MALDI MS Sample Preparation by Using Paraffin Wax Film

Anal. Chem. 2008, 80, 491-500

Technical Notes

MALDI MS Sample Preparation by Using Paraffin Wax Film: Systematic Study and Application for Peptide Analysis Junhua Wang, Ruibing Chen, Mingming Ma, and Lingjun Li*

School of Pharmacy and Department of Chemistry, University of WisconsinsMadison, 777 Highland Avenue, Madison, Wisconsin 53705-2222

Recently developed sample preparation techniques employing hydrophobic sample support have improved the detection sensitivity and mass spectral quality of matrixassisted laser desorption/ionization mass spectrometry (MALDI MS). These methods concentrate the samples on target by minimizing the sample area via the solvent repellent effect of the target surface. In the current study, we employed the use of paraffin wax film (Parafilm M) for improved MALDI MS analysis of low-abundance peptide mixtures, including neuronal tissue releasate and protein tryptic digests. This thin film was found to strongly repel polar solvents including water, methanol, and acetonitrile, which enabled the application of a wide range of sample preparation protocols that involved the use of various organic solvents. A “nanoliter-volume deposition” technique employing a capillary column has been used to produce tiny (∼400 µm) matrix spots of 2,5-dihydroxybenzoic acid on the film. By systematically optimizing the sample volume, solvent composition, and film treatment, the Parafilm M substrate in combination with the nanoliter-volume matrix deposition method allowed dilute sample to be concentrated on the film for MALDI MS analysis. Peptide mixtures with nanomolar concentrations have been detected by MALDI time-of-flight and MALDI Fourier transform ion cyclotron resonance mass spectrometers. Overall, the use of Parafilm M enabled improved sensitivity and spectral quality for the analysis of complex peptide mixtures. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has become one of the primary tools for peptide and protein analysis in complex mixtures since its initial introduction.1-4 A key aspect of MALDI MS analysis is the * To whom correspondence should be addressed. Tel: (608)265-8491. Fax: (608)262-5345. E-mail: [email protected]. (1) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (2) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151-153. (3) Caldwell, R. L.; Caprioli, R. M. Mol. Cell. Proteomics 2005, 4, 394-401. (4) Hummon, A. B.; Amare, A.; Sweedler, J. V. Mass Spectrom. Rev. 2006, 25, 77-98. 10.1021/ac701614f CCC: $40.75 Published on Web 01/15/2008

© 2008 American Chemical Society

development of suitable sample preparation methods that enable better incorporation of analytes into matrix crystals, which is important for the desorption and ionization process.5,6 Typically, the sample preparation is performed on a metallic target using a “dried-droplet” method.1 With this technique, matrix and analyte solutions are simply mixed and slowly air-dried to form analytematrix “cocrystals”. However, when the sample solution spreads over a large area on the MALDI sample plate during sample application creating a spot with larger surface area, it will yield decreased signal intensity. The dried samples typically cover a 5-15-mm2 area, but only a minute portion (0.002-0.03 mm2) is laser irradiated for ion generation.7 In addition, the prepared samples frequently suffer from heterogeneity of crystallization that hampers the analysis of specific compounds due to differential incorporation of different analytes in a complex mixture.8-10 A number of methods have been developed to overcome these problems. For example, several hydrophobic supports have been developed to cover the standard stainless steel targets for sample deposition. The sample droplet spreading can be minimized during liquid evaporation on a hydrophobic surface. The shrinking droplets will produce a concentration effect of analytes with greatly reduced spot size on the target surface. Both disposable hydrophobic foil11 and the 3M product Scotch Guard tape12 have been reported as alternative MALDI sample plate surfaces. Furthermore, a wide range of polymeric materials including poly(tetrafluoroethylene) (Teflon),13-16 polyurethane,17 polyethylene, (5) Dreisewerd. K. Chem. Rev. 2003, 103, 395-425. (6) Knochenmuss, R. Analyst 2006, 131, 966-986. (7) Schuerenberg, M.; Luebbert, C.; Eickhoff, H.; Kalkum, M.; Lehrach, H.; Nordhoff, E. Anal. Chem. 2000, 72, 3436-3442. (8) Dai, Y.; Whittal, R. M.; Li, L. Anal. Chem. 1996, 68, 2494-2500. (9) Luxembourg, S. L.; McDonnell, L. A.; Duursma, M. C.; Guo, X.; Heeren, R. M. A. Anal. Chem. 2003, 75, 2333-2341. (10) Garden, R. W.; Sweedler, J. V. Anal. Chem. 2000, 72, 30-36. (11) Rechthaler, J.; Allmaier, G. Rapid Commun. Mass Spectrom. 2002, 16, 899902. (12) Owen, S. J.; Meier, F. S.; Brombacher, S.; Volmer, D. A. Rapid Commun. Mass Spectrom. 2003, 17, 2439-2449. (13) Hung, K. C.; Ding, H.; Guo, B. Anal. Chem. 1999, 71, 518-521. (14) Yuan, X.; Desiderio, D. M. J. Mass Spectrom. 2002, 37, 512-524. (15) Botting, C. H. Rapid Commun. Mass Spectrom. 2003, 17, 598-602. (16) Moyer, S. C.; Budnik, B. A.; Pittman, J. L.; Costello, C. E.; O’Connor, P. B. Anal. Chem. 2003, 75, 6449-6454.

