Ionization-Mass Spectrometry Using Self

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Matrix-Free Laser Desorption/Ionization-Mass Spectrometry Using Self-Assembled Germanium Nanodots Teruyuki Seino,†,‡,§ Hiroaki Sato,*,‡ Atsushi Yamamoto,| Atsushi Nemoto,‡ Masaki Torimura,‡ and Hiroaki Tao‡

New Energy and Industrial Technology Development Organization (NEDO), 1310 Omiya, Saiwai, Kawasaki 212-8554, Japan, Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba, Ibaraki 305-8569, Japan, and Energy Technology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan

A novel ionization platform for matrix-free laser desorption/ionization-mass spectrometry (LDI-MS) was developed using self-assembled germanium nanodots (GeNDs) of uniform size (∼150-200-nm width and ∼50-nm height) grown on a silicon wafer produced by molecular beam epitaxy. The performance of LDI-MS using GeNDs (GeND-MS) was investigated through measurements of a broad range of analytes, including peptides, proteins, synthetic oligomers, and polymer additives. Mass spectra of tryptic digests were clearly observed even for the mass range lower than m/z 800 without obstructive peaks. A detection limit of subfemtomole level was achieved for angiotensin-I. The upper limit of detectable mass range was ∼17 kDa (myoglobin). GeND-MS also has potential for application to the characterization of industrial compounds. Almost accurate molecular weight distribution was obtained for a nonionic surfactant (Triton X-100) and for poly(ethylene glycol) oligomer. Furthermore, a brominated flame retardant, tetrabromobisphenol-A bis(2,3dibromopropyl ether), was successfully ionized with less fragmentation, a result not obtainable by matrix-assisted laser desorption/ionization-mass spectrometry or desorption/ionization on porous silicon-mass spectrometry. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has been widely used in the field of biological analyses and synthetic polymer characterization. Originally, the discovery by Tanaka et al.1 of cobalt ultrafine powder combined with liquid glycerol as a matrix opened the door to the effective soft laser desorption/ionization-mass spectrometry (LDI-MS) of macromolecules. The success since then of MALDI-MS using organic matrixes, introduced by Karas and Hillemkamp,2 has * To whom correspondence should be addressed. Fax: +81 (29) 861-8308. E-mail: [email protected]. † NEDO. ‡ Research Institute for Environmental Management Technology, AIST. § Present address: Hakodate National College of Technology, 14-1 Tokura, Hakodate, Hokkaido 042-8501, Japan. | Energy Technology Research Institute, AIST. (1) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151-153. (2) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. 10.1021/ac062216a CCC: $37.00 Published on Web 06/02/2007

© 2007 American Chemical Society

somewhat eclipsed LDI using a cobalt matrix. MALDI-MS using organic matrixes has, however, several disadvantages. A suitable matrix needs to be selected depending on the analyte. Furthermore, strong background peaks originating from the matrix frequently interfere with MALDI mass spectra in the lower mass range under m/z ∼800 and sometimes exceed several thousand, depending on the matrix, causing a serious impediment to the measurement of lower molecular weight compounds. To avoid generating the obstructive peaks caused by the use of organic matrixes, interest has refocused on several kinds of metal and metal oxide particles, such as Al, Mn, Mo, Si, Sn, TiO2, W, WO3, Zn, and ZnO,3 sol-gel-deposited TiO2,4 ordered mesoporous WO3-TiO2,5 and gold nanoparticles.6 MALDI has also been achieved by using inorganic carbons such as graphite microparticles,7 C60,8 and carbon nanotubes.9-11 These techniques using nonvolatile inorganic matrixes are also referred to as surfaceassisted laser desorption/ionization (SALDI).5,13 At the present time, however, inorganic matrixes have not been widely adopted, probably for reasons of low sensitivity, less versatility, and the risk of laser irradiation scattering the particles inside the ionization chamber of the MS instrument. The use of the functional surfaces of a sample substrate as an ionization platform, namely, matrix-free LDI-MS, has been proposed (see review by Peterson).12 The typical method adopted in this approach is known as desorption/ionization on porous silicon(3) Kinumi, T.; Saisu, T.; Takayama, M.; Niwa, H. J. Mass Spectrom. 2000, 35, 417-422. (4) Chen, C.-T.; Chen, Y.-C. Rapid Commun. Mass Spectrom. 2004, 18, 19561964. (5) Yuan, M. Y.; Shan, Z.; Tian, B.; Tu, B.; Yang, P.; Zhao, D. Microporous Mesoporous Mater. 2005, 78, 37-41. (6) McLean, J. A.; Stumpo, K. A.; Russell, D. H. J. Am. Chem. Soc. 2005, 127, 5304-5305. (7) Sunner, J.; Dratz, E.; Chen, Y. C. Anal. Chem. 1995, 67, 4335-4342. (8) Cornett, D. S.; Amster, I. J.; Duncan, M. A.; Rao, A. M.; Eklund, P. C. J. Phys. Chem. 1993, 97, 5036. (9) Xu, S.; Li, Y.; Zou, H.; Qiu, J.; Guo, Z.; Guo, B. Anal. Chem. 2003, 75, 61916195. (10) Ren, S.-F.; Guo, Y.-L. Rapid Commun. Mass Spectrom. 2005, 19, 255-260. (11) Ren, S.-F.; Zhang, L.; Cheng, Z.-H.; Guo, Y.-L. J. Am. Soc. Mass Spectrom. 2005, 16, 333-339. (12) Peterson, D. S. Mass Spectrom. Rev. 2007, 26, 19-34. (13) Dattelbaum, A. M.; Iyer, S. Expert Rev. Proteomics 2006, 3, 153-161.

