40 mg/mL nitrocellulose in pure acetone in a 1:1 (v/v) ratio. After this solution was mixed with 2-propanol in a 1:1 (v/v) ratio, 0.5 µL of this mixture was deposited on the target plate to obtain a thin layer. Between 0.2 and 0.5 µL of the acidified sample solution was spotted on the matrix layer and dried. A 0.5-µL sample of a second layer of matrix (a saturated solution of R-CHCA or SA in acetonitrile/water 1:1 (v/v), 0.1% TFA was then added. Just before complete crystallization, the sample was washed with 1020 µL of cold 0.1% aqueous TFA. Sample Preparation for MALDI after Fixation and Immunodetection. Cells from a single X-organ previously characterized by immunohistochemistry were dissociated by adding trypsin to the PBS buffer for 30 min at room temperature. After manual dissociation, the labeled cells containing cHH peptides were aspired into a thin glass capillary and deposited on the stainless steel target. The excess of PBS was removed with the same capillary. After a quick wash with 1 µL of a saturated solution of DHB22 in 0.1% TFA, a 0.5-µL drop of the same matrix solution was added and the resultant mixture was dried at room temperature. A second 0.5-µL drop of matrix was then added and dried (19) Vestal, M. L.; Juhasz, P.; Martin, S. A. Rapid Commun. Mass Spectrom. 1995, 9, 1044-1050. (20) Roepstorff, P.; Nielsen, H. R.; Larsen, M. R.; Haebel, S.; Jensen, C.; Palm, L.; Krogh, T.; Mirgorodskaya, E.; Nordhoff, E.; Kussman, M. Proceedings of the 44th ASMS Conference on Mass Spectrometry and Allied Topics (Portland, OR) 1996; p 1357. (21) Vinh, J.; Loyaux, D.; Redeker, V.; Rossier, J. Anal. Chem. 1997, 69, 39793985. (22) Strupat, K.; Karas, M.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1991, 111, 89-102.

in the same way. The target was then immediately inserted in the mass spectrometer. Sample Recovery for ELISA after MALDI Analysis. After MALDI-TOF analysis by at least two acquisitions of 256 laser shots, each sample spot deposited with the dried droplet method was redissolved in 1-1.5 µL of one of the following solutions: water or 0.1% TFA for the sample in DHB, and acetonitrile/water 50:50 or 30:70 (v/v), 0.1% TFA for the samples in R-CHCA or SA. The solution was deposited in a microtitration plate well and the spot was washed again twice with 1-1.5 µL of solution. The standards used for the MALDI-TOF analysis were recovered from the target and used as a negative control for the ELISA. Aliquots of the same volume of extract used for MALDI analysis were spotted directly on the ELISA plate, as a positive control. Direct ELISA. Detection of immunoreactivity in the MALDI samples was performed by direct ELISA. After drying the microtitration plate by vacuum, 100 µL of coating buffer (sodium bicarbonate buffer, 0.1 M, pH 9.6) was added to each well and incubation steps were performed as described in Meusy and Soyez.23 The primary serum was a rabbit anti-O. limosus cHH antiserum24 diluted at a 1:1000 ratio, and the secondary antiserum was a polymeric alkaline phosphatase conjugate (goat anti-rabbit IgG-alkaline phosphatase) used at a 1:1000 dilution. Immunohistochemical Methods. Medulla terminalis containing the X-organs and the sinus glands were fixed in a 4% paraformaldehyde solution in PBS (0.01 M, pH 7.2) overnight at 4 °C. The anti-O. limosus cHH antiserum was diluted at a 1:100 ratio in PBS (0.01 M) containing 5% normal goat serum (NGS) and 0.5% Triton X-100. After several washes in PBS, the whole medulla was incubated in this solution. After 16-18 h, tissues were removed and washed in PBS/NGS/Triton before incubating overnight with a secondary goat anti-rabbit peroxidase conjugate antibody diluted at a 1:500 ratio in PBS/NGS/Triton. Then, the medulla were carefully washed in PBS. Detection of peroxidase activity was obtained with DAB, which produces a brown-black stain. All of these steps were performed at room temperature. After staining, the samples were kept in PBS at 4 °C until further analysis. RESULTS AND DISCUSSION Matrix Comparison for the Peptide Profiling by MALDITOF MS. We have compared the efficiency of desorption/ ionization and detection of the procHH-related products (cHH and cPRPs, Figure 1) using three different matrixes: R-cyanohydroxycinnamic acid, sinapinic acid, and dihydroxybenzoic acid. Three sample preparation methods were used: the dried droplet method, and both the thin-layer and the sandwich preparations in the presence of nitrocellulose.20 Good signal-to-noise ratios were obtained with the dried droplet preparation both with SA and with DHB and with the sandwich preparation both with R-CHCA/RCHCA and with R-CHCA/SA. Representative spectra of cellular extracts prepared with different matrixes are shown (Figure 2). Though the dried droplet preparation in SA was especially sensitive for the cHH at 8401.7 Da in Figure 2a, the best peptide maps were obtained in DHB, with a complete coverage of the analyzed mass range (500-10 000 Da) (Figure 2b). With R-CHCA, (23) Meusy, J. J.; Soyez, D. Gen. Comp. Endocrinol. 1991, 81, 410-418. (24) Keller, R. In Immunological Techniques in Insect Biology; Gilbert, L. I., Miller, T. A., Eds.; Springer-Verlag: New York; 1988; pp 253-271.

