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In an extension of previous work by Li and co-workers (Kutz, K. K.; Schmidt, J. J.; Li, L. Anal. Chem. 2004, 76, 5630−5640), and with a focus on the...
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Anal. Chem. 2005, 77, 3594-3606

Matrix-Assisted Laser Desorption/Ionization Fourier Transform Mass Spectrometry for the Identification of Orcokinin Neuropeptides in Crustaceans Using Metastable Decay and Sustained Off-Resonance Irradiation Elizabeth A. Stemmler,*,† Heather L. Provencher,† Maureen E. Guiney,† Noah P. Gardner,† and Patsy S. Dickinson‡

Department of Chemistry and Department of Biology, Bowdoin College, Brunswick, Maine 04011

Vacuum UV matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance mass spectrometry (FTMS) has been applied to the direct analysis of crustacean neuronal tissues using in-cell accumulation techniques to improve sensitivity. In an extension of previous work by Li and co-workers (Kutz, K. K.; Schmidt, J. J.; Li, L. Anal. Chem. 2004, 76, 5630-5640), and with a focus on the Maine lobster, Homarus americanus, we report that many peaks appearing in direct tissue spectra from crustaceans result from the metastable decay of aspartate-containing neuropeptides with localized protonation sites. We report on mass spectral characteristics of crustacean neuropeptides under MALDI-FTMS conditions and show how fragments formed by Asp-Xxx cleavages can be used to advantage for the identification of orcokinin peptides, a ubiquitous family of crustacean neuropeptides with a highly conserved N-terminus sequence. We show that predicted fragment ion fingerprints (FIFs) can be used to screen internally calibrated direct tissue spectra to provide highconfidence identification of previously identified orcokinin peptides. We use FIFs, identified based upon characteristic neutral losses, to screen for new members of the orcokinin family. Sustained off-resonance irradiation of y-series fragment ions is used to sequence the variable C-terminus. We apply these techniques to the analysis of CoG tissues from Cancer borealis and Panulirus interruptus and show that orcokinins in P. interruptus were misidentified in a previous MALDI-TOF study. Neuropeptides play an important role in the modulation of vertebrate and invertebrate nervous systems. To better understand the physiological and biochemical role of these chemical messengers and modulators, more complete information is needed regarding the range of neuropeptides present in nervous systems, their location within neuronal cells, tissues, and nerves, and inter* To whom correspondence should be addressed. Facsimile: (207) 725-3017. E-mail: [email protected]. † Department of Chemistry. ‡ Department of Biology.

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or intraspecies variations in peptide identity or sequence.1 The detection, identification, and spatial localization of neuropeptides has been significantly advanced through the use of a variety of mass spectrometric techniques,1-5 with matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry most commonly applied to direct peptide profiling of tissue and single-cell samples. When MALDI is used as the ionization technique, tissues can be analyzed immediately after dissection, which minimizes analyte degradation or chemical alterations that may occur as a result of extractive isolation procedures.3 Even when impurities reach problematic levels, as in marine invertebrate tissues with high extracellular salt concentrations, simple tissue washing procedures have been developed to circumvent this problem.3,6 By appropriate selection of the MALDI matrix solvent, on-probe peptide extraction from tissues occurs as part of the matrix crystallization process7 to produce samples with peptide- and lipid-rich regions.8 By selectively ionizing in a region near, but spatially resolved from, the tissue it is possible to determine neuropeptides with minimal interference from cell phospholipids.8 Using MALDI-TOF with postsource decay (PSD)9,10 or other collision-induced dissociation (CID) techniques,11,12 peptides in tissue extracts or those directly (1) Dahlgren, R. L.; Page, J. S.; Sweedler, J. V. Anal. Chim. Acta 1999, 400, 13-26. (2) van Veelen, P. A.; Jimenez, C. R.; Li, K. W.; Wildering, W. C.; Geraerts, W. P. M.; Tjaden, U. R.; Vandergreef, J. Org. Mass Spectrom. 1993, 28, 15421546. (3) Li, L.; Garden, R. W.; Sweedler, J. V. Trends Biotechnol. 2000, 18, 151160. (4) Jime´nez, 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. (5) Chaurand, P.; Schwartz, S. A.; Caprioli, R. M. Anal. Chem. 2004, 76, 86A93A. (6) Garden, R. W.; Moroz, L. L.; Moroz, T. P.; Shippy, S. A.; Sweedler, J. V. J. Mass Spectrom. 1996, 31, 1126-1130. (7) Li, L.; Romanova, E. V.; Rubakhin, S. S.; Alexeeva, V.; Weiss, K. R.; Vilim, F. S.; Sweedler, J. V. Anal. Chem. 2000, 72, 3867-3874. (8) Garden, R. W.; Sweedler, J. V. Anal. Chem. 2000, 72, 30-36. (9) Li, L.; Garden, R. W.; Romanova, E. V.; Sweedler, J. V. Anal. Chem. 1999, 71, 5451-5458. (10) Jespersen, S.; Chaurand, P.; van Strien, F. J.; Spengler, B.; van der Greef, J. Anal. Chem. 1999, 71, 660-666. (11) Li, K. W.; Kingston, R.; Dreisewerd, K.; Jime´nez, C. R.; van der Schors, R. C.; Bateman, R. H.; Geraerts, W. P. M. Anal. Chem. 1997, 69, 563-565. 10.1021/ac0502347 CCC: $30.25