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mass spectrometry (DIOS-MS).14 DIOS-MS using porous silicon as an ionization platform has been widely applied to the analysis of small molecules such as peptides, drugs, surfactants, and synthetic polymers (oligomers).15-23 DIOS, however, also has several disadvantages. To maintain surface performance, chemical modifications of the etched silicon surface are needed. Nevertheless, surface activation is further required by immersion in organic solvents, typically methanol or 2-propanol overnight prior to measurement. Moreover, the porous silicon structure is too brittle and fragile for even slightly higher laser irradiation. Several recent designs of ionization substrates for matrix-free approaches are based on the nanotechnology techniques used in semiconductor processing. Cuiffi et al.24 have applied column/ void-network silicon thin films prepared by plasma-enhanced chemical vapor deposition in matrix-free LDI-MS. Go et al.25 reported matrix-free LDI-MS using single-crystal silicon nanowires (SiNWs) deposited on silicon wafers. In their report, SiNWs required significantly lower laser power than MALDI or DIOS, therefore reducing background noise peaks from the SiNWs surface. Finkel et al.26 evaluated an ordered arrayed silicon nanocavity that was lithographically fabricated on a silicon wafer for soft ionization by LDI-MS. Okuno et al.27 also studied an ionization substrate with ordered arrays of submicrometer groove structures. In this paper, we propose a novel ionization substrate for matrix-free LDI-MS using a germanium nanodot (GeND) chip. GeNDs are nanoscale crystalline dots made by molecular beam epitaxy (MBE). GeNDs with a few nanometers in size, known as typical “quantum dots,” are believed to have potential for use in future electronic and optical devices.28,29 Germanium atoms slowly deposited on a silicon single-crystal surface by MBE self-assemble into nanoscale dots due to the 4.2% lattice mismatch between germanium and silicon.30 Since the resulting interface between (14) Wei, J.; Buriak, J. M.; Siuzdak, G. Nature 1999, 399, 243-246. (15) Shen, Z.; Thomas, J. J.; Averbuj, C.; Broo, K. M.; Engelhard, M.; Crowell, J. E.; Finn, M. G.; Siuzdak, G. Anal. Chem. 2001, 73, 612-619. (16) Thomas, J. J.; Shen, Z.; Crowell, J. E.; Finn, M. G.; Siuzdak, G. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4932-4937. (17) Kruse, R. A.; Li, X.; Bohn, P. W.; Sweedler, J. V. Anal. Chem. 2001, 73, 3639-3645. (18) Tuomikoski, S.; Huikko, K.; Grigoras, K.; Ostman, P.; Kostiainen, R.; Baumann, M.; Abian, J.; Kotiaho, T.; Franssila, S. Lab Chip 2002, 2, 247253. (19) Lewis, W. G.; Shen, Z.; Finn, M. G.; Siuzdak, G. Int. J. Mass Spectrom. 2003, 226, 107-116. (20) Thomas, J. J.; Shen, Z.; Blackledge, R.; Siuzdak, G. Anal. Chim. Acta 2001, 442, 183-190. (21) Okuno, S.; Shimomae, Y.; Ohara, K.; Fujiwara, H.; Ohyama, J.; Ohmoto, M.; Wada, Y.; Arakawa, R. J. Mass Spectrom. Soc. Jpn. 2004, 52, 142-148. (22) Arakawa, R.; Shimomae, Y.; Morikawa, H.; Ohara, K.; Okuno, S. J. Mass Spectrom. 2004, 39, 961-965. (23) Seino, T.; Sato, H.; Torimura, M.; Shimada, K.; Yamamoto, A.; Tao, H. Anal. Sci. 2005, 21, 485-490. (24) Cuiffi, J. D.; Hayes, D. J.; Fonash, S. J.; Brown, K. N.; Jones, A. D. Anal. Chem. 2001, 73, 1292-1295. (25) Go, E. P.; Apon, J. V.; Luo, G.; Saghatelian, A.; Daniels, R. H.; Sahi, V.; Dubrow, R.; Cravatt, B. F.; Vertes, A.; Siuzdak, G. Anal. Chem. 2005, 77, 1641-1646. (26) Finkel, N. H.; Prevo, B. G.; Velev, O. D.; He, L. Anal. Chem. 2005, 77, 1088-1095. (27) Okuno, S.; Arakawa, R.; Okamoto, K.; Matsui, Y.; Seki, S.; Kozawa, T.; Tagawa, S.; Wada, Y. Anal. Chem. 2005, 77, 5364-5369. (28) Pchelyakov, O. P.; Bolkhovityanov, Y. B.; Dvurechenskii, A. V.; Nikiforov, A. I.; Yakimov, A. I.; Voigtla¨nder, B. Thin Solid Films 2000, 367, 75-84. (29) Brunner, K. Pept. Prog. Phys. 2002, 65, 27-72. (30) Eaglesham, D. E.; Cerullo, M. Phys. Rev. Lett. 1990, 64, 1943-1946.

