Detection of In Situ Derivatized Peptides in Microbial Biofilms by

Time-of-flight MS can be used for rapid screening but often does not allow .... Finally, imaging MS of AAC-tagged ERGMT within intact derivatized biof...
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Anal. Chem. 2007, 79, 508-514

Detection of In Situ Derivatized Peptides in Microbial Biofilms by Laser Desorption 7.87 eV Postionizaton Mass Spectrometry Praneeth D. Edirisinghe,† Jerry F. Moore,‡ Kelly A. Skinner-Nemec,§ Carl Lindberg,§ Carol S. Giometti,§ Igor V. Veryovkin,| Jerry E. Hunt,⊥ Michael J. Pellin,| and Luke Hanley*,†

Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois 60607-7061, Materials Science, Chemistry, and Biological Sciences Divisions, Argonne National Laboratory, Argonne, Illinois 60439, and MassThink, Naperville, Illinois 60565-3123

A novel analytical method based on laser desorption postionization mass spectrometry (LDPI-MS) was developed to investigate the competence and sporulation factorsa pentapeptide of amino acid sequence ERGMTs within intact Bacillus subtilis biofilms. Derivatization of the neat ERGMT peptide with quinoline- and anthracenebased tags was separately used to lower the peptide ionization potential and permit direct ionization by 7.87eV vacuum ultraviolet radiation. The techniques of mass shifting and selective ionization of the derivatized peptide were combined here to permit detection of ERGMT peptide within intact biofilms by LDPI-MS, without any prior extraction or chromatographic separation. Finally, imaging MS specific to the derivatized peptide was demonstrated on an intact biofilm using LDPI-MS. The presence of ERGMT in the biofilms was verified by bulk extraction/LC-MS. However, MALDI imaging MS analyses were unable to detect ERGMT within intact biofilms. Biofilms are the natural and favorable environment for certain bacteria.1 It has been reported that 65% of human microbial infections are caused by bacterial growth on surfaces.2 Individual bacteria communicate within biofilms by using signaling molecules in a process referred to as quorum sensing. Both Gram-positive and Gram-negative bacteria use quorum sensing to regulate competence, sporulation, biofilm formation, and other developmental processes.3 Small peptides that behave as quorum sensing species are found extracellularly within Gram-positive bacterial biofilms.4,5 Understanding the identity, distribution, and activity of quorum sensing species will help develop strategies for controlling health problems that arise from biofilms. * Corresponding author. E-mail: [email protected]. † University of Illinois at Chicago. ‡ MassThink. § Biological Sciences Divisions, Argonne National Laboratory. | Materials Science, Argonne National Laboratory. ⊥ Chemistry, Argonne National Laboratory. (1) Microbial Biofilms; Ghannoum, M., O’Toole, G. A., Eds.; ASM Press: Washington, DC, 2004. (2) Costerton, J. W.; Stewart, P. S.; Greenberg, E. P. Science 1999, 284, 1318. (3) Miller, M. B.; Bassler, B. L. Annu. Rev. Microbiol. 2001, 55, 165. (4) Pottahil, M.; Lazazzera, B. Front. Biosci. 2003, 8, 32. (5) Abraham, W.-R. Curr. Med. Chem. 2006, 13, 1509.