© 2005 American Chemical Society Published on Web 05/03/2005

desorbed from tissues or single cells have been characterized by full or partial sequencing. The stomatogastric nervous system (STNS) of decapod crustaceans has served as an ideal system to study the activity of central pattern generators,13,14 which control rhythmic motor movements such as respiration and locomotion. The relatively simple STNS is composed of four ganglia, the stomatogastric ganglion (STG), the paired commissural ganglia (CoG), and the oesophageal ganglion together with connecting and motor nerves. The system is also exposed to peptides through neurohormones released by neurohaemal organs, including the X-organ/sinus gland (SG) complex in the eyestalk, the pericardial organs, and the postcommissural organ.13 Using the STNS, physiological studies have probed the effects of many neuropeptides, including proctolin,15 RPCH,16,17 and CabTRP.18,19 While many peptides have been initially localized to the STNS using immunocytochemistry, MALDI-TOF has played an important role in the identification of neuropeptides in the STNS of decapod crustaceans.20-23 The orcokinins are a family of conserved peptides in crustaceans,24-27 with the first identified member, NFDEIDRSGFGFN, named orcokinin after being isolated from the crayfish, Orconectes limosus.26 Other family members have been identified in tissue extracts20,28,29 and in the direct analysis of tissue samples.20-22 Immunocytochemical studies20-22,26 have shown that members of this family are found throughout the STNS. The recent isolation of a new orcokinin from the cockroach, Blattella germanica, and identification of noncrustacean proteins homologous to two orcokinin precursor proteins30 suggests that orcokinins may be more widely distributed. Table 1 contains a listing of orcokinin and orcomyotropin-related peptides detected in the lobster, H. americanus. In crustaceans and the cockroach, the N-terminus sequence of this family is conserved, with variations occurring most commonly at the C-terminus sequence at residue 13. Variations at residues 8 and 9 have also been reported. A (12) El Filali, Z.; Hornshaw, M.; Smit, A. B.; Li, K. W. Anal. Chem. 2003, 75, 2996-3000. (13) Skiebe, P. J. Exp. Biol. 2001, 204, 2035-2048. (14) Marder, E.; Bucher, D. Curr. Biol. 2001, 11, R986-R996. (15) Hooper, S. L.; Marder, E. J. Neurosci. 1987, 7, 2097-2112. (16) Dickinson, P. S.; Marder, E. J. Neurophysiol. 1989, 61, 833-844. (17) Dickinson, P. S.; Hauptman, J.; Hetling, J.; Mahadevan, A. J. Neurophysiol. 2001, 85, 1424-1435. (18) Christie, A. E.; Lundquist, C. T.; Nassel, D. R.; Nusbaum, M. P. J. Exp. Biol. 1997, 200, 2279-2294. (19) Thirumalai, V.; Marder, E. J. Neurosci. 2002, 22, 1874-1882. (20) Li, L.; Pulver, S. R.; Kelley, W. P.; Thirumalai, V.; Sweedler, J. V.; Marder, E. J. Comp. Neurol. 2002, 444, 227-244. (21) Skiebe, P.; Dreger, M.; Meseke, M.; Evers, J. F.; Hucho, F. J. Comp. Neurol. 2002, 444, 245-259. (22) Skiebe, P.; Dreger, M.; Borner, J.; Meseke, M.; Weckwerth, W. Cell. Mol. Biol. 2003, 49, 851-871. (23) Li, L.; Kelley, W. P.; Billimoria, C. P.; Christie, A. E.; Pulver, S. R.; Sweedler, J. V.; Marder, E. J. Neurochem. 2003, 87, 642-656. (24) Stangier, J.; Hilbich, C.; Burdzik, S.; Keller, R. Peptides 1992, 13, 859864. (25) Bungart, D.; Dircksen, H.; Keller, R. Peptides 1994, 15, 393-400. (26) Dircksen, H.; Burdzik, S.; Sauter, A.; Keller, R. J. Exp. Biol. 2000, 203, 2807-2818. (27) Yasuda-Kamatani, Y.; Yasuda, A. Gen. Comp. Endocrinol. 2000, 118, 161172. (28) Bungart, D.; Hilbich, C.; Dircksen, H.; Keller, R. Peptides 1995, 16, 6772. (29) Huybrechts, J.; Nusbaum, M. P.; Bosch, L. V.; Baggerman, G.; De Loof, A.; Schoofs, L. Biochem. Biophys. Res. Commun. 2003, 308, 535-544. (30) Pascual, N.; Castresana, J.; Valero, M. L.; Andreu, D.; Belles, X. Insect Biochem. Mol. Biol. 2004, 34, 1141-1146.

Table 1. Orcokinin and Orcomyotropin-Related Peptides Previously Reported in the STNS of H. Americanus

peptide

sequence

expected mass, [M + H]+

[His13]-orcokinin22

NFDEIDRSGFGFH NFDEIDRSGFGFN NFDEIDRSGFGFV NFDEIDRSGFGF NFDEIDRSGFG FDAFTTGFGHN (XXX)DMDR[I/L]GFGFNa

1540.681 53 1517.665 55 1502.691 03 1403.622 62 1256.554 21 1213.527 26 1474.626 72

(1) (2) [Asn13]-orcokinin20,22 (3) [Val13]-orcokinin20,22 (4) Orc [1-12] 20,22 (5) Orc [1-11]20,22 (6) FDAFTTGFGHN22 (7) Hom-orcokinin22 a

Sequence reported by Skiebe et al.22

summary of currently identified orcokinin peptides in crustaceans has been published by Skiebe et al.22 Structure-activity studies,28 directed at orcokinin and analogues, showed that activity in the hindgut bioassay for O. limosus was completely lost when more than one amino acid at the N-terminus was removed. Changes at the C-terminus showed smaller effects, and C-terminus amidation resulted in no change in activity. The C-terminus variability exhibited by this peptide family and the limited ability of immunocytochemistry to distinguish structurally similar peptides makes mass spectrometry an important technique for the characterization and detection of orcokinin peptides. Compared with other mass spectrometric techniques, Fourier transform ion cyclotron resonance mass spectrometry (FTMS)31 provides the highest resolution,32,33 critical for revealing overlapping peaks in the analysis of complex mixtures. Mass accuracy is also high (errors in the low- to sub-parts-per million range34,35), with internal calibration techniques36,37 and other approaches used to compensate for space charge effects. Furthermore, these measurements can be made with high sensitivity (attomole detection),38-40 assisted by the use of external41,42 or internal36,37 ion accumulation techniques. FTMS permits multistage CID experiments using a range of activation methods, including sustained off-resonance irradiation (SORI),43,44 for structural characterization. Notably, CID experiments can be carried out with (31) Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S. Mass Spectrom. Rev. 1998, 17, 1-35. (32) He, F.; Hendrickson, C. L.; Marshall, A. G. Anal. Chem. 2001, 73, 647650. (33) Bossio, R. E.; Marshall, A. G. Anal. Chem. 2002, 74, 1674-1679. (34) Easterling, M. L.; Mize, T. H.; Amster, I. J. Anal. Chem. 1999, 71, 624632. (35) Li, Y.; McIver, R. T., Jr.; Hunter, R. L. Anal. Chem. 1994, 66, 2077-2083. (36) O’Connor, P. B.; Costello, C. E. Anal. Chem. 2000, 72, 5881-5885. (37) Mize, T. H.; Amster, I. J. Anal. Chem. 2000, 72, 5886-5891. (38) Moyer, S. C.; Budnik, B. A.; Pittman, J. L.; Costello, C. E.; O’Connor, P. B. Anal. Chem. 2003, 75, 6449-6454. (39) Solouki, T.; Marto, J. A.; White, F. M.; Guan, S.; Marshall, A. G. Anal. Chem. 1995, 67, 4139-4144. (40) Baykut, G.; Jertz, R.; Witt, M. Rapid Commun. Mass Spectrom. 2000, 14, 1238-1247. (41) O’Connor, P. B.; Budnik, B. A.; Ivleva, V. B.; Kaur, P.; Moyer, S. C.; Pittman, J. L.; Costello, C. E. J. Am. Soc. Mass Spectrom. 2004, 15, 128-132. (42) Brock, A.; Horn, D. M.; Peters, E. C.; Shaw, C. M.; Ericson, C.; Phung, Q. T.; Salomon, A. R. Anal. Chem. 2003, 75, 3419-3428. (43) Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Anal. Chim. Acta 1991, 246, 211-225. (44) Mirgorodskaya, E.; O’Connor, P. B.; Costello, C. E. J. Am. Soc. Mass Spectrom. 2002, 13, 318-324.