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GeNDs and silicon substrate forms a continuous crystal lattice, GeNDs are rigid and ideally do not exfoliate from the silicon substrate. A nanodot sample target made of SiO2 with Pt nanodots has already been proposed for improved DNA analysis by MALDIMS.31 This nanodot plate was, however, simply used as a support of sample/matrix crystal for MALDI analysis. We have expected that the high UV absorptivity of GeNDs assists the LDI process of analyte molecules deposited on the GeND surfaces. In this study, the performance of matrix-free LDI-MS using GeNDs, which we term GeND-MS, was demonstrated through measurements of a broad range of analytes including biomolecules and synthetic polymers and additives. EXPERIMENTAL SECTION Materials. Angiotensin-I ([M + H]+ ) m/z 1296.7 monoisotopic), insulin from bovine pancreas ([M + H]+ ) m/z 5734.5 average), and myoglobin from horse heart [M + H]+ ) m/z 16 952.3 average) were purchased from Sigma (St. Louis, MO). A vial of tryptic digests of bovine serum albumin (BSA) was purchased from SeQuant (Umea, Sweden). As synthetic polymer sample, octylphenol polyethoxylate, which is a typical nonionic surfactant known as Triton-X 100 (Aldrich, Milwaukee, WI), and poly(ethylene glycol) (PEG; Aldrich) with number-average molecular weight (Mn) of ∼1900-2200, as stated by the manufacturer, were used. Tetrabromobisphenol-A bis(2,3-dibromopropyl ether) (TBBPA-dbpe), which is a typical brominated flame retardant, was purchased from Japan Science Information (Nagoya, Japan). Preparation of GeND Chip. The GeND chips were prepared using an in-house-built MBE apparatus.32 GeNDs were formed during the epitaxial growth of germanium atoms on an n-type silicon wafer (Furuuchi Chemical, Tokyo, Japan, undoped; resistivity 1000 Ω·cm; crystal orientation (100); diameter 2 in.; thickness 0.25 mm). Prior to germanium deposition, the surface of the silicon wafer was chemically cleaned using the standard Shiraki process,33 following predeposition of silicon buffer layer (∼20 nm) at 400 °C to make an atomically flat and clean silicon surface. The temperature of the germanium source (a Knudsen cell) was kept at 1295 °C to maintain the germanium growth rate at 0.01 nm/s. In this study, three GeND chips (A-C) with different dot sizes were prepared by changing the germanium irradiation time and the temperature of the silicon substrate: 300 s and 540 °C for chip A, 150 s and 800 °C for chip B, and 600 s and 540 °C for chip C. The processed wafers were then cut to 10 × 10 mm to form the ionization chip. The surface morphology of the GeND chips was characterized by a Hitachi (Tokyo, Japan) S-4500 field emission scanning electron microscope (FE-SEM) and a Seiko Instruments (Chiba, Japan) SPI 3800N + SPA-300HV atomic force microscope (AFM). GeND-MS Measurements. Angiotensin-I, insulin, myoglobin, and BSA digests were dissolved in a mixture of methanol and deionized water (30/70 v/v) containing 0.1% trifluoroacetic acid (TFA). The concentration or amount of each peptide and protein sample is reported in the related text. PEG, Triton-X, and TBBPAdbpe samples were dissolved in tetrahydrofuran (THF) to a (31) Honda, A.; Sonobe, H.; Ogata, A.; Suzuki, K. Chem. Commun. 2005, 53405342. (32) Yamamoto, A.; Ohta, T.; Miki, K.; Sakamoto, K.; Kato, H.; Matsui, T. J. Jpn. Inst. Met. 1999, 63, 1386-1392. (33) Ishizaka, A.; Shiraki, Y. J. Electrochem. Soc. 1986, 133, 666-671.