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This paper presents a novel analytical method based on laser desorption postionization mass spectrometry (LDPI-MS) to investigate a known quorum sensing peptide within intact Bacillus subtilis biofilms. B. subtilis microbe is a widely studied Grampositive microbe known to produce short peptides as quorum sensing species.4-6 A pentapeptide with the amino acid sequence ERGMT was chosen here as the analyte since it is a nonposttranslationally modified peptide that plays an important quorum sensing role in B. subtilis biofilms.4 ERGMT regulates B. subtilis biofilm competence and sporulation,4,6,7 as well as gene expression through interaction with the aspartyl-phosphate phosphatase intracellular receptors. ERGMT was first identified from planktonic cells (those free floating in media) by growing B. subtilis in media, chemically inducing the peptide, then solidphase extracting and analyzing the peptide by liquid chromatography (LC) and MS.6 Small-molecule identification in biofilms by mass spectrometry is usually preceded by bulk extraction and chromatography.4,8 However, separation methods generally do not preserve information on the spatial distribution of the analyte within the biofilm, undermining attempts to elucidate how quorum sensing species regulate the highly structured, multicellular biofilm. Electron and optical microscopies have been widely used for molecular imaging of biofilms.9,10 Conventional fluorescence and scanning electron microscopy preclude the analysis of thick specimens such as biofilms because they do not distinguish depth information, only probe the biofilm surface, or require disruptive fixation strategies. Fluorescence microscopic imaging of specific molecular analytes requires either that the target species already be identified (i.e., to develop the immunoassay) or that it be part of a general class of compounds (i.e., amine-containing).11 Fluorescence detection also has relatively low chemical resolution, (6) Solomon, J. M.; Lazazzera, B. A.; Grossman, A. D. Genes Dev.. 1996, 10, 2014. (7) Kleerebezem, M.; Quadri, L. E.; Kuipers, O. P.; de Vos, W. M. Mol. Microbiol. 1997, 24, 895. (8) Okada, M.; Sato, I.; Cho, S. J.; Iwata, H.; Nishio, T.; Dubnau, D.; Sakagami, Y. Nat. Chem. Biol. 2005, 1, 23. (9) Hickey, P. C.; Swift, S. R.; Roca, M. G.; Read, N. D. Methods Microbiol. 2005, 34, 63. (10) Neu, T. R.; Lawrence, J. R. Methods Microbiol. 2005, 34, 89. (11) Haugland, R. P. The Handbook: A Guide to Fluorescent Probes and Labeling Technologies, 10th ed.; Invitrogen Corp: Eugene, OR, 2005. 10.1021/ac0615605 CCC: $37.00

© 2007 American Chemical Society Published on Web 12/09/2006

making it difficult to determine the exact chemical speciation of the fluorescing species. Finally, fluorescence measurements can suffer from nonspecific binding and background interferences. Imaging MS is widely applied to study the spatial distribution of proteins, peptides, and small molecular compounds in animal and plant tissues.12-18 For example, diffusion of the antimicrobial chlorhexidine digluconate was detected in Candida albicans biofilms by imaging time-of-flight (ToF) secondary ion mass spectrometry (SIMS), detecting the presence of an antimicrobial agent, the concentration of nutrients, and the viability of the cell population.19 Intact bacteria have also been characterized by wholecell matrix-assisted laser desorption/ionization (MALDI) MS via a proteomic approach, usually by coating intact microbes with a solution of matrix compound or by selectively binding them onto functionalized glass slides.20-23 These various methods in imaging MS are widely applicable, but do not address interference between different analytes, matrix compounds in complex samples,24 or both, nor other issues of chemical complexity or noise in MS. Time-of-flight MS can be used for rapid screening but often does not allow positive molecular identification due to insufficient resolution in complex samples. One solution to these problems is improved selectivity in the MS step by use of Fourier transform MS or tandem MS. However, low biomarker concentrations can defeat such detection strategies. Bacterial biofilms present their own difficulties for MALDI-MS, as discussed further below. Another strategy for molecular imaging MS is to improve detection by use of postionization. Vacuum ultraviolet photoionization (VUV) photon energies are in the range of ionization potentials (IPs) for molecular species, allowing single photon ionization (SPI), which reduces ion fragmentation compared with other laser ionization schemes and produces ion signal intensity directly proportional to the analyte concentration.25 The molecular fluorine laser, which outputs at 157 nm, is currently the only commercially available, high-intensity (∼1 mJ/pulse) VUV source and has several advantages compared to other VUV sources, as previously discussed.26 However, the 7.87-eV photon energy of (12) Cannon, D. M. J.; Pacholski, M. L.; Winograd, N.; Ewing, A. G. J. Am. Chem. Soc. 2000, 122, 603. (13) Todd, P. J.; Schaaff, T. G.; Chaurand, P.; Caprioli, R. M. J. Mass Spectrom. 2001, 36, 355. (14) Roddy, T. P.; Cannon, D. M., Jr.; Meserole, C. A.; Winograd, N.; Ewing, A. G. Anal. Chem. 2002, 74, 4011. (15) Ostrowski, S. G.; Van Bell, C. T.; Winograd, N.; Ewing, A. G. Science 2004, 305, 71. (16) Rohner, T. C.; Staab, D.; Stoeckli, M. Mech. Age. Dev. 2005, 126, 177. (17) Reyzer, M. L.; Caprioli, R. M. MS imaging: New technology provides new opportunities. In Using Mass Spectrometry for Drug Metabolism Studies; Korfmacher, W. A., Ed.; CRC Press: Boca Raton, FL, 2005; pp 305. (18) Rubakhin, S. S.; Jurchen, J. C.; Monroe, E. B.; Sweedler, J. V. Drug Discovery Today 2005, 10, 823. (19) Tyler, B. J.; Rangaranjan, S.; Mo ¨ller, J.; Beumer, A.; Arlinghaus, H. F. Appl. Surf. Sci. 2006, 252, 6712. (20) Ramirez, J.; Fenselau, C. J. Mass Spectrom. 2001, 36, 929. (21) Williams, T. L.; Andrzejewski, D.; Lay Jr., J. O.; Musser, S. M. J. Am. Soc. Mass Spectrom. 2003, 14, 342. (22) Alfonso, C.; Fenselau, C. Anal. Chem. 2003, 75, 694. (23) Wilkins, C. L.; Lay, J. O. Identification of Microorganisms by Mass Spectrometry; Wiley: New York, 2006; Vol. 169. (24) McCombie, G.; Knochenmuss, R. Anal. Chem. 2004, 76, 4990. (25) Hanley, L.; Kornienko, O.; Ada, E. T.; Fuoco, E.; Trevor, J. L. J. Mass Spectrom. 1999, 34, 705. (26) Edirisinghe, P. D.; Moore, J. F.; Calaway, W. F.; Veryovkin, I. V.; Pellin, M. J.; Hanley, L. Anal. Chem. 2006, 78, 5876.