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high-resolution isolation of precursor ions using stored waveform inverse Fourier transformations45 or correlated harmonic excitation fields (CHEF),46,47 and with high mass accuracy for product ions.46,48 A recent study by Li and co-workers49 has shown that MALDIFTMS, with applications of in-cell accumulation (ICA) techniques, holds significant advantages over MALDI-TOF for direct tissue analysis. Applied to the analysis of tissue samples from the Jonah crab, Cancer borealis, the work by Li’s group demonstrated significant improvements in signal-to-noise ratio, ability to acquire wide mass range spectra, and improved mass measurement accuracy (2 ppm).49 SORI-CID for peptide sequencing from tissue samples was also possible with the ICA-enhanced sensitivity. In this paper, we describe an extension of the work by Li’s group, with a focus on peptide sequence-dependent metastable decay and its exploitation for peptide determination. EXPERIMENTAL SECTION Instrumentation. A HiResMALDI Fourier transform mass spectrometer (IonSpec, Irvine, CA) with a Cryomagnetics (Oak Ridge, TN) 4.7 T actively shielded superconducting magnet was used for all measurements. Ions were generated using a pulsed nitrogen laser (337 nm) and were transported from the external ion source to the closed cylindrical ICR cell using a quadrupole ion guide. For single-shot experiments, ions were trapped in the cell by holding the rear trapping plate at 20 V and raising the front trapping plate from 0 to 20 V after a variable delay following desorption (gated trapping). Inner trapping ring potentials were held at 0.5 V throughout the experiment. The ion guide rf potential and trapping delay time were optimized to transmit and trap ions of a selected mass range. A pulse of argon was introduced to the vacuum system during trapping to transiently elevate the system pressure for collisional cooling. For spectra measured using ion accumulation techniques, ions generated from successive laser shots (typically 7) were accumulated in the cell by lowering the front trapping plate potential to 6 V prior to applying gated trapping for each shot. Stitched pulse sequences, initially described by O’Connor and Costello,36 were used for calibration and some SORI-CID experiments. For SORI-CID experiments, Ar was used as the collision gas, the frequency offset was set equal to -1.8% of the reduced cyclotron frequency,44 and the voltage amplitude was in the range of 6-8.5 Vbp. A delay of 5-10 s preceded ion detection, which occurred with analyzer pressures of (1-2) × 10-10 Torr. For all measurements, ions were detected after ramping the outer trapping plates to 0 V with the inner trapping rings remaining at 0.5 V. Ions were excited prior to detection using rf swept excitation (m/z ) 100-9000, 160 V, 1 ms or m/z ) 300-20 000, 20 V, 15 ms). Most broadband spectra were measured using quadrupole rf potential and the gated trapping instrument setting for m/z 1500. Signals were sampled at 500 kHz with low mass limits of m/z 300. These conditions were adjusted to accommodate the desired mass range and spectral resolution. (45) Guan, S. H.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1996, 158, 5-37. (46) Heck, A. J. R.; Derrick, P. J. Anal. Chem. 1997, 69, 3603-3607. (47) de Koning, L. J.; Nibbering, N. M. M.; van Orden, S. L.; Laukien, F. H. Int. J. Mass Spectrom. 1997, 165, 209-219. (48) Kruppa, G.; Schnier, P. D.; Tabei, K.; Van Orden, S.; Siegel, M. M. Anal. Chem. 2002, 74, 3877-3886. (49) Kutz, K. K.; Schmidt, J. J.; Li, L. Anal. Chem. 2004, 76, 5630-5640.

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Transients from direct tissue spectra were apodized using a Blackman function and zero-filled prior to fast Fourier transformation; SORI-CID spectra were processed without apodization. Samples were prepared on a 10-faceted stainless steel probe tip. Calibration. Samples were calibrated by external or internal calibration. Highest accuracy and precision was obtained with internal calibration using a stitched in-cell accumulation procedure, based upon the internal calibration on adjacent samples (InCAS) technique36 with modifications that included the selective isolation of calibrant peaks and slow, swept excitation conditions. Calibrants were applied to a separate probe face. The stitched in-cell accumulation procedure was initiated by first executing a quench sequence to clear the cell and preset the cell potentials. Next, a calibrant sequence was loaded and executed to introduce the calibrant and isolate the desired calibrant ions with an arbitrary waveform. Two to six calibrant peaks that covered the desired mass range were isolated. The probe was then rotated to the sample location, and the sample accumulation and detection sequence was loaded and executed. To limit the number of ions in the cell, an arbitrary waveform with a window of m/z ) 300-2000 was applied as part of the sample accumulation sequence. The number of accumulated laser shots and the laser power attenuation setting for each sequence were independently adjusted to control the relative intensity of calibrant and sample ions. Using the second, 15-ms rf swept excitation conditions (summarized above), mass accuracies and precisions (standard deviations) were both less than 1 ppm for calibrations based on 2048 K data sets with two zero fills. Materials. 2,5-Dihydroxybenzoic acid (DHB; SigmaAldrich, St. Louis, MO) was sublimed prior to use for most studies. Matrix solutions of DHB (1.0 M) were prepared in 1:1 acetonitrile/water or 1:1 acetonitrile/0.1%TFA water. D-Fructose or fucose (SigmaAldrich) was added to the matrix solution for some experiments; 0.75 M fructose in water was used for tissue washing. As calibrants, we used poly(propylene glycol)s (725 and 2000; SigmaAldrich). The calibrants, dissolved in CHCl3, were applied to a layer of DHB crystals prepared using the thin-film method with acetone as the solvent. The [Val13]- and [Ala13]-orcokinin standards were kind gifts from L. Li and E. Marder (Brandeis University); CapTrp 1a was from M.P. Nusbaum (University of Pennsylvania). Proctolin (SigmaAldrich), RPCH (Peninsula Labs, San Carlos, CA), and [Val1]-SIFamide (GenScript, Piscataway, NJ) were also used as part of this study. Animals/Dissection Details. Experiments were conducted using the American lobster, H. americanus (purchased locally), California spiny lobsters, Panulirus interruptus (Tomlinson Commercial Fishing, San Diego, CA), and the Jonah crab, C. borealis (purchased locally), kept in recirculating seawater at 10-12 °C for up to 4 weeks before dissection. After lobsters were anaesthetized by chilling for 20-40 min on ice, the stomachs were removed and placed in cold Homarus saline (composition, in mM: 479 NaCl, 12.8 KCl, 13.7 CaCl2, 3.9 Na2SO4, 20 MgSO4, and 4.8 HEPES; pH 7.4-7.5), Panulirus saline (composition, in mM: 479 NaCl, 12.8 KCl, 13.7 CaCl2, 3.9 Na2SO4, 10 MgSO4, 11 Trizma base, and 4.8 maleic acid; pH 7.5-7.6), or C. borealis saline (composition, in mM: 442 NaCl, 11 KCl, 13 CaCl2, 26 MgCl, 12 Trizma base, and 1.2 maleic acid; pH 7.4-7.5) for the remainder of the dissection. The STG and the CoG were dissected off the

surface of the stomach and pinned out in a Sylgard-lined dish, where the sheath surrounding each ganglion was completely removed. The sinus glands were dissected from the eyestalks in cold saline. Sample Preparation. The STGs were left intact, while each CoG and each sinus gland was cut into two pieces before being applied to the probe tip. Tissues were removed from the saline with fine forceps, rinsed sequentially in two 25-µL droplets of 0.75 M (135 mg/mL) fructose or 0.065 M (10 mg/mL) DHB, and placed on a face of the probe tip, minimizing cotransfers of solution. The tissue was sliced 10-20 times with a 0.1-mm needle, the tissue was gathered together, and a 0.5-µL droplet of the DHB matrix was added. Matrix crystallization proceeded at ambient temperature. RESULTS AND DISCUSSION Representative MALDI-FTMS spectra for SG, CoG, and STG tissue samples from H. americanus are shown in Figure 1. In contrast with MALDI-TOF spectra, where only the protonated or sodiated molecule is detected, many neuropeptides examined in this study produce intense fragment ions under MALDI-FTMS conditions. Although fragmentation resulting from loss of NH3 was recognized in the previous MALDI-FTMS study applied to tissues from the crab, C. borealis,49 we find that many other peaks in the MALDI-FTMS spectra of tissue samples result from peptide metastable decay. The characterization and exploitation of this fragmentation for neuropeptide identification is the focus of this study. In the following discussion we will first address the sample preparation protocols we have used for our work and then describe work with standards to characterize metastable peptide fragmentation, with a focus on peptides in the orcokinin family. Sample Preparation. The analysis of biological tissues and cells from marine invertebrates is complicated by the high physiological salt concentrations associated with these samples.6 Sweedler and co-workers6,7 have developed MALDI sample preparation techniques for dissected tissue samples that make use of a 0.065 M (10 mg/mL) aqueous solution of the DHB MALDI matrix for rinsing tissues prior to MALDI sample preparation. This approach reportedly stabilizes cell membranes, deactivates endogenous proteolytic enzymes, and reduces the high salt concentration.6,7 We have adapted this approach to make use of an aqueous fructose solution for tissue washing. Tissues are dissected and isolated in physiological saline and are then rinsed twice in 25-µL droplets of 0.75 M (135 mg/mL) aqueous fructose before applying the tissue to the MALDI probe face. We began working with the fructose solution to facilitate tissue handling and in an attempt to minimize peptide loss by washing the tissue in a solution isoosmotic with neuronal cells. Because this solution is significantly less acidic than 10 mg/mL DHB, we compared spectra generated from sinus and CoG tissues using the two washing procedures. Both approaches effectively eliminated the deleterious effects of salts, as no sodium or potassium additions were found with peptides that produced [M + H]+ ions. No significant differences in peptide profiles were observed, showing that the neutral pH of the fructose wash did not significantly enhance or suppress the intensity or number of peptides detected (data not shown). We also tested the acidified methanol washing protocol used in the C. borealis FTMS study49 on a limited number of tissue