Figure 1. Comparison of LDI mass spectra of angiotensin-I (25 pmol) deposited on a silicon wafer (a) and the GeND chip (b).

concentration of 0.5 mg/mL. As a cationization salt, sodium iodide (0.5 mg/mL in acetone) to form sodium adduct ions [M + Na]+ of PEG and Triton X-100 samples, or silver trifluoroacetate (AgTFA) (0.5 mg/mL in THF), for TBBPA-dbpe to form [M + Ag]+, were used. The solutions of cationization salt and sample were mixed at a ratio of 1/2 (v/v). Each sample solution (∼1.0 µL) was directly spotted onto the GeND chip and dried in air. The GeND chip was mounted on a standard MALDI plate using electroconductive adhesive tape. GeND-MS measurements were performed using basically the same operation as MALDI-MS measurements on a Voyager DEPRO time-of-flight mass spectrometer (Applied Biosystems, Framingham, MA) equipped with a pulsed nitrogen laser (λ ) 337 nm, 3-ns pulse width, and 3-Hz frequency) and a delayed extraction ion source. Ions generated by laser desorption were accelerated at 20 kV and then analyzed in the linear (1.4-m flight path) or the reflector (2.0-m flight tube) positive ion mode. Laser beam intensity was set to just above the threshold for analyte ionization. Laser fluence was measured using a J9LP energy sensor (Coherent, Santa Clara, CA). All the mass spectra were collected by averaging 500 individual laser shots. For PSD-MS/ MS measurement, an Axima-CFR plus (Shimadzu, Kyoto, Japan), LDI-TOFMS was also used. MALDI-MS and DIOS-MS Measurements. Comparative measurements were carried out by MALDI-MS and DIOS-MS. The measurement apparatus, conditions, and sample preparations were basically the same as those for GeND-MS. In the case of MALDIMS, R-cyanocinnamic acid (CHCA) in a 30% methanol solution containing 0.1% TFA and dithranol in THF were used as matrix reagents. The concentration of each matrix reagent was ∼10 mg/ mL. DIOS-MS measurements were performed using a commercial MassPREP DIOS target (Waters, Milford, MA). RESULTS AND DISCUSSION Figure 1 shows a comparison of the LDI mass spectra of angiotensin-I (25 pmol) as a model peptide sample deposited on the silicon wafer (a) and the GeND chip C (b). Only strong background peaks are observed when the silicon wafer was used, whereas in the case of the GeND chip, a protonated molecule [M + H]+ peak of angiotensin-I is clearly observed at m/z 1296.7 with almost no fragment peaks. This result clearly indicates that a matrix-free LDI can be achieved using the GeND chip. Here, the peaks observed in the lower mass region of the GeND mass