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polypropylene,18,19 paraffin wax,20-22 nitrocellulose,23,24 silicone,25 and linear poly(methyl methacrylate)26 have also been reported as coating materials on MALDI sample plates. However, many of these coating methods suffer from shortcomings including complex preparation protocols, lengthy drying times (4-10 h), the requirement for use of hazardous solvents (e.g., hexane and THF25), or sample carryover effects due to difficulty of removal via standard plate washing procedures. Prestructured sample supports for MALDI MS were introduced by Schurenberg et al. as an improved strategy for coating the sample probes.7 These MALDI supports are characterized by a strongly solvent repellent coating with small (200-800 µm in diameter) hydrophilic anchors or patches. When sample/matrix mixture is deposited onto the target, the droplets will shrink due to solvent evaporation, thus producing concentrated sample spots on the confined anchor positions. By reducing the sample spot size, a larger portion of the concentrated sample can be irradiated by the laser, desorbed, and ionized, thereby improving the sensitivity. More recently, studies using pairs of hydrophilic anchor surfaces to improve dynamic range and mass accuracy were reported.27 However, the fabrication of sample anchors requires special techniques and apparatus that are usually not available in general chemical and biochemical laboratories. Also, the sample anchors can be contaminated after several uses, limiting the lifetime of the probes. Although such Teflon-coated targets (e.g., AnchorChips and µFocus) are now commercially available,28,29 research is ongoing to explore alternative sample substrates with comparable performance and reduced cost. A paraffin wax film, sold under the trade name of Parafilm “M” that is inexpensive and widely used in many laboratories, was previously used as coating material to improve the analysis of DNA30 and peptides16 by MALDI MS. The Parafilm M coating offers many advantages including (1) more homogeneous coverage of matrix/analytes over the sample surface, (2) low cost and easy sample preparation, (3) inert surface that minimizes side reactions, and (4) easy probe cleaning after analysis. In the current study, we systematically investigated the use of Parafilm M coating for complex peptide mixtures. The hydropho(17) McComb, M. E.; Oleschuk, R. D.; Manley, D. M.; Donald, L.; Chow, A.; O’Neil, J. D. J.; Ens, W.; Standing, K. G.; Perreault, H. Rapid Commun. Mass Spectrom. 1997, 11, 1716-1722. (18) Blackledge, J. A.; Alexander, A. J. Anal. Chem. 1995, 67, 843-848. (19) Worrall, T. A.; Cotter, R. J.; Woods, A. S. Anal. Chem. 1998, 70, 750-756. (20) Terry, D. E.; Umstot, E.; Desiderio, D. M. J. Am. Soc. Mass Spectrom. 2004, 15, 784-794. (21) Tannu, N. S, Wu, J.; Rao, V. K.; Gadgil, H. S.; Pabst, M. J.; Gerling, I. C.; Raghow, R. Anal. Biochem. 2004, 327, 222-232. (22) Ham, B. M.; Jacob, J. T.; Cole, R. B. Anal. Bioanal. Chem. 2007, 387, 889-900. (23) Liu, Y.-H.; Bai, J.; Liang, X.; Lubman, D. M.; Venta, P. J. Anal. Chem. 1995, 67, 3482-3490. (24) Montgomery, H. V.; Dodi, I. A.; Tanaka, K. Mol. Cell. Proteomics 2006, 5 (Suppl. S), S10-S10 60. (25) Redeby, T.; Roeraade, J.; Emmer, A. Rapid Commun. Mass Spectrom. 2004, 18, 1161-1166. (26) Jia, W.; Wu, H.; Lu, H.; Li, N.; Zhang, Y.; Cai, R.; Yang, P. Proteomics 2007, 7, 2497-2506. (27) Sjodahl, J.; Kempka, M.; Hermansson, K.; Thorsen, A.; Roeraade, J. Anal. Chem. 2005, 77, 827-832. (28) http://www.bdal.de/modux3/modux3.php?pid)000,000,000,01,01,006,045,0&rid)000,000,000,01,01,001,001,0&width)1280&height)800#5. (29) http://www.maldiplate.com/product_Product_Images.asp. (30) Hung, K. C.; Rashidzadeh, H.; Wang, Y.; Guo, B. C. Anal. Chem. 1998, 70, 3088-3093.