samples. We found that approach introduced phospholipid contaminant peaks, including m/z 782.57, [M + Na]+, and fragments at m/z 723.49 and 599.50 from phosphatidylcholine (34:1),50 in the mass range of interest in this study. We found tissues easier to handle using the fructose washing protocol and used that approach throughout this study. We have found that the best quality, most persistent, signals result when we use a DHB matrix concentration (154 mg/mL, 1.0 M) that is higher than that typically used for MALDI-TOF studies; the addition of a comatrix (fructose or fucose) is also helpful. On our flat, stainless steel probe tip, the matrix spreads and crystallizes to form needlelike crystals covering a region with a diameter of 1.5-2 mm. The tissues remain concentrated in the center and we desorb from regions along the outer rim, avoiding the central regions where phospholipid-derived signals are produced. With lower matrix concentrations (10-50 mg/mL), the crystal density on the probe tip is low and signals are weak or not observed. In general, our highest quality signals are associated with the formation of a thick area of crystals, and from these regions we can produce signals that are difficult to exhaust. Using our sample preparation protocol, we have measured spectra suitable for direct tissue analysis from ∼95% of the tissues from >35 animals examined as part of this study using 7 in-cell ion accumulated laser shots. Spectra with S/N characteristics in the molecular ion region that are similar to those shown in Figure 1 were obtained from ∼40% of those samples. We believe that the variable size and surface coverage of the crystalline sample is the most significant factor influencing signal intensity. Instrumental Conditions and Measured Mass Accuracy (MMA). On our external source FTMS, we trap ions covering a limited mass range using gated trapping techniques. For the work reported in this study, we have used ICA (7 laser shots) and gated trapping conditions optimized for transmission and trapping the orcokinin peptides that have been a focus of this study. For polymer standards, which produce only [M + Na]+ ions, ions in the range of m/z 600-2500 have been detected under similar conditions, with most efficient trapping occurring for ions at m/z 1700, with lower and higher mass ions being trapped with reduced efficiency. All detection has been in broadband mode, with a low mass limit of m/z 300. This setting, which limits the digitization rate and digital resolution, gives resolving powers in the range of ∼65 000-80 000 (m/∆m50%) for the m/z 1500.639 peak from [Asn13]-orcokinin (4-s transient) on our 4.7-T instrument. Under these conditions, we are routinely able to resolve the [M + H]+ peak of [Val13]-orcokinin at m/z 1502.6910 from the (A + 2),13C2 isotope peak of the [MH - NH3]+ peak of [Asn13]-orcokinin at m/z 1502.6454. For calibration, we have used a modified version of the InCAS technique, introduced by O’Connor and Costello,36 for internal calibration of spectra. We have used poly(propylene glycol) (PPG) 725+2000 as the calibrant, deposited on a separate probe face, and apply an arbitrary waveform to isolate six PPG calibrant ions as part of our stitched accumulation sequence. We use this isolation event to limit the total number of ions introduced to the cell. PPG is used instead of the peptide calibrants used in the work by Li’s group49 because the PPG ions are easily resolved from neuropeptide peaks at the same nominal mass because of (50) Jones, J. J.; Stump, M. J.; Fleming, R. C.; Lay, J. O.; Wilkins, C. L. J. Am. Soc. Mass Spectrom. 2004, 15, 1665-1674.

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Figure 1. MALDI-FTMS peptide profiles from H. americanus tissue samples measured using DHB as the matrix and the in-cell accumulation of seven laser shots. (A) Half of a sinus gland, (B) half of a commissural ganglion, and (C) a single stomatogastric ganglion. See Table 1 for the amino acid sequence for peptides 1-7. Numbers with an apostrophe identify peptide fragment ions. Asp-Xxx cleavages for peptides 1-4 are shown in Scheme 1.

differences in their fractional mass. Following the selective accumulation of calibrant ions, we rotate the probe and introduce ions from the tissue sample into the cell. An important aspect of our internal calibration approach has been the use of a slow (15 ms), m/z 300-20 000, rf swept excitation, which significantly improved the precision and accuracy of our mass measurements. 3598 Analytical Chemistry, Vol. 77, No. 11, June 1, 2005

Representative MMA are given in Table 2 for repeated, internally calibrated peaks in a SG of H. americanus. The average MMA (-0.09 ppm) and the pooled standard deviation (0.97) associated with these measurements on our 4.7-T instrument are lower than those reported on the 7-T instrument used for the previous MALDI-FTMS crustacean neuropeptides study.49

Table 2. Mass Measurement Accuracy for Fingerprint Ions from Orcokinin and Orcomyotropin-Related Neuropeptides Detected in the Sinus Gland of H. Americanus Using InCASa

peptide and fragment ions (1) [His13]-orcokinin, NFDEIDRSGFGFH [M + H]+ [MH - NH3]+ y10+ [y10 - H2O]+ y7+ (2) [Asn13]-orcokinin, NFDEIDRSGFGFN [M + H]+ [MH - NH3]+ [y10 - NH3]+ [y10 - NH3-H2O]+ y7+ [y7 - NH3]+ (3) [Val13]-orcokinin, NFDEIDRSGFGFV [M + H]+ [MH - NH3]+ y10+ [y10 - H2O]+ y7+ (4) Orc [1-12], NFDEIDRSGFGF [M + H]+ [MH - NH3]+ y9+ [y9 - H2O]+ y6+ (5) Orc [1-11], NFDEIDRSGFG [M + H]+ [MH - NH3]+ [y8 - H2O]+ y6+ (6) FDAFTTGFGHN [M + H]+ [MH - NH3]+ y9+ [y9 - NH3]+ calibrant peaksc H(OC3H6)12OH, [M + Na]+ H(OC3H6)15OH, [M + Na]+ H(OC3H6)18OH, [M + Na]+ H(OC3H6)25OH, [M + Na]+ H(OC3H6)28OH, [M + Na]+ average errord rms errord pooled SDd

calculated mass

average measured massb

average error and SD (ppm)b

1540.681 53 1523.654 98 1164.543 24 1146.532 68 807.389 64

1540.682 31 0.5 (0.7) 1523.656 62 1.1 (0.8) 1164.542 35 -0.8 (1.7) 1146.534 14 1.3 (0.3) 807.389 27 -0.5 (0.2)