the fluorine laser is lower than the IP of most peptides and other target analytes.27 Several of the authors have previously demonstrated that covalently bound aromatic tags can reduce the IP of a tagged peptide, allowing them to undergo SPI-initiated postionization by 7.87-eV photons.28 For example, anthracene-tagged hexapeptides were detected intact after 7.87-eV SPI.26,29 Multiphoton ionization of aromatic tagged peptides was also demonstrated,30,31 but there are certain advantages of SPI, as previously discussed.26 Perhaps the primary advantage of 7.87-eV SPI of tagged molecular analytes is the selective ionization of only those species whose IPs are below the photon energy, thereby reducing background and interference peaks in the MS. This reduction in MS chemical noise combined with tagging of the target analyte(s) improves the ability to identify the target. Untagged peptides that contain a tryptophan residue can be directly detected without tagging, although they appear to undergo some fragmentation.29 This paper discusses chemical derivatization combined with laser desorption 7.87-eV postionization (LDPI-MS) for the analysis of the ERGMT quorum sensing peptide in B. subtilis bacterial biofilms. Derivatization of the ERGMT peptide with quinoline and anthracene (AQC and AAC, respectively) tags was separately used to lower the peptide IP and permit its direct detection by 7.87-eV LDPI-MS. Previous applications of quinoline tagging of aminecontaining species in complex mixtures utilized either optical detection methods32,33 or MALDI-MS.34 The techniques of mass shifting and selective ionization of the derivatized peptide were combined here to permit detection of the AQC- or AAC-tagged ERGMT peptide within intact biofilms by LDPI-MS, without any prior extraction or chromatographic separation. Imaging MS was then demonstrated on the derivatized peptide within an intact biofilm using LDPI-MS. The presence of ERGMT in the biofilms was verified by bulk extraction/LC-MS. However, MALDI-MS was unable to detect ERGMT within intact biofilms. EXPERIMENTAL DETAILS LDPI-MS Instrument. Details of the 7.87-eV LDPI-MS experiment were described in detail elsewhere26,28,35 and are only summarized here. Scheme 1 is a diagram of the LDPI-MS experiment, in which the desorption laser fired first, the postionization laser fired ∼3 µs later, and extraction to a reflectron timeof-flight MS occurred 0.5 µs after the postionization laser was fired. These delay times and extraction voltages were chosen to optimize signal and resolution by postionizing the maximum fraction of the desorbed neutral molecular plume. The instrument was capable of mass resolution up to 2500 m/∆m and mass accuracy of ∼100 ppm,26 although many of the spectra reported here were (27) King, B. V.; Pellin, M. J.; Moore, J. F.; Veryovkin, I. V.; Tripa, C. E. Appl. Surf. Sci. 2003, 203/204, 244. (28) Edirisinghe, P. D.; Lateef, S. S.; Crot, C. A.; Hanley, L.; Pellin, M. J.; Calaway, W. F.; Moore, J. F. Anal. Chem. 2004, 76, 4267. (29) Hanley, L.; Edirisinghe, P. D.; Calaway, W. F.; Veryovkin, I. V.; Pellin, M. J.; Moore, J. F. Appl. Surf. Sci. 2006, 252, 6723. (30) Li, L.; Lubman, D. M. Appl. Spectros. 1988, 42, 411. (31) Houston, C. T.; Reilly, J. P. J. Phys. Chem. A 2000, 104, 10383. (32) Busto, O.; Guasch, J.; Borrull, F. J. Chromatogr., A 1996, 737, 205. (33) Ovalles, J. F.; del Rosario Brunetto, M.; Gallignani, M. J. Pharm. Biomed. Anal. 2005, 39, 294. (34) Ullmer, R.; Plematl, A.; Rizzi, A. Rapid Commun. Mass Spectrom. 2006, 20, 1469. (35) Veryovkin, I. V.; Calaway, W. F.; Pellin, M. J. Nucl. Instrum. Methods Phys. Res. A 2004, 519, 353.