Figure 2. Morphology of GeNDs on the GeND chip C. (a) FE-SEM image. (b) AFM image. (c) Three-dimensional image of the same area shown in (b). Table 1. Threshold Laser Fluence and S/N Ratio for the Ionization of Angiotensin-I (4 pmol) Using Three Different GeND Chips

plate A B C

dot size (nm) widtha heightb 20-30 50-100 150-200

∼5 ∼30 ∼50

fluence (µJ/pulse)

S/N ratioc

18.5 7.5 5.5

138 ( 30 536 ( 55 840 ( 109

a Estimated by SEM. b Estimated by AFM. c Three repeated measurements.

spectrum can be attributed to contaminants such as plasticizers and small organic compounds as chemical background. Figure 2 shows the morphology of the GeND chip C observed by FE-SEM and AFM. These images indicate that the size of GeNDs is nearly uniform and that homogeneous in-plane distribution on the silicon surface could be easily achieved. The average size of GeNDs was the width of ∼150-200 nm determined by FE-SEM and the top height of ∼50 nm by AFM. Suitable nanodot size was tentatively evaluated for the ionization of angiotensin-I (4 pmol/spot). Table 1 summarizes the threshold laser fluence and signal-to-noise (S/N) ratio for three different GeND chips (A-C). For chip A with small dots, a higher laser fluence (∼18.5 µJ/pulse) was required, and a poor S/N ratio was obtained. Increasing the dot size reduced the threshold laser fluence to 5.5 µJ/pulse and increased the S/N ratio of chip C with a height of ∼50 nm and a width of ∼150-200 nm. So far, this size is the upper limit produced using our MBE apparatus, since further germanium deposition resulted in continuous island formation. Subsequent demonstrations were therefore performed using chip C. The detection limit was evaluated using angiotensin-I and insulin, as shown in Figures 3 and 4. For angiotensin-I, detection of only 800 amol could be achieved (Figure 3), which was closely Analytical Chemistry, Vol. 79, No. 13, July 1, 2007

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Figure 3. GeND mass spectra of angiotensin-I. (a) 400 fmol and (b) 800 amol.

Figure 6. Mass spectra of BSA digests (1 pmol) observed by MALDI-MS with a CHCA matrix (a) and GeND-MS (b). Assigned peptides are denoted by the start and end amino acid position in the BSA sequence. Mox and CAM indicate oxidation of methionine residue and carbamide methylation of cysteine residue, respectively. Asterisks in (a) correspond to matrix cluster peaks.

Figure 4. Mass spectra of insulin with different sample amounts observed by GeND-MS (a) and DIOS-MS using a commercial chip (b).

Figure 5. GeND mass spectrum of myoglobin observed in positive linear mode.

comparable to that of DIOS-MS (700 amol of des-arg-bradykinin).14 The limit for insulin (m/z 5735) by GeND-MS was lower than 200 fmol (Figure 4a), whereas that by DIOS-MS was above 20 pmol under our experimental conditions (Figure 4b). This result suggests that sensitivity to higher molecular weight compounds is likely to be superior to that of DIOS. So far, the upper limit of detectable mass is m/z 16 952 (myoglobin, 60 pmol) as shown in Figure 5, which was higher than that reported for DIOS-MS using a UV laser (∼12 kDa).14 The analysis of myoglobin by DIOS-MS has required the use of an IR laser.34 Generally, it is hard to ionize high molecular weight compounds (to date the largest compound is ∼24 000)4 by means of matrix-free LDI-MS. The applicable mass range and sensitivity of GeND-MS appear to be at a high level in matrix-free techniques. (34) Rousell, D. J.; Dutta, S. M.; Little, M. W.; Murray, K. K. J. Mass Spectrom. 2004, 39, 1182-1189.