1517.665 55 1500.639 00 1124.500 71 1106.490 20 784.373 66 767.347 11

1517.663 81 1500.638 96 1124.500 45 1106.490 29 784.374 23 767.346 95

1502.691 03 1485.664 49 1126.552 75 1108.542 19 769.399 15

1502.689 88 -0.8 (0.4) 1485.663 20 -0.9 (0.3) 1126.553 20 0.4 (0.3) 1108.541 49 -0.6 (0.1) 769.399 24 0.1 (0.1)

1403.622 62 1386.59 07 1027.484 34 1009.473 77 670.330 74

1403.624 18 1.1 (0.3) 1386.597 18 0.8 (0.3) 1027.484 93 0.6 (0.3) 1009.473 80 0.03 (0.1) 670.330 51 -0.3 (0.2)

-1.1 (0.9) -0.03 (0.3) -0.2 (0.2) 0.08 (0.2) 0.7 (0.1) -0.2 (0.1)

1256.554 21 ND 1239.527 66 1239.526 53 -0.9 (0.7) 862.405 36 862.404 81 -0.6 (0.3) 523.262 32 523.261 33 -1.9 (0.2) 1213.527 26 1213.529 51 1.9 (0.3) 1196.500 71 1196.502 87 1.8 (0.2) 951.431 91 951.431 91 0.004 (0.2) 934.405 36 934.404 70 -0.7 (0.1) 737.502 16 737.502 11 -0.07 (0.13) 911.627 76 911.628 12 0.40 (0.06) 1085.753 35 1085.752 71 -0.6 (0.5) 1492.046 41 1492.047 55 0.8 (0.5) 1666.172 00 1666.171 60 -0.2 (0.1) -0.09 0.70 0.97

a PPG was used as the internal calibrant. Peptide signals from a single acquisition with seven accumulated laser shots. b Results from three individually calibrated spectra were averaged to give the reported masses, errors and standard deviations. c PPG ions isolated for calibration. d Average error, root-mean-squared (rms) error, and pooled standard deviation from 109 MMA measurements, 3 calibrated spectra.

Mass Spectral Characteristics of Representative Crustacean Peptides. For MALDI-FTMS measurements, the time required for ion trapping and pump down for low-pressure detection can range from 4 to >10 s, which greatly exceeds the low-microsecond time scale associated with detection by a TOF mass analyzer. Under low-pressure MALDI-FTMS conditions spectra, fragile biomolecules, including some peptides, undergo

metastable decomposition.41,51-53 MALDI-FTMS experiments, as well as MALDI-ion trap54 and TOF results,55,56 have demonstrated that peptide metastable decomposition is influenced by amino acid composition, matrix, laser irradiance, and the time delay before detection. As part of this study, we have characterized the MALDIFTMS behavior of six crustacean neuropeptide standards, including proctolin (RYLPT), RPCH (pELNFSPGWamide), CabTRP 1a (APSGFLGMRamide), [Val13]-orcokinin, [Ala13]-orcokinin, and [Val1]-SIFamide (VYRKPPFNGSIFamide). [M + H]+ ions were the most commonly observed form for the molecular ion; however, we found that one peptide standard, RPCH, formed almost exclusively [M + Na]+ and, to a lesser extent, [M + K]+ ions. We also found that the standards showed significant differences in the extent of metastable decay. For some peptides (proctolin, RPCH, CabTRP 1a, [Val1]-SIFamide) the direct MALDI-FT mass spectra show the [M + H]+ (or [M + Na]+, for RPCH) ion as the base peak in the spectrum, with less abundant peaks resulting from loss of NH3 or H2O. For example, [Val1]-SIFamide, a new member of the SIFamide family identified in our laboratory, is very stable toward loss of NH3 and other forms of metastable decay (see Figure 2A). In tissue sample preparations, this peptide retains the same stability characteristics and appears as the dominant peak in the spectra of H. americanus STG tissue samples (see Figure 1C). More fragmentation is induced if the laser fluence is increased or if “hotter” matrixes, such as sinapinic acid or R-cyano-4-hydroxycinnamic acid, are used. In contrast, the [Val13]- and [Ala13]- orcokinin standards underwent extensive fragmentation that can be somewhat reduced, but not eliminated, under similar conditions (see Figure 2B for [Val13]-orcokinin). These orcokinin peptides produce more intense peaks resulting from the loss of NH3 and also produce abundant y-series ions resulting from cleavage on the C-terminus side of the aspartate residue (Asp-Xxx cleavage;54,57-59 see Scheme 1 for the orcokinins appearing in Figure 1). These characteristic cleavages have been well documented in MALDIion trap studies54 and in MS/MS experiments,57-60 where it has been found that cleavage occurs to a lesser extent C-terminus to glutamate residues. The orcokinin peptides are particularly susceptible to this mode of fragmentation, as a single arginine residue is present to sequester the ionizing proton,58 and the location of the arginine results in the exclusive formation of y-series ions. For the orcokinin peptides, a [y10 - H2O]+ peak consistently appears as a more abundant fragment relative to y10+. We attribute this preference to a cyclization reaction to pyroglutamate from the N-terminal glutamate residue. (51) Castoro, J. A.; Wilkins, C. L. Anal. Chem. 1993, 65, 2621-2627. (52) Stemmler, E. A.; Hettich, R. L.; Hurst, G. B.; Buchanan, M. V. Rapid Commun. Mass Spectrom. 1993, 7, 828-836. (53) Ho, Y. P.; Fenselau, C. J. Mass Spectrom. 2000, 35, 183-188. (54) Qin, J.; Chait, B. T. J. Am. Chem. Soc. 1995, 117, 5411-5412. (55) Spengler, B.; Kirsch, D.; Kaufmann, R. Rapid Commun. Mass Spectrom. 1991, 5, 198-202. (56) Yu, W.; Vath, J. E.; Huberty, M. C.; Martin, S. A. Anal. Chem. 1993, 65, 3015-3023. (57) Tsaprailis, G.; Nair, H.; Somogyi, A.; Wysocki, V. H.; Zhong, W. Q.; Futrell, J. H.; Summerfield, S. G.; Gaskell, S. J. J. Am. Chem. Soc. 1999, 121, 51425154. (58) Wysocki, V. H.; Tsaprailis, G.; Smith, L. L.; Breci, L. A. J. Mass Spectrom. 2000, 35, 1399-1406. (59) Tsaprailis, G.; Somogyi, A.; Nikolaev, E. N.; Wysocki, V. H. Int. J. Mass Spectrom. 2000, 196, 467-479. (60) Qin, J.; Chait, B. T. Int. J. Mass Spectrom. 1999, 191, 313-320.

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Figure 2. Direct MALDI-FTMS spectra of neuropeptide standards. (A) [Val1]-SIFamide and (B) [Val13]-orcokinin (3). Spectra measured using DHB as the matrix.