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Scheme 1. Schematic of the Laser Desorption Postionization Experiment

collected at lower resolution for improved sensitivity. Ions formed by LDPI typically display kinetic energy distributions much smaller than for MALD ions, thereby necessitating different pulsing and extraction parameters. These experiments used a N2 laser (spot size 25 µm2, 337 nm, 20 µJ) for desorption and an F2 laser (cross section 10 mm2, 157 nm, 500 µJ) as the postionization laser. The postionization laser beam was not tightly focused since the LDPI-MS instrument was designed to extract ions from a large volume. The reflectron TOF MS was developed to optimize useful yield (atoms detected per atom consumed) by the use of efficient extraction from a large postionization volume.35 This strategy rendered a large fraction of the desorbed species available for detection and gave a useful yield of >25% for neutral Mo atoms sputtered by secondary neutral mass spectrometry. This instrument was also equipped with a sample motion stage (Ampro, Bloomington, MN) which had ∼100-nm lateral resolution in three orthogonal axes and allowed imaging by rastering the analysis spot with LDPI-MS, SIMS, or SIMS with postionization. Synthesis of AAC and Peptide Derivatization. AQC was obtained commercially (also known as AccQTag Reagent, purchased from Waters, Milford, MA). AAC was synthesized using the same procedure developed for AQC,36 as shown in Scheme 2, from the 2-aminoanthracene (Sigma Aldrich, St. Louis, MO) precursor. The AAC structure was confirmed by MS and nuclear magnetic resonance. Synthetic ERGMT peptide was used as purchased without further purification (Sigma-Genosys, St. Louis, MO). Neat peptides were derivatized by reaction with AQC or AAC dissolved in acetonitrile,32,33,36 leading to the derivatization reaction depicted in Scheme 2. Biofilm Growth and Derivatization. A B. subtilis 168 culture was grown to an optical density of 1.0 in Luria-Bertani (LB) media37 on a desktop shaker. Fresh LB media was put into Petri dishes and inoculated to an optical density of 0.01 from the B. subtilis 168 culture. The dishes were incubated at 32 °C under static conditions for 2 days to allow the biofilm to form at the air-water interface. Part of these biofilms were used for control measurements as nonstarved biofilms. The media was then changed to MSgg,37 a minimal media for starvation. These starved biofilm samples were taken after 1 and 2 days for experiments. The biofilms were derivatized by transferring to an aluminum sample plate, drying in air for several minutes, and then rehy(36) Cohen, S. A.; Michaud, D. P. Anal. Biochem. 1993, 211, 279. (37) Kearns, D. B.; Chu, F.; Branda, S. S.; Kolter, R.; Losick, R. Mol. Microbiol. 2005, 55, 739.