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Figure 3. MALDI-TOF MS spectrum of a single X-organ extract. A single X-organ was sonicated in 10 µL of solvent and 0.3 µL was analyzed with the dried droplet method in DHB. The volume analyzed corresponds to only one cHH expressing cell. The peptide profiling was obtained over a large mass range: (a) from 1500 to 5500 Da and (b) from 5500 to 15 000 Da. The spectrum confirms the presence of the cPRP Ser and Asn peptides as well as the cHH peptide. The respective experimental masses were 3519.11, 3546.22, and 8401.68 Da. Several unidentified peaks are observed.

Figure 2. MALDI-TOF MS spectra of a sinus gland extract analyzed with different matrixes and sample preparations. One sinus gland was sonicated in 10 µL of solvant and 0.3 µL was analyzed with each of the following sample preparations: (a) dried droplet SA, (b) dried droplet DHB, (c) dried droplet R-CHCA, (d) sandwich R-CHCA/ R-CHCA, and (e) sandwich SA/ R-CHCA. The solvent and other parameters used are described in the Experimental Section.

the peptide mapping also covered the complete analyzed mass range, but the signal was much weaker than with either DHB or SA (Figure 2c). The thin-layer method was tested but it did not significantly increase the signal (data not shown). The sandwich method gave better results, with the R-CHCA/R-CHCA mixture giving stronger signals than the R-CHCA/SA preparation (Figure 2d,e). To our knowledge, a direct peptide fingerprinting of a crustacean neurohemal organ by mass spectrometry has not been reported previously. Figure 2 shows a large number of ions corresponding to masses that cannot be related to known sinus gland peptides. Some of these unknown peptides may correspond to unidentified neurohormones. The dried droplet method with DHB gave the best results in the linear MALDI-TOF MS analysis. Consequently, it was selected for further analysis of single glands and cells. This sample preparation was first used to analyze a single X-organ. Since one X-organ was resuspended and sonicated in 10 µL of solution (see Experimental Section), the sample (0.2-0.5 µL) deposited on the target was estimated to roughly correspond to 0.6-1.5 cHH cells. From HPLC purification data (not shown), the amount of cHH 1808 Analytical Chemistry, Vol. 70, No. 9, May 1, 1998