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bic property of the film makes it repellent to several commonly used solvents including water, methanol, and acetonitrile, thereby creating smaller and more concentrated sample spots. This feature makes the support more compatible with various sample preparation protocols including microscale desalting, solid-phase extraction, HPLC elution, or direct tissue extraction techniques that use various compositions of organic solvents. Additionally, the treatment (soaking) of the film with different acidic/alkaline solvents prior to coating, such as acetic acid or ammonium hydroxide, was found to further improve its performance. In combination with the nanoliter-volume deposition technique to produce tiny spots on the film serving as hydrophilic anchors, we can fabricate the “anchor”-type probes in-house with very low cost. These customized nanoliter-volume anchor array probes together with the chemically treated Parafilm M coating offer improved sample preparation and analytical sensitivity for MALDI MS analysis. As the most diverse and complex group of signaling molecules, neuropeptides induce and regulate many important physiological processes throughout the animal kingdom.31,32 Knowledge of complete neuropeptide profiles at the cellular and network levels is critical for a better understanding of peptidergic signaling in a neuronal circuit. Mass spectrometry has been proven as a powerful tool for neuropeptide investigation.4 Several research groups have pioneered the use of various mass spectrometric techniques for different aspects of neuropeptide research.33-36 We recently developed a number of improved sample preparation and instrumentation protocols that enabled us to map peptide distribution and sequence neuropeptides directly from neuronal tissue samples using MALDI FTMS.37-39 However, several technical challenges still exist for the study of neuropeptides. For example, these signaling molecules are active at very low concentrations (pMnM level in vivo), so attomole detection limits are needed for microliter sample volumes. In addition, the analysis is often further complicated by interference of the high salt concentration in circulating fluids. Improved sample processing and preparation methods such as using a self-assembled monolayer40 and a monolayer of glass beads embedded in Parafilm M41 for the study of neuropeptides by MALDI MS have been reported. Here, various aspects of sample preparation for neuropeptide analysis using a Parafilm M substrate, including the nanoliter-volume matrix depositing technique, solvent composition, and choice of film treatment solvent, were optimized using model neuropeptide standards. The utility and improved analytical performance of the (31) Taghert, P. H., and Veenstra, J. A. Adv. Genet. 2003, 49, 1-65. (32) Schwartz, M. W.; Woods, S. C.; Porte, D.; Jr., Seeley, R. J.; Baskin, D. G. Nature 2000, 404, 661-671. (33) Nilsson, C. L.; Karlsson, G.; Bergquist, J.; Westman, A.; Ekman, R. Peptides 1998, 19, 781-789. (34) Baggerman, G.; Cerstiaens, A.; De Loof, A.; Schoofs, L. J. Biol. Chem. 2002, 277, 40368-40374. (35) Li, L. J.; Kelley, W. P.; Billimoria, C. P.; Christie, A. E.; Pulver, S. R.; Sweedler, J. V.; Marder, E. J. Neurochem. 2003, 87, 642-656. (36) Li, L. J.; Pulver, S. R.; Kelley, W. P., Thirumalai, V, Sweedler, J. V.; Marder, E. J. Comp. Neurol. 2002, 444, 227-244. (37) Kutz, K. K.; Schmidt, J. J.; Li, L. J. Anal. Chem. 2004, 76, 5630-5640. (38) Fu, Q.; Kutz, K. K.; Schmidt, J. J.; Hsu, Y. W. A.; Messinger, D. I.; Cain, S. D.; De la Iglesia, H. O.; Christie, A. E.; Li, L. J. J. Comp. Neurol. 2005, 493, 607-626. (39) Fu, Q.; Christie, A. E.; Li, L. J. Peptides 2005, 26, 2137-2150. (40) Wei, H. Dean, S. L., Parkin, M. C., Nolkrantz, K.; O’Callaghan, J. P.; Kennedy, R. T. J. Mass Spectrom. 2005, 40, 1338-1346. (41) Monroe, E. B.; Jurchen, J. C.; Koszczuk, B. A.; Losh, J. L.; Rubakhin, S. S.; Sweedler, J. V. Anal. Chem. 2006, 78, 6826-6832.