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The advantages of higher sensitivity and avoidance of obstructive peaks are suited to protein identification by peptide mass fingerprint (PMF) analysis. Figure 6 illustrates the mass spectra of BSA digests observed by MALDI-MS with CHCA matrix (a) and GeND-MS (b) as a preliminary example. In the MALDI mass spectrum, a considerable number of matrix cluster peaks up to m/z ∼1100 were caused by conventional sample treatment, with the result that only the three digests could be observed in the mass range under m/z 1100. To avoid generating interfering peaks, careful pretreatments and measurement procedures are likely to be needed. On the other hand, the GeND mass spectrum is free from interfering peaks, resulting in many digests being distinctly observed. Sequence coverage of 34% by MALDI-MS could increase up to 47% by GeND-MS. The detection of lower molecular weight peptides by GeND-MS would be effective not only to enhance the reliability of identification results but also to give a good clue to characterize post-translational modifications. Protein identification has also been performed by MS/MS measurements of tryptic digests. For MALDI-MS/MS measurements, however, the matrix cluster ions generate numerous and strong fragment peaks due to intense laser irradiation, interfering with product ions. In this case, GeND-MS/MS measurements would also be effective. Figure 7 illustrates postsource decay (PSD) MS/MS spectrum of the BSA digest at m/z 689.4 as a precursor ion. Sufficient numbers of product ions serve to determine the amino acid sequence AWSVR by a Mascot MS/ MS ion search engine. In the field of polymer characterization, it is important to observe accurate molecular weight distribution. Figure 8 shows mass spectra of octylphenol polyethoxylate (Triton X-100) observed by MALDI-MS with a 2,5-dihydroxybenzoic acid (DHB) matrix and GeND-MS. Here, sodium iodide was used as a cationization salt to form sodium adduct ions of the analyte [M + Na]+. A comparable peak distribution of Triton X-100 was observed in the range from about m/z 400 to 1000 separated by 44 Da with the peak maximum at around m/z 625. Figure 9 shows the GeND mass spectrum of PEG. A Gaussian shape peak distribution with

Table 2. Comparison of Average Molecular Weights and Polydispersity Indices of Polymers by GeND-MS and MALDI-MS samples Triton X-100 PEG 2000

Figure 7. PSD-MS/MS spectrum of a tryptic digest of BSA at m/z 689.4 as a precursor ion. The mass spectrum was observed by Axima-CFR plus.

Figure 8. GeND mass spectrum of Triton X-100 observed by MALDI-MS with a DHB matrix (a) and GeND-MS (b).

Figure 9. GeND mass spectra of PEG.

a maximum at ∼2000 can be observed. Table 2 summarizes the number- and weight-average molecular weight (Mn and Mw) and polydispersity index (PDI) determined by GeND-MS and MALDIMS for Triton X-10035 and PEG samples. The values determined by GeND-MS are in very good agreement with those by MALDIMS. Since the accuracy of the molecular weight distribution (35) Sato, H.; Shibata, A.; Wang, Y.; Yoshikawa, H.; Tamura, H. Polym. Degrad. Stab. 2001, 74, 69-75.

ionization methods GeND MALDId GeND MALDI

Mna ((28)c

621 619 ((8) 2088 ((9) 2057 ((3)

Mwb

PDI

644 ((28) 646 ((8) 2130 ((12) 2092 ((4)

1.04 1.04 1.02 1.02

a Number-average molecular weight. b Weight-average molecular weight. c Three repeated measurements. d In ref 35.