was evaluated to be close to 40 pmol in one sinus gland, where the hormones are stored in large amounts before release. This amount corresponds to at least 6000-fold more cHH than in one cHH cell. Thus, one single cHH cell contains approximately 1-5 fmol of cHH. The very high sensitivity of MALDI-TOF MS allowed the peptide fingerprinting of a single cHH cell of 30 µm in diameter, containing only a few femtomoles of cHH. The peptide profiling obtained from the X-organ extract showed a number of peptides ranging in mass from 1500 to 15 000 Da (Figure 3). A comparison of the full spectra of peptides obtained from the sinus gland and X-organ extracts (Figures 2b and 3) reveals some differences in peptide content. Peptides corresponding to procHHs at ∼12 200 Da were never identified in the samples analyzed. These data, together with previous results obtained by direct MALDI-MS mapping of tissues which showed induction and repression of peptide expression,25 offer interesting perspectives for the fine analysis of hormone synthesis and release at the organ or cellular level. Peptide Recovery Following Peptide Profiling by MALDIMS. Cerpa-Poljak et al.26 have reported previously that the residual analyte after MALDI analysis could be recovered for radioimmunoassay (RIA) analysis. Using a peptide sample prepared with the dried droplet method in R-CHCA, these authors showed that the amount of sample consumed during the MALDIMS analysis was negligible and that the unconsumed sample retained full immunoreactivity for further radioimmunoassay analysis. In the present study, the possibility of recovering samples from different matrixes for further immunoassay was studied at the (25) Uttenweiler-Joseph, S.; Moniatte, M.; Lambert, J.; Van Dorsselaer, A.; Bulet, P. Anal. Biochem. 1997, 247, 366-375. (26) Cerpa-Poljak, A.; Jenkins, A.; Duncan, M. W. Rapid Commun. Mass Spectrom. 1995, 9, 233-239.

Figure 4. Effect of MALDI matrixes on peptide immunoreactivity determined by ELISA. Each sample contained the same amount of peptide (2 × 10-4 sinus gland equivalent) and was analyzed with the following: (Ca) 50% acetonitrile in 0.1% aqueous TFA; (Cw) 0.1% TFA; (DHBw) DHB in 0.1% aqueous TFA; (DHBa) DHB in 50% acetonitrile, 0.1% aqueous TFA; (CHCA) CHCA in 50% acetonitrile, 0.1% aqueous TFA; (SA) SA in 50% acetonitrile, 0.1% aqueous TFA. Each bar represents the mean of two measurements. The optical density at 405 nm was measured 30 min after the addition of the phosphatase substrate.

cellular level. Recovery was performed on samples containing similar volumes of the same sinus gland or X-organ extract and tested with the three different matrixes commonly used for MALDI-TOF MS experiments: DHB, R-CHCA, and SA. The immunodetection was done by direct ELISA using the anti-O. limosus cHH antiserum. Analysis of organ extracts and cells showed that the results of the ELISA were related to the signal obtained for the cHH and/ or the cPRP peptides in the MALDI spectra: when no cPRPs were detected by MALDI, the ELISA consistently gave negative results and vice versa. Comparison of the ELISA results from samples containing the same peptide amount but mixed with the different matrixes indicated that the recovery from preparations with SA or R-CHCA was very high (about 80-120% of the controls), whereas the immunoreactivity was much lower with DHB (2040% of the controls) (Figure 4). The use of 50% acetonitrile improved both the resolubilization of the peptide sample and the binding of the peptide to the ELISA plate, particularly for the DHB sample (Figure 4). Moreover, it also allowed the recovery of sandwich preparations (data not shown). Thus, acetonitrile should be used for the recovery of samples whatever matrix is used. No effect of the matrix was observed on control peptides analyzed in the presence or in the absence of matrix. These results show that an efficient recovery of samples for subsequent immunological analysis after MALDI recording is possible with a number of commonly used matrixes. This kind of approach may offer not only qualitative information but also interesting quantitative results that are generally difficult to ascertain by MALDI analysis alone. Peptide Profiling Following Immunohistochemistry. The feasability of direct MALDI-TOF analysis after immunohistochemical analysis was investigated. Immunohistochemical treatment includes the fixation of the tissues with 4% paraformaldehyde and the use of enzymes (peroxidase or phosphatase) for immunodetection (see Experimental Section). Among the different matrix

Figure 5. Comparison of the peptide profiling obtained from (a) a fresh sinus gland and (b) a sinus gland after immunocytochemical treatment. The sinus glands were directly deposited onto the MALDI target and analyzed by MALDI-TOF MS after the addition of DHB. Although the relative intensity of some peaks appears to be different, qualitative analysis shows that most of the peptides are still present after immunocytochemical treatment. The major changes are (i) the absence of cHH after fixation and immunodetection and (ii) the apparition of mass increments of 12 Da clearly observed in the cPRP peptides.