new method was demonstrated for the MS analysis of a range of complex peptide mixtures, including protein tryptic digest, signaling peptide releasate, and fractionated neuronal tissue extract. EXPERIMENTAL SECTION Chemicals and Materials. Methanol, acetonitrile (ACN), ammonium hydroxide, trifluoroacetic acid (TFA), and acetic acid were purchased from Fisher Scientific (Pittsburgh, PA). Acetyl chloride was purchased from Sigma-Aldrich (St. Louis, MO). 2,5-Dihydroxybenzoic acid (DHB) was obtained from ICN Biomedicals Inc. Parafilm M was obtained from Pechiney Plastic Packaging (Menasha, WI). C18 Ziptips were manufactured by Millipore, and all water used in this study was doubly distilled on a Millipore filtration system (Bedford, MA). The physiological saline consisted of the following (in mM): NaCl, 440; KCl, 11; MgCl2, 26; CaCl2, 13; Trizma base, 11; maleic acid, 5; pH 7.45. Acidified methanol was prepared using 90% methanol, 9% glacial acetic acid, and 1% water. Neuropeptide Standards. The peptides P1 PFCNAFTGCamide (crustacean cardioactive peptide, 956.38 Da), P3 CYFQNCPRGamide ([Arg8] vasopressin, 1084.44 Da), P5 IARRHPYFL (kinetensin, 1172.67 Da), and P7 APSGAQRLYGFGLamide (allatostatin I, 1335.72 Da) were purchased from the American Peptide Co. (Sunnyvale, CA). P2 SGGFAFSRPLamide (1037.55 Da), P4 GAHKNYLRF (1105.59 Da), and P6 SGKWSNLRGAWamide (1260.66 Da) were synthesized at the Biotechnology Center of the University of Wisconsin at Madison. P8 NFDEIDRSGFGFA ([Ala13] orcokinin, 1474.66 Da) and P9 NFDEIDRSGFGFV ([Val13] orcokinin 1502.70 Da) were synthesized at the Biotechnology Center of the University of Illinois at Urbana-Champaign. Animals. Jonah crabs, Cancer borealis, were shipped from Marine Biological Laboratories (Woods Hole, MA) and maintained without food in an artificial seawater tank at 10-12 °C. Details of the animal treatment and dissection were described previously.37,38 Briefly, animals were cold-anesthetized by packing in ice for 1530 min prior to dissection and sinus glands (SGs) were dissected in chilled physiological saline. Sample Preparation Procedure. For collecting constitutive releasate, two freshly dissected C. borealis SGs were bathed in 10 µL of physiological saline at 4 °C for 1 h. The resulting solution was desalted by ZipTipC18 and eluted in 3 µL of 50% acetonitrile/ 50% water containing 0.1% TFA. The desalted solution was loaded onto the MALDI sample plate for analysis. For the peptide extraction study, 50 freshly dissected SGs were combined and homogenized and peptides were extracted using ice-cold acidified methanol. Twenty microliters of the extract was injected onto a reversed-phase column (2.1-mm i.d., 250-mm length, 5-µm particle size, Alltech Assoc. Inc., Deerfield, IL). The peptides were separated using a Rainin HPLC system and eluted with mobile phase B of 5-95% gradient over 120 min (A, 0.1% TFA in water; B, 0.1% TFA in acetonitrile). Fractions were collected every 1 min and evaporated under vacuum to dryness. Fractions were then reconstituted in 10 µL of 0.1% formic acid and loaded onto the MALDI sample plate for analysis. Bovine Serum Albumin (BSA) Tryptic Digestion. BSA was digested by trypsin (Promega) according to the procedures provided by the supplier. Briefly, 200 µg of BSA was dissolved in 200 µL of 6.0 M urea in 200 mM sodium acetate buffer. The solution was then mixed with 10 µL of 200 mM dithiolthreitol

Figure 1. Schematic diagram of the capillary deposition device for matrix spotting. The 0.5-mL DHB matrix was loaded in a 2.5-mL syringe. A 10-cm-long capillary with 50-µm i.d. was inserted into the needle. The capillary was bent to form a circle and fixed onto the needle tube with a 3-cm protruding end to contact the target surface and leave a droplet of matrix.

(DTT, Promega) and incubated at 37 °C for 1 h. An aliquot of 8.0 µL of 1.0 M iodoacetamide (ICN Biomedicals, Inc.) was added, and the solution was kept in dark and incubated for 1 h at room temperature. An aliquot of 40 µL of 200 mM DTT was then added to quench the alkylation reaction. The solution was diluted with 10% ACN in water to 1.2 mL to bring the BSA concentration to 1/6 µg µL-1 (2.4 µM) and the urea concentration to 1.0 M. The sample was incubated with 4 µg of trypsin at 37 °C overnight. Film Treatment and Coating. A piece of Parafilm M was cut to the size of 2.5 cm (length) × 0.4 cm (width) and stretched to ∼8 cm in length while keeping the same width. The obtained sheet was directly placed onto the probe for sample deposition.30 Alternatively, the film was softened by treatment with solution. Briefly, the stretched film was dipped in the treatment solution for 1 h or longer (less than 24 h). Acetic acid (100%), ammonium hydroxide (28.8%), and 1% TFA aqueous solution were tested as the soaking solvent, respectively. The droplets of sample solution were observed to form a nice nonspreading “bead” on the surface when using acetic acid and ammonium hydroxide for film treatment, whereas the droplet would spread severely on the surface by using 1% TFA treatment. The soaked film was then removed from solution using forceps, allowed to completely dry, and further stretched to form an ultrathin film (∼16 cm in length) prior to being placed onto the target. For MALDI TOF experiments, a 26-well plate with 3-cm diameter was used, and for MALDI FTMS experiments, a 192-well sample plate in 4.5 cm × 4.5 cm dimensions was used (Applied Biosystems). The stretched film was ∼20 µm in thickness. When it was placed over the two edges of the MALDI probe, the film automatically affixed onto the sample probe surfaces without the need of adhesive tape. After each use, the film can be easily removed from the target without any washing procedure. Matrix Spotting and Sample Preparation. The on-film matrix spotting was performed using a capillary deposition technique. Briefly, 0.5 mL of 150 mg/mL DHB, freshly prepared in 1:1 methanol/water, was loaded in a 2.5-mL syringe, with a 10-cm-long, 50-µm (i. d.) capillary section placed in the needle (see Figure 1). The capillary was bent toward the syringe to form a circle and fixed on the needle tube with a 3-cm protruding end. When the solution was pushed from the needle (in a horizontal position), the matrix flowed out of the capillary and deposited perpendicularly onto the target (or film). A series of DHB matrix spots of 50-100 nL was produced manually at a flow rate of 20 nL/s with a 2.5-5-s interval. The matrix spot size was ∼400800 µm due to the solvent repellent property of the film. The typical size of DHB spots used for sample application was ∼400 µm in diameter in the following experiments (Figure 2A1). Analytical Chemistry, Vol. 80, No. 2, January 15, 2008

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Figure 2. Photographs showing the sample applying and drying process on Parafilm M (no. 1 well) and bare target (no. 2 well). In each well, ∼50 nL of DHB was deposited by a capillary and then 0.6 µL of sample was applied. (A) Predeposited DHB spots, (B) after applying samples, and (C) after samples dried. The diameters of the wells and spots were determined and labeled.