observed by MALDI-MS has been established when the PDI value of a given sample is lower than 1.1,36 these results indicate that GeND-MS can provide a fairly accurate molecular weight distribution of polymers. Analysis of brominated flame retardants has become an important subject from the viewpoint of risk management. So far, however, few MALDI mass spectra of brominated flame retardants have been reported to our knowledge, probably due to the lack of a suitable matrix and their sensitivity to laser heat and light. Figure 10 shows mass spectra of TBBPA-dbpe, which is a typical brominated flame retardant used for polyolefins and polystyrene, observed by MALDI-MS with a dithranol matrix (a), DIOS-MS (b), and GeND-MS (c), respectively. Here, to form silver adduct ions of the analyte [M + Ag]+, AgTFA as a cationization salt was added in all cases. In the MALDI and DIOS mass spectra, it is difficult to find [M + Ag]+ ions around m/z 1050, but many fragment peaks are generated, some of which might be due to debromination. Laser power at 12.2 µJ/pulse for MALDI-MS and at 7.0 µJ/pulse for DIOS-MS were set for the generation of [M + Ag]+ ions. When more intense laser light was irradiated, no [M + Ag]+ peaks caused by fragmentation were observed. In the MALDI-MS measurements, other typical matrixes for hydrophobic industrial polymers such as DHB, 2-(4-hydroxyphenylazo)benzoic acid, and trans-3-indoleacrylic acid, were also examined, but they showed no [M + Ag]+ ion peaks. On the other hand, GeND-MS gives clear silver-cationized TBBPA-dbpe ([M + Ag]+) with characteristic isotope distribution mainly reflecting the composition of eight bromine atoms (79Br/81Br ) 51/49 atom %) and a silver cation (107Ag+/109Ag+ ) 52/48 atom %) as shown in the inset to Figure 10c. The laser power setting was the same as for DIOS-MS (7.0 µJ/pulse). The predominant formation of [M + Ag]+ ions of TBBPA-dbpe suggests that GeND-MS is more suitable for analyzing brominated compounds. As demonstrated above, GeND-MS was able to perform soft desorption/ionization of various compounds, not only hydrophilic biomolecules and water-soluble polymers but also hydrophobic synthetic compounds with little or no fragmentation. We have also successfully analyzed other compounds, including oligosaccharides and various types of synthetic polymers and additives. A further advantage of GeND is its high resistance to demanding treatment. When a slightly higher laser power is irradiated on conventional SALDI substrates, there is a risk that (36) Montaudo, G.; Montaudo, M. S.; Samperi, F. Matrix-Assisted Laser Desorption Ionization/Mass Spectrometry of Polymers (MALDI-MS). In Mass Spectrometry of Polymers; Montaudo, M., Lattimer, R. P., Eds.; CRC Press: Boca Raton, FL, 2001; p 419.

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On the other hand, it has been proposed that the ion formation mechanism in SALDI is similar to that in field desorption, which achieves charge separation in local, high electric fields in a protruding surface structure.37 We have reported that a pyroelectric ceramic, lead lanthanum zirconate titanate (PLZT), with no nanostructures could successfully perform soft LDI for peptides, oligosaccharides, and synthetic oligomers.38 We speculated that the strong electric field generated near the flat surface of PLZT by UV laser irradiation might assist the LDI process. Okuno et al.27 have investigated the requirements for LDI on submicrometer structures. They concluded that LDI could be implemented on submicrometer structures with a metal-coated surface, irrespective of the basal materials. In the light of these above-mentioned reports, it appears that electric fields in the vicinity of the GeND surface induced by laser irradiation might contribute to desorption/ionization together with (or instead of) rapid thermal desorption. Further investigations are currently in progress to elucidate the desorption/ionization mechanisms in GeND.

Figure 10. Mass spectra of TBBPA-dbpe observed by MALDI-MS with a dithranol matrix (a), DIOS-MS (b), and GeND-MS (c).

inorganic particles from the matrix will be scattered inside the ionization chamber, or brittle nanostructures on the ionization substrate will be easily damaged. GeNDs, on the other hand, can withstand even higher laser irradiation, even exceeding 100 µJ, the full power of our instruments, since they are covalently bonded with the silicon substrate at the interface to form a continuous crystal lattice. Is the mechanism of desorption/ionization of GeND-MS the same as for DIOS and other matrix-free techniques? In DIOS-MS, it has been reported that the properties of porous silicon, such as high surface area, low thermal conductivity, and high UV absorbance, are required for successful soft ionization.14,15,17 The GeNDs have similar properties. Countless nanodots on a silicon substrate can offer a sufficiently large area for sample deposition with a thin molecular layer. The lower thermal conductivity of germanium (∼60 W/mK) than that of silicon (∼150 W/mK) is more favorable to localized heating on the GeNDs, which is likely to assist the desorption/ionization of analytes. (37) Alimpiev, S.; Nikiforov, S.; Karavanskii, V.; Minton, T.; Sunner, J. J. Chem. Phys. 2001, 115, 1891-1901. (38) Sato, H.; Seino, T.; Yamamoto, A.; Torimura, M.; Tao, H. Chem. Lett. 2005, 34, 1178-1179.

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CONCLUSIONS A novel matrix-free LDI-MS, which we term GeND-MS, using germanium nanodots grown on a silicon monocrystalline substrate using the MBE method has been developed. GeND-MS has successfully enabled the soft desorption/ionization of a wide range of samples, including not only hydrophilic biomaterials, such as peptides and proteins, but also hydrophobic synthetic compounds. For peptide samples, clear mass spectra in the lower mass region (