preparations that gave a good signal with the cell extracts, the dried droplet method offered one major advantage: it was possible to increase stepwise the amount of matrix deposited and to homogenize the sample spot several times with a pipet at each matrix addition. This procedure together with the DHB matrix gave the best signal-to-noise ratio with clear peptide peaks. This may be due to the efficient breakage of the cells during the successive matrix depositions and to the high concentration of the analyte in the matrix crystal needles. With the dried droplet preparation in DHB, we obtained informative results by the direct analysis of samples at both the single-gland level and the singlecell level. The peptide profiling obtained for a sinus gland after histochemical fixation with 4% paraformaldehyde and immunodetection by peroxidase was compared to the peptide profiling obtained for a fresh untreated sinus gland (Figure 5). The immunocytochemical treatment did not drastically affect the peptide fingerprinting obtained by MALDI-TOF MS since a number of peptides were found in both conditions though the glands were from two different animals (Figure 5). However, one clear difference was observed for the cHH peak at 8401.7 Da; it was not detected after the immunohistochemical treatment. This observation could be explained by several hypotheses involving different steps of the immunohistochemical treatment that could be critical for the detection of the cHH peptide: (i) the formation of a large complex composed of the cHH peptide and the antibodies (the anti-cHH antibody and the secondary antibody) could have induced a sterical hindrance that retained the cHH inside of the cell; (ii) the presence of a noncovalent complex may not have been detected by the MALDI conditions used, since they were adapted neither to high masses nor to noncovalent complexes analysis;27 (iii) the cHH peptide may have been modified by both the fixation and the immunodetection. However, after (27) Moniatte, M.; van der Goot, F. G.; Buckley, J. T.; Pattus, F.; van Dorsselaer, A. FEBS Lett. 1996, 384, 269-272.

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Figure 7. Photograph of a DHB sample spot after crystallization of the matrix (2-mm diameter) containing a single cHH expressing cell (30-µm diameter) indicated by an arrow. The MALDI spot was obtained by twice adding 0.5 µL of DHB solution to the cell. An enlargement of the cell is presented in the lower part of the figure.

Figure 6. Comparison, in MALDI-TOF MS reflectron mode, of peptides before (a, c) and after (b, d) paraformaldehyde incubation. Both cPRP peptides Ser and Asn were analyzed directly from (a) a fresh sinus gland and (b) a sinus gland after immunocytochemical treatment. Two mass increments of 12 Da were observed after treatment. Angiotensin I was analyzed (c) without any treatment and (d) after overnight incubation in a 4% paraformaldehyde/PBS solution at 4 °C, corresponding to the conditions used for the paraformaldehyde fixation of the tissue. For angiotensin I (d), two mass increments of 12 Da were also observed.

immunodetection of the cHH peptides, the absence of cHH detection in the MALDI analysis did not pose a problem since the latter was essentially based on the detection of the cPRP peptides. A second difference was observed for a number of peptides and particularly for the major cPRP peptides presenting mass increments of 12 Da (Figure 5b). This increment of 12 Da, observed in linear mode and confirmed in the reflectron mode (Figures 5 and 6), suggested that a chemical modification consisting of the addition of one carbon atom to the molecule occurred during the immunocytochemical procedure. Indeed, the 4% paraformaldehyde fixation step, performed in a salt buffer, offers favorable conditions for a chemical reaction between free primary amine groups and the formaldehyde molecules present 1810 Analytical Chemistry, Vol. 70, No. 9, May 1, 1998

in the paraformaldehyde solution. This reaction, known to be activated by salt addition,28 results in the chemical modification of amine groups in imines and leads to a 12-Da increase in mass. This hypothesis was confirmed by the incubation of a number of synthetic peptides commonly used as standards for MALDI-TOF MS analysis (angiotensin I, ACTH clip 18-39, neurotensin, substance P) in 4% paraformaldehyde solution, both in PBS buffer and in water. This incubation resulted in 12-Da mass increments (from one to three according to the peptide analyzed). These increases in mass were considerably enhanced in the presence of PBS buffer (see spectrum obtained for angiotensin in Figure 6d). By postsource decay MALDI-TOF MS analysis29 of the modified peptides, the chemical addition of 12 Da was localized to the N-terminal residue (data not shown). When several 12-Da units were added, it was observed that the lateral amino groups of the arginine and lysine residues could also be modified. One should therefore take into account this chemical modification when interpreting MALDI-TOF MS peptide profiling obtained from paraformaldehyde-fixed tissues. Application to the Double Characterization of Single Cells. The same procedure was then applied to the analysis of single cells prepared by the dissociation of X-organ tissues previously fixed with 4% paraformaldehyde and immunostained. After this treatment, it was possible to sort the cells under a dissecting microscope, because the red coloration exclusively marked the cHH-producing cells. After dissection and sorting, one single cHH positive cell was deposited with a glass capillary onto the dried target. The cell stuck to the metallic target, so that it was possible to wash it with 1 µL of DHB solution as described by Garden et al.30 This rinsing step was necessary to eliminate the sodium and the phosphate present in the dissociation buffer, prior to matrix (28) Carey, A.; Sundberg, R. J. Advanced Organic Chemistry, 3rd ed.; Plenum Press: New York, 1990; Part B, p 31. (29) Kaufman, R.; Spengler, B.; Lu ¨tzenkirchen, F. Rapid Commun. Mass Spectrom. 1993, 7, 902-910.