An aliquot of 0.3-1.2 µL of sample was then applied onto the dried DHB spots on the sample probe (Figure 2B1) while evaporating the solvent to dryness using gentle ventilation to yield highly concentrated sample/DHB spots on the film-coated MALDI plate (Figure 2C1). The conventional on-target MALDI sample preparation was performed by spotting an aliquot of 0.3-1.0 µL of analyte solution onto an individual well on the bare MALDI sample plate followed by mixing with 0.5 µL of 150 mg/mL DHB and letting it dry at room temperature. Unless noted otherwise, all of the “on target” experiments mentioned below refer to this method (i.e., no film was used). Instrumentation. MALDI TOFMS experiments were performed on a Bruker Reflex II time-of-flight mass spectrometer (Billerica, MA) equipped with a pulsed nitrogen laser (337 nm). All spectra were recorded in the positive ion reflectron mode, with delayed ion extraction (∼150 ns) and a 25-kV accelerating voltage. MALDI FTMS experiments were performed on a Varian IonSpec Fourier transform mass spectrometer (Lake Forest, CA) equipped with a 7.0-T actively shielded superconducting magnet. The FTMS instrument consisted of an external high-pressure MALDI source. A 355-nm Nd:YAG laser (Laser Science, Inc., Franklin, MA) was used to create ions that can be accumulated in the external hexapole storage trap before being transferred through a quadrupole ion guide to the ICR cell. All mass spectra were collected in positive ion mode. The ions were excited prior to detection with an rf sweep beginning at 7050 ms with a width of 4 ms and amplitude of 150 V base to peak. The filament and quadrupole trapping plates were initialized to 15 V, and both were ramped to 1 V from 6500 to 7000 ms to reduce baseline distortion of peaks. Detection was performed in broadband mode from m/z 108.00 to 2500.00. Fragmentation of the peptides was accomplished by sustained off-resonance irradiation and collision-induced dissociation (SORI-CID). An arbitrary waveform with a (10 Da isolation window was introduced to isolate the ion of interest during the period of 2000-2131 ms. Ions were excited with a SORI burst excitation (2.648 V, 2500-3000 ms). A pulse of N2 was introduced through a pulse valve from 2500 to 2750 ms to elevate the pressure to 10-6 Torr that induced collision activation leading to subsequent fragmentation. 494

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RESULTS AND DISCUSSION Systematically Optimizing the Method of Using Parafilm M as Sample Support. For conventional on-target MALDI sample preparation, usually an aliquot of 0.3-0.5 µL of analyte solution was applied and mixed with an equal volume of matrix on target. In this case, a sample is often spread during the matrix cocrystallization process, thus resulting in a large sample area and reduced sensitivity because only a very small fraction (0.110%) of this area can be irradiated and ionized for detection.7 This phenomenon, as a result, has greatly limited the volume of the sample one could load onto the MALDI sample plate for analysis. In this study, the Parafilm M coating was found to be strongly repellent to several polar solvents, including water, methanol, and acetonitrile, among others. This property enables the shrinking of a much larger volume of sample solution onto the predeposited tiny DHB matrix spots that serve as the hydrophilic anchors on the hydrophobic film surface without spreading extensively. We observed ∼3-fold improvement in S/N using the Parafilm M method when a regular sample volume of 0.3-0.5 µL was applied (see Figure S-1 Supporting Informaion, SI). Nevertheless, the optimal loading volume was found to be 0.6-1.2 µL for the Parafilm M coating substrate, which was determined with varying sample solution composition containing different amounts of water or organic solvent. The size and morphology of matrix spots could also affect the volume of sample to be loaded. We examined a range of DHB concentrations at 50, 75, 100, 125, and 150 mg/mL in different methanol/water composition solutions. It was found that a resulting spot of ∼400 µm diameter in a spherical shape was ideal for sample application (Figure 2A1), with a DHB concentration at 150 mg/mL in 1:1 methanol/water yielding the best MS performance among others. The effect of solvent composition was reported to be critical for sample preparation in MALDI MS. A number of approaches have been developed to improve the solubility of different classes of analytes according to their polarity (hydrophilicity) and acidity for MALDI analysis.42-44 In the present report, the sample prepared in doubly distilled water (DDW) was also found to behave differently on the Parafilm M from that in solvent containing different ACN compositions. We tested solvents containing 25, 50, and 75% ACN for sample preparation and found that 75% ACN enabled the fastest evaporation while 25% ACN produced sample spots with the smallest diameter. Overall, the samples prepared in 30-50% ACN yielded the best signal (see Figure S-3 SI). Soaking the Parafilm M in weak acid or base solution was found to greatly increase the expandability of the film, thus enabling the production of a thinner film for coating via stretching. Both acetic acid (100%) and ammonium hydroxide solution (28.8%) are good alternatives for the soaking treatment. The soaking of 1-24 h exhibits nearly identical peptide ion signals and spectral quality; thus, 1-h soaking time was used for the remainder of this study. Soaking can also minimize contamination from the film via washing and drying procedures. An unknown peak at m/z 1199.80 that may correspond to the wax paraffin occasionally appeared in (42) Hoteling, A. J.; Erb, W. J.; Tyson, R. J.; Owens, K. G. Anal. Chem. 2004, 76, 5157-5164. (43) Breaux, G. A.; Green-Church, K. B.; France, A.; Limbach, P. A. Anal. Chem. 2000, 72, 1169-1174. (44) Loo, R. R. O.; Loo, J. A. Anal. Chem. 2007, 79, 1115-1125.