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Scheme 2. (a) Reaction of 6-Aminoquinolyl-N-hydroxysuccinimidyl Carbamate (AQC) with Primary or Secondary Amines To Form Derivatized Species (i.e., AQC-tagged Peptides), (b) Reaction of AQC with Water To Form AMQ, and (c) Preparation of 2-Aminoanthracenyl-N-hydroxysuccinimidyl Carbamate (AAC)

drating with pH 9 phosphate buffer. After heating the sample holder with biofilm to 50 °C, AQC dissolved in acetonitrile was added,32,33,36 leading to derivatization (see Scheme 2). The biofilms were kept solvated with acetonitrile for another 15 min, then dried in a vacuum desiccator, and placed in the ultrahigh vacuum environment of the LDPI-MS instrument. Excess, unreacted AQC should have reacted with water to form 6-aminoquinoline (AMQ, see Scheme 2), which was expected to sublime off the surface under vacuum. A similar procedure was used to derivatize biofilms with AAC. RESULTS AND DISCUSSION B. subtilis biofilms grown in rich LB media did not express the pentapeptide of amino acid sequence ERGMT, but ERGMT was expressed after 1 day of culturing in starvation conditions.6 Finally, the levels of ERGMT dropped dramatically after 2 days of culturing in starvation conditions, after which time the biofilms reverted to a group of spores. These changes in ERGMT levels were confirmed by bulk extraction and LC-MS of large areas of intact biofilms (see Supporting Information). Next, the direct detection of ERGMT in an intact biofilm by MALDI-MS was attempted and failed (see Supporting Information). LDPI-MS was therefore used to detect neat ERGMT that had been derivatized with either AQC or AAC chemical tags. Next, the background signal from an intact biofilm was evaluated during LDPI-MS prior to derivatization. Intact B. subtilis biofilms cultured to enhance or suppress ERGMT production were then derivatized and analyzed by LDPI-MS. Tagged ERGMT, amino acids, and smaller peptides were variously detected within the intact biofilms, depending upon whether or not the biofilm was cultured to produce ERGMT. Finally, imaging MS of AAC-tagged ERGMT within intact derivatized biofilms was demonstrated using LDPI-MS. LDPI-MS of Derivatized Peptides. Anthracene-based tagging was shown particularly effective in lowering the IP of small peptides, permitting 7.87-eV LDPI, shifting the peptide parent ion peak to a higher mass, and overall reducing chemical noise in the relevant region of the MS.26,29 However, the previously

Figure 2. Laser desorption mass spectra background of biofilms (a) with (LDPI-MS) and (b) without postionization (LDMS). (c) Sublimation postionization mass spectra (Sublimated PI MS), without desorption laser.

Figure 1. (a) 7.87-eV LDPI-MS and (b) LDMS of AQC-derivatized ERGMT peptide. Two amine groups allowed two potential derivatization sites, as shown schematically. The AQC- and AAC-ERGMT parent ion peaks appear at m/z 764 and 813, respectively.

described derivatization techniques were found to be incompatible with microbial biofilms whose slightly basic environment was inconsistent with the strongly basic conditions required for derivatization (data not shown). Therefore, the AAC- and AQCbased tagging strategies shown in Scheme 2 were developed to derivatize peptides under slightly basic conditions. Figure 1 (trace a, top) displays the 7.87-eV LDPI-MS of the AQC-ERGMT derivatized pure peptide and shows a strong signal for the derivatized parent ion peak at m/z 764 with no fragmentation or almost any other signal above m/z 200. AQC can bind to any free primary or secondary amine group present in the peptide.32 The two viable AQC binding sites for ERGMT are in arginine (R) or glutamic acid (E), as depicted schematically in Figure 1. The reaction of AQC with water also formed AMQ (see Scheme 2), which appeared as the peak at m/z 144, despite the volatility of this compound. AQC binding also enhanced the desorption/ionization yield of glycopeptides in the presence of nonglycosylated peptides during MALDI-MS,34 and AQC may have similarly enhanced the desorption yield here as well. Derivatization had the added advantage of shifting the mass of the derivatized compound to a higher mass region that had less chemical noise. Figure 1 (trace b, top) displays the laser desorption mass spectrum