Scheme 1. Asp-Xxx Cleavages for the Orcokinins in H. Americanus

The fragmentation observed under direct MALDI-FTMS conditions is replicated when the [M + H]+ ion is isolated and dissociated by SORI-CID (data not shown). The products that will be the focus of this study (y10+, [y10 - H2O]+, y7+) are also observed when the [MH - NH3]+ ions from [Val13] and [Ala13] standards are activated by SORI-CID, showing that NH3 loss occurs predominantly at the N-terminus for these peptides and that the loss of NH3 does not impact the masses of the y-series products. Orcokinins with C-terminus asparagine residues, found in tissue samples, show additional y-series products at [y10 NH3]+, [y10 - NH3 - H2O]+, and [y7 - NH3]+ ions. For these peptides, loss of NH3 from the C-terminus asparagine residue becomes important. Metastable decomposition of the orcokinin peptides takes place immediately following desorption, during transport to the cell, or during the time delay (∼10 s) introduced to reduce the cell 3600 Analytical Chemistry, Vol. 77, No. 11, June 1, 2005

pressure to the low 10-10 Torr range prior to excitation and detection. Figure 3 shows plots of relative ion intensity as a function of the time delay before detection for two orcokinin peptides, [Asn13]-orcokinin and the truncated Orc [1-12], desorbed from a CoG tissue sample. The [Asn13]-orcokinin, with asparagine residues at both the N- and C-termini, is the most fragile orcokinin we have encountered, showing extensive losses of NH3, as well as Asp-Xxx cleavages. In contrast, when the C-terminus residue is removed (Orc [1-12]) or changed to Val, Ala, or His, the [M + H]+ ion intensities are enhanced relative to the other fragments. Instrumentally we can reduce the extent of decomposition by working with the lowest laser irradiance and increasing the peak argon pressure used for collisional stabilization. We have also evaluated the impact of sample preparation on this metastable decay. Working with peptide standards, we have found that metastable decomposition can be dramatically reduced using 3-hydroxypicolinic acid (HPA), a matrix commonly used for the analysis of oligonucleotides. With HPA, the MALDI-FTMS spectra of [Val13]- and [Ala13]-orcokinin standards show intense [M + H]+ ions with weak or undetectable fragment ions; however, our attempts to apply this matrix system to direct tissue analysis have been less successful because HPA has a strong propensity for

Figure 4. Relative ion abundance for various ions (m/z 767, 670, 1280, 934, 1423) plotted as a function of the relative abundance of m/z 1500 for spectra measured at different delay times from a CoG. The m/z 1500 is the [MH - NH3]+ fragment from [Asn13]-orcokinin. The m/z 767 and 670 are fragment ions; m/z 1280, 934, and 1423 are assigned as [M + H]+ ions.

Figure 3. Relative ion intensity as a function of the time delay before ion excitation and detection for ions generated upon desorption from an SG tissue sample using DHB as the matrix. (A) [Asn13]-orcokinin, NFDEIDRSGFGFN (2) and (B) Orc [1-12], NFDEIDRSGFGF (4).

clustering with salt-derived metal ions. High-pressure MALDIFTMS41,61 offers another means of reducing metastable decomposition, but our instrument does not have this capability. The facile fragmentation of aspartate-containing peptides under vacuum MALDI-FTMS conditions presents challenges and opportunities for tissue characterization. The opportunities, described below, derive from the rich, peptide-specific information that is present in the direct tissue spectra. The challenge is distinguishing the [M + H]+ ions of intact peptides from those ions produced by metastable decay. For example, the y-type ions derived from orcokinin peptides are equivalent to N-terminally truncated versions of the precursor. In the absence of other data, we have used two characteristics to help distinguish fragments from genuine, intact peptides. First, we look for the onset or enhanced abundance of lower mass ions using gated trapping conditions optimized for trapping higher mass precursors. If observed, this suggests that the lower mass ions are produced from higher m/z precursors following transport and trapping in the cell. Second, we can evaluate the variations in ion intensity as a function of delay time before ion detection to look for evidence of precursor ions that fragment to yield product ions. For example, in Figure 3 we showed how the relative abundance of y7+ and y10+ ions increases with delay time relative to [M + H]+ and [MH - NH3]+ precursors. Using a full data set of tissue data composed of all peptide ion signals measured at different delay times, we can correlate the relative abundance of uncharacterized peaks with the abundance of a known peptide peak. (61) O’Connor, P. B.; Mirgorodskaya, E.; Costello, C. E. J. Am. Soc. Mass Spectrom. 2002, 13, 402-407.

For example, Figure 4 shows the relative abundance of five ions detected at different trapping times in the spectrum of a CoG sample, plotted as a function of the [Asn13]-orcokinin (2) [MH - NH3]+ ion relative abundance. From this display, we clearly see that the m/z 767.35 peak shows an inverse relationship with the [Asn13]-orcokinin [MH - NH3]+ ion. This is expected for the m/z 767.35 peak, the [y7 - NH3]+ fragment from the ionized [Asn13]-orcokinin, but this is also a general behavior for other orcokinin fragments. For example, m/z 670.33, the y6+ fragment from truncated Orc [1-12] shows a similar correlation. In contrast, the more stable molecular ion from CabTRP 1a (m/z 934.49) shows a small positive correlation, reflecting the stability of this peptide relative to the [Asn13]-orcokinin [MH - NH3]+ ion. Two other peaks, which we have identified as [Val1]-SIFamide, (VYRKPPFNGSIFamide, m/z 1423.78) and (VY)GPRD[I/L]AN[I/L]Y (m/z 1280.66), also show a positive correlation, suggesting that these peptides are also more stable than the [MH - NH3]+ reference and are not being produced or degraded by metastable decay. This behavior supports our conclusion that the observed peaks are [M + H]+ ions stable with respect to fragmentation. Identification of Orcokinin Family Peptides Applied to H. americanus. The characteristic Asp-Xxx fragmentation patterns produced by orcokinin neuropeptides can be exploited as a tool to identify this family of peptides in tissue samples. The 12 currently identified members of this family of peptides, summarized by Skiebe et al.,22 have a conserved N-terminus sequence (NFDEIDR), with amino acid variations appearing at the C-terminus (see Scheme 2). Because the N-terminus region is lost during metastable decomposition, information regarding the variable C-terminus is retained in the charged fragments, which then serve as useful fragment ion fingerprints (FIFs) for the original peptide. For example, Figure 5A-C shows expansions of three regions from a H. americanus SG sample where peaks from orcokinin peptides with 13-amino acid residues appear. Figure 5A shows the molecular ion region, which can be aligned with the regions containing [y10 - H2O]+ peaks (Figure 5B) and y7+ peaks (Figure 5C). We identify the orcokinin peptides appearing in these regions as [His13], [Asn13], and [Val13]Analytical Chemistry, Vol. 77, No. 11, June 1, 2005

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Scheme 2. Orcokinin Fragmentation Pathways

orcokinin, based upon accurate mass measurements and their characteristic fingerprints. These three orcokinin peptides have been previously reported in H. americanus tissue samples.20,22 Our results are the first report from eyestalk SG tissues. Table 2 provides a summary of mass measurements, based on replicated calibration measurements (n ) 3), for orcokinins 1-5, and the orcomyotropic-related peptide 6 for a H. americanus SG sample. For each internally calibrated spectrum, prior to averaging, no individual measurement error exceeded 2 ppm and the pooled standard deviation for the full data set was 0.97 ppm, with an average measured mass error of -0.09 ppm. These highly accurate and precise measurements, associated with the calibration of both weak and intense peaks, makes it possible to apply high-resolution screens to identify peptides in the direct tissue spectra. While accurate mass measurements are useful for the determination of signals derived from [M + H]+ ions, the information provided by metastable decay, used in combination with accurate mass measurements, provides a much higher degree of certainty regarding peptide identity. While this level of information can be provided by SORI-CID experiments, the fact that the information is present in the direct tissue spectra eliminates the need for more time-consuming acquisition and analysis of MS/MS data and facilitates the efficient analysis of direct tissue samples. Using our understanding of orcokinin peptide fragmentation, coupled with accurately calibrated mass spectral measurements, we have applied two data analysis tools to identify orcokinin peptides in crustacean tissue samples from direct tissue spectra. The first tool is a screen for known neuropeptides using an inhouse database of known crustacean neuropeptides and their experimentally determined or predicted FIFs, which we currently search using Microsoft Excel. As applied to a second set of seven internally calibrated SG samples from H. americanus, we searched the calibrated spectra using a 2 ppm tolerance and detected mass fingerprints for all five previously detected orcokinins (1-5) in all spectra, based upon the detection of a minimum of four fingerprint peaks per peptide. The [M + H]+ peak for [Val3]orcokinin (m/z 1502.6910), which must be resolved from 13C2 isotope peak from the [MH - NH3]+ ion of [Asn13]-orcokinin (m/z 1502.6454), was detected in five out of seven spectra. Our screen also consistently picked up the orcomyotropin peptide 6. In contrast, we did not detect any peaks in any spectra for [Ala13]3602