the spectra with a low to moderate intensity scale. This peak was suppressed to very low level or completely eliminated by the soaking treatment (see Figure S-5 SI). The ammonium hydroxidetreated film was found to be more effective and yielded a slightly better sensitivity than acetic acid-treated film (see Figures S-4 and S-5 SI). The peptides with concentration down to 5 nM were detected on treated film (Figure 3D). Based on the above optimized conditions, a group of model neuropeptide standards, P1-P9, was chosen to evaluate the performance of the Parafilm M method. Figure 3 compares the MALDI FTMS spectra of the nine-standard mixture at a concentration of 5-16 nM using conventional bare target deposition and the Parafilm M-coated sample support method. Using the conventional on-target dried-droplet deposition method, most of these peptides were undetectable (P1, P2, and P5 were barely detectable); only P3 and P4 were detected at a decent intensity scale (Figure 3A). The same peptide standard mixture yielded much more intense ion signal profiles on the Parafilm M-coated sample plate (Figures 3B-D). For example, at least 5-fold improvement in S/N was achieved for the peptides (P1-P5) commonly detected in both Figures 3A and B (film coated). Furthermore, three additional peptides including P7-P9 were readily detected with film-coated sample plate, which were missing in the spectrum collected from the bare target deposition method. The S/N was further improved by applying a larger volume of sample (1.2 and 0.9 µL instead of 0.6 µL) (see Figure S-2 in the Supporting Information). In addition, the effect of solvent composition on MS performance and signal coverage was examined. As shown, a new peptide peak P6 was detected at a considerable intensity in the same peptide mixture prepared in 30% ACN/70% water (A/W) (Figure 3C) that was missing in the DDW solution (Figure 3B). A 2-fold dilution of the sample in 30% ACN/70% water with each analyte at 5-8 nM concentration still revealed the detection of all of the nine peptide peaks (Figure 3D). However, the same dilution prepared in pure water (DDW) only yielded three most abundant peptide peaks, P2-P4 (data not shown). This result indicated that using an organic containing solvent may provide better extraction and coverage of a wider range of peptides with varying physical and chemical properties in a complex mixture than using pure aqueous solvent. Keller and Li previously reported a similar method to use organic solvent for enhanced extraction of hydrophobic analyte.45 Furthermore, the volatile components are considered to yield more rapid solvent evaporation that enables better incorporation of analyte into matrix crystals as compared with the slower drying samples.46-48 For our predeposited DHB method used here, the organic component can dissolve the matrix top layer during the evaporation process and, thus, form an even and thin cocrystal layer facilitating the desorption/ionization process of MALDI.49 Analysis of BSA Tryptic Digest Using Parafilm M-Coated Sample Plate. To further evaluate the utility and analytical (45) Keller, B. O.; Li, L. Anal. Chem. 2001, 73, 2929-2936. (46) Figueroa, I. D.; Torres, O.; Russell, D. H. Anal. Chem. 1998, 70, 45274533. (47) Koomen, J. M.; Russell, W. K.; Hettick, J. M.; Russell, D. H. Anal. Chem. 2000, 72, 3860-3866. (48) Vorm, O., Roepstorff, P., Mann, M. Anal. Chem. 1994, 66, 3281-3287. (49) McCombie, G.; Knochenmuss, R. J. Am. Soc. Mass Spectrom. 2006, 17, 737-745.