(LDMS) of peptide-derivatized AQC (without postionization) and showed no ion signal of any significance. The LDPI-MS of ERGMT peptide derivatized with AAC in Figure 1 (trace c, bottom) displayed a strong parent ion at m/z 813, analogous to that seen for AQC-ERGMT. However, LDPI of AAC-ERGMT also produced fragment ions between m/z 400 and 600 in addition to the strong ion signal observed at lower masses. Tests showed AAC was less soluble in water compared to AQC, presumably due to the larger nonpolar group in AAC. However, the reactivity of AAC was expected to be similar to AQC and allowed use of a similar amounts of either reagent for derivatization. Background Signal during 7.87-eV LDPI-MS. One important question is whether LDPI-MS produces any significant background ion signal from intact biofilms, especially in the mass range of interest to chemical derivatization. Figure 2 displays the 7.87-eV LDPI-MS (trace a), laser desorption without postionization (trace b labeled LDMS), and postionization only of sublimed species (trace c labeled sublimated PI MS) recorded for an underivatized B. subtilis biofilm grown in LB media. The LDMS showed no signal of significance heresonly peaks below m/z 170 due to direct desorption/ionization of very low molecular weight species, fragmentation of higher molecular weight ions, or both. These biofilms were mostly water, and even though they were dried prior to LDPI-MS analysis, they continued to sublime water and other species in the vacuum chamber. However, 7.87-eV postionization of these sublimed species did not show any peaks above the background noise. The LDPI-MS showed much higher signal than the other two spectra in Figure 2 but still displayed no distinguishable peaks above m/z 250. The low-mass species observed in the LDPI-MS might have been tryptophan-containing small peptides, aromatic amines, or a few other species known to ionize via SPI with these low photon energies.26,27,29,38 Whatever biofilm species to which these low molecular weight ions were attributed, the absence of higher mass signal indicated there were no problematic interferences in the underivatized biofilms in the mass range of m/z 550-850 of relevance to the detection of both tagged and untagged ERGMT. (38) Finch, J. W.; Toerne, K. A.; Schram, K. H.; Denton, M. B. Rapid Commun. Mass Spectrom. 2005, 19, 15.

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Figure 3. 7.87-eV LDPI-MS of AQC-derivatized B. subtilis biofilm, grown in LB media (conditions where no ERGMT peptide was expressed). Inset shows expanded upper mass range.

Biofilm Characterization by AQC and AAC Derivatization LDPI-MS. B. subtilis biofilms grown in LB media did not express ERGMT peptide while those grown in starved media for 1 day did express this quorum sensing species.6 AQC derivatization of LB media grown biofilms confirmed this as the m/z 764 peak of AQC-ERGMT was not observed in the LDPI-MS shown in Figure 3. However, this biofilm did display many other low-mass ion peaks during LDPI-MS, which were attributed to AQC-derivatized aminecontaining species.34,36,39,40 There were many possible assignments for these peaks (see Supporting Information), but a definitive identification could not be provided due to the relatively low resolution under which these spectra were collected. However, the most intense peaks in the m/z 200-350 range were attributed to amino acids from dried cells and digested proteins that reacted with AQC. For example, the peak near m/z 300 was attributed to AQC-derivatized asparagine, aspartic acid, isoleucine, or leucine. The m/z 375-450 peaks were attributed to dipeptides present in the biofilm while those in the range of m/z 600-650 were likely tripeptides. AQC derivatization followed by 7.87-eV LDPI-MS allowed experimental monitoring of a variety of amino acids and small peptides within intact biofilms with much higher chemical specificity would be available from fluorescence detection. For example, AQC derivatization could be used to detect the majority of amino acids present in B. subtilis biofilms that have not yet been identified.41 Next, the starved biofilm known to express ERGMT was derivatized with AQC and measured by LDPI-MS. The LDPI-MS of the derivatized starved biofilm shown in Figure 4 (trace a) displayed an m/z 764 peak for AQC-ERGMT (corroborated by the spectrum of the pure AQC-ERGMT shown in trace c), as expected from the LC-MS results (see Supporting Information). Derivatization of ERGMT with AQC shifted its mass upward by m/z 171, thereby moving it above the range of many of these unidentified peaks. Figure 5 (trace a) presents the LDPI-MS of an AAC-derivatized, 1-day-starved biofilm and showed the m/z 813 peak that clearly (39) Bosch, L.; Alegria, A.; Farre, R. J. Chromatogr., B 2006, 831, 176. (40) Oreiro-Garcia, M. T.; Vazquez-Illanes, M. D.; Sierra-Paredes, G.; SierraMarcuno, G. Biomed. Chromatogr. 2005, 19, 720. (41) Stein, T. Mol. Microbiol. 2005, 56, 845.