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orcokinin, previously identified in H. americanus,20 but recently shown22 to be a variant (Hom-orc) that appears at the same nominal mass. Our screen also consistently picked up two additional peptides. The first peptide, with an experimentally determined mass of m/z 1280.663 64 ((0.000 02, n ) 3) was identified in our laboratory as (VY)GPRD[I/L]AN[I/L]Y (1280.663 36, calculated), based upon SORI-CID data and sequence similarity to a linker peptide in pre-proorcokinin A and B from Procambarus clarkii,27 VYVPRYIANLY. The second peptide was Hom-orc, 7, whose partial sequence has been determined by PSD and ESI Q-TOF measurements.22 Although a peak appears at m/z 934, this peak was consistently (and correctly) identified as the m/z 934.4054 [y9 NH3]+ fragment from FDAFTTGFGHN, not CapTRP 1a (934.4927), using our 2 ppm tolerance. While CapTRP 1a was detected in CoG and STG samples, it was not detected in SG tissues. The second data analysis tool we used was designed to screen for orcokinin peptide FIFs, based upon mass differences corresponding to the neutral loss of NFD (376.1383 Da), NFD - H2O (394.1489 Da), or NFDEID (733.2919 Da) (see Scheme 2). We also looked for neutral loss of NH3 to confirm molecular ion identity. Microsoft Excel was again used for this analysis. As applied to the analysis of peaks from internally calibrated direct tissue spectra from H. americanus, searched with a 5 ppm tolerance, this screen resulted in the identification of orcokinin fingerprint masses for orcokinins 1-4. The truncated orcokinin (5), with a molecular ion peak that was not detected, was not flagged unless the mass difference screen also included the possibility of differences originating from the [MH - NH3]+ ion (i.e., each of the neutral loss masses was reduced by the mass of NH3). With this modification, orcokinin 5 was detected. This second approach to searching the data for FIFs was developed for the analysis of tissues that may contain orcokinins that have not been previously identified, and the utility of this approach has been demonstrated in applications to P. interruptus, described below. With these two tools, searching for previously identified orcokinin FIFs or for characteristic neutral loss FIFs, it is possible to efficiently evaluate highly complex spectra and retrieve information regarding previously identified or new orcokinin peptides. For orcokinin peptides, SORI-CID can also be used to provide additional confirmation of peptide identity, targeting the variable C-terminus of the peptide. For example, Figure 6 shows isolation and SORI-CID spectrum of the m/z 769, y7+ fragment from the [Val13]-orcokinin from a H. americanus SG tissue sample. The spectrum shows only b-series fragment ions, reflecting the position of the arginine residue at the N-terminus of the y7+ ion precursor. The spectrum shows an intense [b5 + H2O]+ fragment, observed in other studies involving arginine residues at or near the N-terminus,62 and charge localized ions.63 The [b5 + H2O]+ fragment serves to identify the C-terminus amino acid (loss of 99.068, valine, in the example shown). The b3+ ion is often weak or not observed, which may reflect a bias toward reduced fragmentation C-terminal to glycine, a spectra feature that has been correlated with charge-localized ions.64 Another notable (62) Thorne, G. C.; Ballard, K. D.; Gaskell, S. J. J. Am. Soc. Mass Spectrom. 1989, 1, 249-257. (63) Gu, C. G.; Tsaprailis, G.; Breci, L.; Wysocki, V. H. Anal. Chem. 2000, 72, 5804-5813.

Figure 5. Direct MALDI-FTMS spectra of H. americanus SG tissue sample. (A) Expansion of the [M + H]+ ion region, (B) Expansion of the [y10 - H2O]+ region, (C) Expansion of the y7+ ion region. Spectra measured using DHB as the matrix.

feature of the spectrum is the presence of neutral losses of CH2O, in combination with loss of H2O, which we attribute to a sidechain neutral loss from the serine residue. Figure 6 also illustrates (64) Kapp, E. A.; Schutz, F.; Reid, G. E.; Eddes, J. S.; Moritz, R. L.; O’Hair, R. A. J.; Speed, T. P.; Simpson, R. J. Anal. Chem. 2003, 75, 6251-6264.

an advantage of MALDI-FTMS dissociation experiments, namely, the ability to achieve monoisotopic isolation of the m/z 769 ion (Figure 6A) in the presence of the intense m/z 767, [y7 - NH3]+ fragment from [Asn13]-orcokinin (see Figure 5C) using an arbitrary waveform, followed by a CHEF,47 ion isolation event. The SORI Analytical Chemistry, Vol. 77, No. 11, June 1, 2005

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Figure 6. SORI-CID of the y7+, m/z 769.399, from the SG of H. americanus. (A) Isolation of m/z 769.399 using an arbitrary waveform and CHEF ion isolation. (B) SORI-CID spectrum. The m/z 769 peak (y7+) is formed by metastable decay following desorption and ion trapping. [Val13]-orcokinin, NFDEIDRSGFGFV, is assigned as the precursor to y7+ with [M + H]+ at m/z 1502.69.

spectrum from the tissue sample shows good agreement with that measured using the [Val13]-orcokinin standard. We can also use SORI-CID to establish connections between ions produced by metastable decay and their precursors. For example, we identified the m/z 670.33 ion in the SG MALDI-FTMS direct tissue spectrum (see Figure 1A) as the y6+ product from the truncated Orc [1-12], based upon predicted Asp-Xxx cleavages (see Scheme 1). Isolation and SORI-CID of the [MH NH3]+ ion at m/z 1386.60 yields a spectrum showing the expected aspartate-directed cleavages that produce the y9+, [y9 - H2O]+ and y6+ products observed in the spectrum (see Figure S-1A, Supporting Information). While the selective Asp-Xxx directed cleavages limit the sequence information that can be obtained from the spectrum, SORI-CID of the y6+ ion provides more complete sequence information for the variable C-terminus. For example, SORI-CID for the m/z 670, y6+ ion, yields a series of b-ions that supports the amino acid sequence (see Figure S-1C). We again observed the neutral losses of CH2O, attributed to the serine residue, and observe an intense [b5 + H2O]+ ion reflecting the loss of a phenylalanine residue from the C-terminus. By starting with the y6+ ion produced by metastable decay, instead of the isolation and dissociation of a SORI-CID product, we are able to obtain SORI-CID spectra with higher S/N characteristics. Orcokinin Peptides in the CoG of C. borealis and P. interruptus. We have applied the approach described above to the analysis of tissue samples from the Jonah crab, C. borealis, and the California spiny lobster, P. interruptus. Although a detailed comparison of neuropeptides from these two crustaceans is beyond the scope of this paper, results from our FIF screen, as applied to CoG direct tissue spectra from the two species, serve to illustrate the utility of this fingerprinting approach for the detection of previously reported orcokinins and for the identification of new members of this family of neuropeptides. Figure 7A-C shows direct tissue spectra from the CoG of H. americanus, C. borealis, and P. interruptus, respectively, expanded to show the region where y6+ and y7+ ions appear. The spectrum of the CoG of H. americanus shows the y6+ and y7+ (and higher mass) fingerprint ions for the same orcokinins detected in SG 3604 Analytical Chemistry, Vol. 77, No. 11, June 1, 2005