performance of the Parafilm M sample deposition method for complex peptide mixture analysis, a BSA tryptic digest was used. The resulting BSA tryptic digest with a starting concentration at 2.4 µM (containing 1.0 M urea/NaAc) and its 50-400-fold dilutions were applied to bare target and Parafilm M support for analysis, respectively (Figure 4 and Table S-2 SI). Figure 4A shows the result of un-desalted sample directly from the digest products using a conventional bare target deposition method. As seen, 14 peaks corresponding to the BSA tryptic fragments were detected with the amino acid coverage as low as 27%. In addition, many unassigned peaks were seen in the spectrum, which may come from the salt adduct and noise. The signals were improved dramatically after desalting with ZipTip, with 24 peaks identified and amino acid sequence coverage improved to ∼44% (Figure 4B). The un-desalted BSA digest was diluted by 50-, 100-, 200-, and 400-fold and subjected to analysis on both bare target and Parafilm M support. As we can see from Figure 4C, a 50-fold (48.0 nM)diluted sample yielded a result comparable to the desalted BSA tryptic digest at its original concentration in terms of detected peptide peak numbers (21) and amino acid sequence coverage (41.2%). Further reduced concentrations of digested samples (>100-fold dilution) on the bare target yielded fewer peaks or almost no signals, presumably due to insufficient sensitivity (data not shown). In contrast, by using the Parafilm M method, the >100-fold dilution tryptic samples still yielded high-quality mass spectra. As an example, the result of 400-fold-diluted (6.0 nM) sample is shown in Figure 4D. The intensity scale is comparable to the 50-fold-diluted sample deposited on bare target, with much reduced salt adduct and noise peaks. In total, 33 tryptic peptide peaks including more low-mass peptides were identified and assigned to the BSA tryptic fragment, with the highest sequence coverage (48%) among all four preparations (Table S-2 SI), demonstrating a very high concentrating factor on the Parafilmcoated sample surface in comparison to bare target deposition method. The total amount of BSA used here was ∼3.6 fmol (0.6 µL at 6.0 nmol/L), which showed nearly 30-fold improvement to that (100 fmol) reported by Moyer et al.16 using the same Parafilm M support but with a conventional matrix deposition method. Furthermore, the sensitivity improvement for BSA analysis using our new protocol was 4.5-fold better than that using an imprinted hydrophobic polymer support as recently reported by Jia et al.26 Profiling of Constitutive Release from C. borealis Sinus Glands. The crustacean SG is a well-defined neuroendocrine structure that produces numerous hemolymph-borne agents including many neuropeptides. Previously, we examined neuropeptide profiles of the individual SGs and pooled tissue extract of the red rock crab, Cancer productus, by a combination of anatomical and mass spectrometric methods.37-39, 50 In an ongoing effort to investigate the putative hormonal roles of the peptides present in the SGs, we performed a mass spectrometric assay for the peptides secreted constitutively in physiological saline following tissue dissection. Figure 5 shows the MALDI TOF mass spectra of the C18 ZipTip desalted sample on the bare target (Figure 5A) and on the film (Figure 5B). It can be seen from the spectra that the signals of most peaks were improved by 310-fold on film compared to those on the bare target. There were (50) Fu, Q.; Goy, M. F.; Li, L. J. Biochem. Biophys. Res. Commun. 2005, 337, 765-778.

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Figure 3. MALDI-FT mass spectra of 9-peptide standard mixtures. (A) On bare target; (B-D) on Parafilm M with predeposited DHB. (A, B) Sample in DDW, (C, D) sample in 30%/70% ACN/H2O (A/W). Peptide concentrations (in nM) in (A-C) are P1 12, P2 11, P3 16, P4 14, P5 15, P6 16, P7 10, P8 10, and P9 10. (D) Two-fold-diluted sample. An aliquot of 0.6 µL of sample was applied to each substrate. One scan was taken after the accumulation of the ions from 50 laser shots with the same laser power. Noise peaks are noted with asterisks.

11 major peaks detected in the mass range of m/z 800-4500 from the sample spotted on the bare target. Many of these peaks were 496 Analytical Chemistry, Vol. 80, No. 2, January 15, 2008

detected with higher abundance from the sample spotted on the film. In addition, seven putative peptide peaks were uniquely

Figure 4. MALDI FT mass spectra of BSA tryptic digest. (A-C) on bare target. (D) On Parafilm M with predeposited DHB. Sample details are labeled on each spectrum. An aliquot of 0.6 µL of sample was applied to each substrate. One scan was taken after the accumulation of the ions from 100 laser shots with the same laser power. Noise and unidentified peaks are noted with asterisks and number signs, respectively.

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Figure 5. MALDI-TOF mass spectra of C. borealis SG lysate on (A) target or (B) Parafilm M with predeposited DHB. (C) The product ion spectrum of the precursor MH+ 844.5 by MALDI TOF/TOF. An aliquot of 0.5 µL of sample was applied to both support substrates. The peptides were eluted in 50% ACN aqueous solution with ZipTip for desalting. The intensity scales of (A) and (B) were 4000 and 12 000, respectively. Seven additional peaks were detected on the Parafilm M-coated support (B), as underlined. The six peaks noted with asterisks corresponded to previously identified neuropeptides: HIGSLYRamide (MH+ 844.48), [Ala13]-orcokinin (MH+ 1474.66), [Val13]-orcokinin (MH+ 1502.70), and CHH precursor-related peptides (CPRPs) including CPRP-I (MH+ 3963.05), CPRP-II (MH+ 3977.07), and CPRP-III (MH+ 3991.08), respectively.

detected in the sample spotted on the film-coated target. Among these putative peptide peaks, six corresponded to previously known neuropeptides in SG tissue including HIGSLYRamide (MH+ 844.48),51 [Ala13]-orcokinin (MH+ 1474.66), [Val13]-orcokinin (MH+ 1502.70), and crustacean hyperglycemic hormone (CHH) precursor-related peptides (CPRPs) including CPRP-I (MH+ 3961.05), CPRP-II (MH+ 3977.07), and CPRP-III (MH+ 3992.08).50 It is notable that [Val13]-orcokinin and CPRP-III were not detected using the conventional dried-droplet method on the bare target. (51) DeKeyser, S. S.; Kutz-Naber, K. K.; Schmidt, J. J.; Barrett-Wilt, G. A.; Li, L. J. Proteome Res. 2007, 6, 1782-1791.