Figure 8. MALDI-TOF spectrum of a single cHH expressing cell. After paraformaldehyde fixation and immunolabeling with the specific anti-cHH antibody, the tissue was dissected and the cells were dissociated. The red labeled cells corresponding to cHH expressing cells were identified and sorted. The spectrum shows that in one single cHH cell both isoforms of the procHH are expressed since both cPRP Ser and Asn peptides are detected.

deposition. A 0.5-µL aliquot of DHB solution was then added twice to the cell, and the mixture was homogeneized with a pipet and dried at room temperature. A photograph of a typical single cell preparation is shown in Figure 7. The cell is surrounded by DHB crystal needles. MALDI analysis gave a significant peptide profiling as presented in Figure 8. For the cPRP peptides, the same pattern was obtained consistently in at least five cells. The analysis of single cells with the different MALDI matrixes was performed directly after dissection and showed that the only significant signal was obtained with DHB. The direct analysis of peptides has previously been done on large single neurons and tissues from snails9,11 and marine molluscs.30 In these studies, the cells analyzed had a much larger diameter (∼100 µm) than the 30-µm-diameter cHH cells used in the present work. Moreover, in the current study, single sinus glands as well as single cells have been successfully analyzed by mass spectrometry even after immunohistochemical treatment. This is, to our knowledge, the first report of peptide profiling by MALDI analysis after immunohistochemical analysis involving paraformaldehyde fixation and immunostaining. (30) Garden, R. W.; Moroz, L. L.; Moroz, T. P.; Shippy, S. A.; Sweedler, J. V. J Mass Spectrom. 1996, 31, 1126-1130.

By the development of a combination of two powerful analytical chemistry tools, we have successfully characterized a single neurosecretory cell in two complementary ways. First immunocytochemistry was used to identify and select the cHH-containing cells and then MALDI-TOF MS was used to record directly the peptide profiling of a single cHH cell. Detection of both cPRP peptides in the same cell demonstrates unambiguously that the cell produces both procHH isoforms. This study demonstrates that peptide mass fingerprinting by MALDI-MS can be used in combination with immunological characterization not only on the same organ or on the same cell preparation but also on a single cell. Direct MALDI mass spectrometry analysis of single cells offers a valuable approach to gain insight into the full spectrum of neuropeptide diversity which is closely related to cellular diversity. The ability to compare the peptide content of a sample with its immunological characterization using specific antibodies offers a powerful approach for the chemical analysis of cellular diversity at the single-cell level. ACKNOWLEDGMENT This work was supported by the Association pour la Recherche contre le Cancer (ARC, France). J.V. received a predoctoral fellowship from both the Centre National de la Recherche Scientifique (CNRS, France) and Synthe´labo Recherche (France) and support from the Association pour le De´veloppement de la Formation par la Recherche Biome´dicale (ADFRB, France). Thanks to F. Maurel for his judicious advice about the chemical modification induced by paraformaldehyde, and to Pr. R. Lafont and J.P. Le Caer for critical reading of the manuscript. Thanks to Jennifer Brunton, Jim Porter, and Anthony Frankfurter for critical corrections of the manuscript. Thanks to Prof. R. Keller (University of Bonn, Germany) for the generous gift of the anti-cHH antiserum.

Received for review December 4, 1997. February 11, 1998.

Accepted

AC971309C

Analytical Chemistry, Vol. 70, No. 9, May 1, 1998

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