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Figure 4. LDPI-MS of AQC derivatized B. subtilis, starved biofilm, and ERGMT spiked AQC-derivatized biofilm and AQC-ERGMT peptide standard.

Figure 5. LDPI-MS of (a) AAC-derivatized, starved B. subtilis biofilm (b), AAC-derivatized ERGMT peptide (no biofilm), and (c) biofilm without derivatization.

indicated the presence of AAC-ERGMT. By contrast, the underivatized biofilm did not display this peak (trace c of Figure 5). The LDPI-MS of neat AAC-ERGMT was also shown for comparison in Figure 5 (trace b). Both AQC- and AAC-derivatized biofilms detected by LDPIMS displayed less chemical noise and fewer peaks compared to MALDI-MS of underivatized films (see Supporting Information). Furthermore, both tags showed identifiable fragments in LDPIMS. The AQC-derivatized biofilm LDPI-MS displayed m/z 457 assigned to AQC-b2 and m/z 429 assigned to AQC-a2, both of which were standard peptide fragments43 to which the tag was attached. LDPI-MS of AAC-derivatized biofilms showed analogous fragments including m/z 478 assigned to AAC-a-2, m/z 535 assigned to AAC-a3, m/z 563 assigned to AAC-b3, and m/z 666 assigned to AAC-a4. The relatively low mass resolution made these fragment assignments tentative, but they were nonetheless supported by previous work that found similar tag-attached fragments of peptides during 7.87-eV LDPI-MS.26,28,29 Various xn, yn, and zn fragments were prominent in 7.87-eV photodissociation of independently produced peptide ions,42 but many of those photodis(42) Thompson, M. S.; Cui, W.; Reilly, J. P. Angew. Chem., Int. Ed. 2004, 43, 4791. (43) Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 11, 601.

damage effects did not perturb the data from adjacent laser spots. Previous work on nonbiological samples found that LDPI-MS images gave better contrast and quantitation of the surface concentration compared with direct laser desorption experiments (without added matrix), although comparisons with MALDI-MS were not performed.44,45 Attempts to detect ERGMT peptide within the intact biofilm by MALDI-MS failed (see Supporting Information), so a sensitivity comparison could be made between MALDIMS and LDPI-MS beyond stating that the latter was more sensitive for these intact biofilms. The signal enhancement with LDPI-MS may be its ability to utilize the many laser desorbed neutrals not detected in MALDI-MS25 and the absence of surface charging effects when neutrals rather than ions are desorbed from a surface.

Figure 6. 7.87-eV LDPI-MS contour images of the (a) m/z 813 ( 1 and (b) 177 ( 1 peaks of AAC-derivatized, 1-day-starved biofilms deposited on a gold grid. Tick marks are spaced by 50 µm, the laser spot size was ∼5-µm diameter, and the image was collected by rastering the laser by 25-µm steps. The total image field is 500 × 300 µm, and the units on the color coding are total ion counts.

sociation peaks appeared in the high-noise, lower mass range of the spectra, which were highly congested in LDPI-MS. Furthermore, the spectra presented here were collected under single photon ionization conditions where photodissociation of photoions via a multiphoton process should not occur. The high sensitivity of LDPI-MS was indicated by the observation that the spectrum was recorded from a ∼100-µm2 area of a biofilm. By contrast, the LC-MS identification of ERGMT discussed above required extraction from ∼60 cm2 of biofilms (see Supporting Information). A direct comparison of the film areas required for analysis at a reasonable signal-to-noise ratio indicates an ∼107 higher sensitivity for LDPI-MS compared with LC-MS of bulk biofilm extracts. Adding a known concentration solution of synthetic peptide to the dried, AQC-derivatized biofilm led to ∼20% increase in the m/z 764 peak intensity. This single-point, standard addition experiment was replicated and indicated an average ERGMT concentration of 10 ( 4 pM in 1-day-starved biofilms. The observation of picomolar concentrations of ERGMT within a ∼100-µm2 area of a biofilm indicates the high sensitivity of derivatization followed by 7.9-eV LDPI-MS. The impressive sensitivity for LDPI-MS indicated the feasibility of imaging with LDPI-MS. Figure 6 is the 7.87-eV LDPI-MS image of an AAC-derivatized, starved biofilm. The m/z 813 ( 1 image (part a) corresponded to the AAC-ERGMT parent ion, and the high-intensity region of the spectrum in the center-right side of Figure 6 overlapped with a group of several microbes observed optically (optical data not shown), indicating that those cells released the peptide. The m/z 177 peak shown in part b of Figure 6 was a control and corresponded to signal from unreacted AAC, the smallest ion that was highly available throughout the biofilm and showed a different spatial distribution. The LDPI-MS instrument was capable of rastering the sample with 100-nm resolution compared with the laser position, but the resolution of the image was determined by the laser spot size, which was ∼5 µm in diameter, and possible laser damage effects. The images in Figure 6 were taken at 25-µm steps so that laser