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The identities of these putative peptide peaks were verified by peptide sequencing experiments performed either with ESIquadrupole time-of-flight or MALDI TOF/TOF instruments. Figure 5C shows a representative product ion spectrum of the precursor ion MH+ 844.5 by MALDI TOF/TOF collisional-induced dissociation (see ref 51 for the MALDI TOF/TOF instrument and setting information). As shown, sequence-specific fragment ions and several immonium ions that are indicative of specific amino acid residues are detected, confirming the sequence assignment. Numerous other peaks were detected for the first time in this report, possibly due to the degradation of large proteins or

Figure 6. MALDI-FT mass spectra of HPLC fraction 65 from C. productus SG extract. (A) On Parafilm M with predeposited DHB. (B) On bare target. An aliquot of 1.0 µL of sample was applied to both support substrates. The m/z 1098.52 and 1256.55 peaks correspond to previsouly identified neuropeptides EIDRSGFGFA and NFDEIDRSGFG, respectively. (C) SORI-CID fragmentation spectrum of MH+ 1256.55 of (A) with b/y ions and internal fragment ions labeled. The derived amino acid sequence is shown above the spectrum. Noise peaks are noted with asterisks.

released peptides. Although some of the tandem mass spectra (e.g., MH+ 1458.6) were available, they do not match any known neuropeptides and more detailed structural characterization and sequence verification is underway. Analysis of HPLC Fractions from C. productus SG Tissue Extract. HPLC fractions of pooled C. productus SG tissue extract were analyzed by MALDI FTICR MS. Putative peptide signals detected from these fractions were improved by 3-5-fold with the Parafilm M method compared to those obtained on the bare target. Panels A and B in Figure 6 show the representative mass spectra of a 1-min fraction collected from an HPLC separation prepared on two support substrates. Two known peptides, EIDRSGFGFA (MH+ 1098.52) and NFDEIDRSGFG (MH+ 1256.55), were detected in this fraction. As seen in Figure 6, the ion signals of both peptides increase by 3-4-fold on the NH4OH-treated Parafilm M (A) compared to the bare target (B). This signal improvement

enabled fragmentation analysis by SORI-CID for sequence confirmation for spectrum collected on Parafilm M substrate (A). As shown in Figure 6C, most of the major fragment ions were detected by performing a single-scan SORI-CID experiment and can be assigned to sequence-specific product ions. This result highlights the utility of the film-coated sample support for improved analysis of various biological samples ranging from HPLC fractions to tissue extract. CONCLUSIONS In summary, this study has demonstrated an improved sample preparation method using a Parafilm M support for MALDI MS analysis of complex peptide mixtures. The spectra obtained with the Parafilm M coating are superior to those obtained on a stainless steel target, in terms of spectral quality and sensitivity. Compared to other reported prestructured targets, Parafilm M Analytical Chemistry, Vol. 80, No. 2, January 15, 2008

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with predeposited DHB matrix allows sensitive measurements of peptides at femtomole levels without any extra cost or labor. The film coating process is straightforward and is widely applicable to various types of sample plates and instruments. These features make it an attractive alternative for routine sample preparations in various laboratories. Furthermore, Parafilm M with an array of predeposited DHB spots offers the potential to interface microscale separation methods such as capillary LC or capillary electrophoresis offline with MALDI MS for high-throughput screening applications. ACKNOWLEDGMENT We thank Xin Wei from the Li laboratory for taking photographs used in Figure 2 and Stephanie Cape for critical reading of the manuscript and helpful discussions on the use of Parafilm M support. Dr. Andrew E. Christie (Department of Biology, University of Washington) is acknowledged for providing C. productus sinus gland tissue used in HPLC fractionation. The authors thank the UW School of Pharmacy Analytical Instrumentation Center for access to the MALDI FTMS instrument. We also thank Dr. Martha M. Vestling for initial training and access to the MALDI TOF instrument (Department of Chemistry Mass Spectrometry Facility at UWsMadison). The authors thank the

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University of Wisconsin-Biotechnology Center Mass Spectrometry Facility, Dr. Amy Harms, and Dr. Mike Sussman for access to the MALDI-TOF/TOF instrument. This work was supported in part by the School of Pharmacy and Wisconsin Alumni Research Foundation at the University of WisconsinsMadison, a National Science Foundation CAREER Award (CHE-0449991), and National Institutes of Health through Grant 1R01DK071801. L.L. acknowledges an Alfred P. Sloan Research Fellowship. SUPPORTING INFORMATION AVAILABLE The method optimization using large-volume vs small-volume samples; aqueous solvent vs organic/aqueous solvents; acetic acid vs ammonium hydroxide-treated Parafilm M. The control experimental data and further discussions on the Parafilm M method vs premixed dried droplet method and using the film without stretching or without treatment. The results of BSA digests are summarized in Table S-2. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review July 31, 2007. Accepted October 17, 2007. AC701614F