tissues (see Figure 7A). As applied to the spectra of C. borealis, peaks flagged in our FIF screen for previously identified orcokinin peptides included Orc [1-12], [Ala13]-, [Val13]-, [Ser9-Asn13]-, and [Ser9-Val13]-orcokinin. The y6+ or y7+ peaks from these peptides are identified in Figure 7B, and fingerprint ions in the [y10 - H2O]+ and molecular ion region were also observed. For [Ser9-Asn13]orcokinin, no [M + H]+ ion was observed but we did detect the [MH - NH3]+ ion. Because [Ser9-Asn13]-orcokinin has a C-terminus asparagine residue, loss of NH3 is expected to be significant, as was observed for [Asn13] in H. americanus. This facile loss provides an explanation for the missing molecular ion peak for [Ser9-Asn13]-orcokinin. Facile loss of NH3 also correlates with the detected fingerprint ions, which were found at [y7 - NH3]+ and [y10 - NH3 - H2O]+. For C. borealis, the orcokinins detected by our screen show good agreement with previous reports of orcokinins in the Jonah crab. Orc [1-12], [Val13]-, and [Ala13]orcokinin were identified in the previous MALDI-FTMS study applied to C. borealis.49 [Ser9-Asn13]-orcokinin was found in the CoG of C. borealis in a previous study;20 [Ser9-Val3]-orcokinin has been detected in the brain and thoracic ganglion of C. borealis.29 We did not detect the previously reported Orc [1-11]. Our characteristic neutral loss FIF screen did not reveal any new orcokinin peptides. We also examined tissues from the California spiny lobster, P. interruptus. A previous MALDI-TOF and immunocytochemistry study reported that five orcokinin peptides (2-5 and [Ala13]orcokinin) were present in tissue samples from P. interruptus.20 In contrast with the analysis of H. americanus and C. borealis direct tissue spectra, our search for previously identified orcokinin peptides resulted in no hits, although we did detect a number of other previously identified peptides, such as CapTRP 1a. This result calls into question the orcokinin peptide identifications of the previous MALDI-TOF study20 and shows that P. interruptus tissues contain new variants of the orcokinin family. Analysis of the data in terms of orcokinin neutral loss FIFs resulted in fingerprint ions for seven new orcokinin peptides. The y6+ and y7+ mass region for the CoG of P. interruptus is shown in Figure 7C. We have used exact mass measurements and SORI-CID to

Figure 7. Direct MALDI-FTMS spectra of the y6+ and y7+ regions of tissue samples. (A) CoG of H. americanus, (B) CoG of C. borealis, and (C) CoG of P. interruptus. Spectra measured using DHB as the matrix.

determine the full sequence of the m/z 620.351 peak (see Figure S-2, Supporting Information), and have found that this is the y6+ ion from a new, truncated orcokinin with the sequence NFDEIDRAG[I/L]GF. Exact mass measurements from internally calibrated spectra for the full set of FIF ions are given in Table 3. The characterization of the remaining new members of the orcokinin family is under investigation in our laboratory. A final observation regarding peptide metastable decay under MALDI-FTMS conditions is relevant. The CoG spectra in Figure 7A-C show a common peak at m/z 817.483, an ion with the sequence (HV)FLRFamide, confirmed by SORI-CID experiments,

and exact mass measurements. Analysis of delay before detect data, such as that shown in Figure 5, indicates that this peak is a fragment ion formed by metastable decay, and we believe that the precursors are variants of a SchistoFLRFamide peptide, such as the PELDHVFLRFamide detected in C. borealis.29 The m/z 817.483 fragment is another example of a fragment produced by an Asp-Xxx cleavage. In this case, the C-terminus sequence is retained in formation of the m/z 817.483 fragment, the conserved HVFLRF sequence for this peptide family. Thus, detection of the m/z 817.483 peak provides a common fragment that provides a clear indication that a member of this peptide family is present, Analytical Chemistry, Vol. 77, No. 11, June 1, 2005

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Table 3. Mass Measurements for Pan Orc [1-12] Detected in the CoG of P.Interuptus Calibrated Using InCASa peptide and fragment ions

calculated mass

average measured massb

average error and SD (ppm)b

Pan Orc [1-12], NFDEIDRAG[I/L]GF [M + H]+ [MH - NH3]+ y9+ [y9 - H2O]+ y6+

1353.643 36 1336.616 81 977.505 08 959.494 52 620.351 48

1353.644 90 1336.616 70 977.507 18 959.493 32 620.351 06

1.1 (0.4) -0.08 (0.5) 2.1 (0.4) -1.3 (0.4) -0.7 (0.4)

a PPG was used as the internal calibrant. Peptide signals from a single acquisition with seven accumulated laser shots. b Results from five individually calibrated spectra were averaged to give the reported masses, errors, and standard deviations.

but strategies to identify the precursor, using MALDI-FTMS data, rely on the determination of the neutral amino acid losses. CONCLUSIONS In this study we have extended previous work49 on the application of MALDI FTMS for neuropeptides analysis and show that many peaks in the previously reported spectra result from Asp-Xxx cleavages characteristic of orcokinin peptides and other susceptible neuropeptides. This metastable fragmentation significantly increases the number of peaks appearing in the MALDIFTMS spectra of direct tissue samples and must be recognized when using these data to estimate the total number of peptides that can be detected in neuronal tissue samples. We have shown that Asp-Xxx cleavages can be exploited to provide confident

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identification of peptides from direct tissue samples and describe the application of two data searching strategies that can be used to target previously identified or new members of the orcokinin family of peptides. The high-resolution and accurate mass measurement capabilities of FTMS, coupled with information-rich spectra, make this approach a powerful and reliable tool for the characterization of neuronal tissue samples. ACKNOWLEDGMENT This work was supported by the National Science Foundation MRI-0116416 (E.A.S.) and IBN-0111040 (P.S.D.). We thank Bowdoin College for support through Coles and Doherty Summer Research Fellowships (to H.L.P., M.E.G. and N.P.G.). We thank L. Li for advice on our early work with direct tissue analysis, and L. Li and E. Marder for the kind gift of the orcokinin standards. SUPPORTING INFORMATION AVAILABLE Figure S-1. SORI-CID spectra for ions attributed to Orco [1-12], NFDEIDRSGFGF (4), from the SG of H. americanus. (A) SORI-CID of m/z 1386.60, [MH - NH3]+ ion, n ) 1. (B) Isolation of m/z 670.33 using an arbitrary waveform and CHEF ion isolation. (C) SORI-CID spectrum of m/z 670.33 (y6+ from Orco [1-12]), n ) 10. Figure S-2. SORI-CID of m/z 620.351 from the CoG of P. interuptus. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review February 7, 2005. Accepted April 1, 2005. AC0502347