CONCLUSIONS These results have significant implications for mass spectrometric imaging and analysis of molecular species in complex biological mixtures. MALDI and SIMS will remain extremely useful as strategies continue to expand upon the capabilities of commercial instrumentation.13,16-18 However, the sensitivity and selectivity that LDPI-MS affords can offer significant analytical advantages in select circumstances. The quorum sensing peptide studied here was not readily detected in intact biofilms using MALDI-MS, yet derivatization followed by LDPI-MS permitted its selective detection. The 7.87-eV photons can be used to detect many organic species via aromatic tags, which serve to localize the initial SPI event.26,28,29 Analyte derivatization followed by 7.87-eV LDPI-MS imparts selectivity to the analysis of complex biological systems and shifts analyte masses upward, thereby reducing spectral complexity. SPI of various pharmaceutical compounds and other analytically interesting compounds were detected by 7.87-eV LDPIMS even without derivatization.27,38 LDPI-MS also utilizes desorbed neutrals rather than ions and can therefore minimize problems of low ion/neutral ratios in desorption plumes, matrix interferences, competitive analyte peak suppression, and sample charging. The LDPI-MS instrument utilized here did not allow MS-MS or sufficient mass resolution for direct peak confirmation, but it is, in principle, possible to introduce a fluorine laser for postionization into any MS instrument with a MALDI source. For example, LDPI using a 10.5-eV VUV source followed by MS-MS was demonstrated in an ion trap almost a decade ago.46 It is envisioned that improved MS analyses will be coupled to these derivatization/VUV postionization strategies in future work. A significant achievement of LDPI-MS is its ability to record a molecule-specific image, which has been used here to image extremely low concentrations of ERGMT peptide in B. subtilis biofilms. Two new tags were demonstrated that allow derivatization of intact bacterial biofilms. The AAC and AQC tags can be used to derivatize and select amine-containing species for LDPIMS detection. AAC and AQC are also effective fluorescence probes for peptides and amino acids, permitting correlation between MS and optical imaging14 of biofilms for monitoring quorum sensing production rates and other phenomena of biofilm development. However, the techniques presented here will also be applicable (44) Savina, M. R.; Lykke, K. R. Trends Anal. Chem. 1997, 16, 242. (45) Savina, M. R.; Lykke, K. R. Anal. Chem. 1997, 69, 3741. (46) Kornienko, O.; Ada, E. T.; Tinka, J.; Wijesundara, M. B. J.; Hanley, L. Anal. Chem. 1998, 70, 1208.

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to selective, small-molecule imaging of animal and plant tissue in cases where experimental issues prevent the successful application of MALDI-MS and SIMS. ACKNOWLEDGMENT P.D.E. was supported in part by subcontract 5-KD73-P-0015300 from Argonne National Laboratory. Additional funding for this work was provided by the University of Illinois at Chicago. The submitted manuscript has been created (in part) by UChicago Argonne, LLC, Operator of Argonne National Laboratory (“Argonne”). Argonne, a U.S. Department of Energy Office of Science laboratory, is operated under Contract DE-AC02-06CH11357 by UChicago Argonne, LLC and the U.S. Government retains for

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itself, and others acting on its behalf, a paid-up nonexclusive, irrevocable worldwide license in said article to reproduce, prepare derivative works, distribute copies to the public, and perform publicly and display publicly, by or on behalf of the Government. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review August 21, 2006. Accepted October 31, 2006. AC0615605