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Spointofof(camairrtheofemployed in the analysis of proteins and peptides [2, 3].Several other nanomaterials, including carbon nano-tubnaareSAphanAuionSAthaingsu(frlitedifan

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Sixqu(Nofoudulaynanomaterials and samples onto the metal plates usedin SALDI-MS. First, one of the nanomaterial solutions (1

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2104does, nanodiamonds, and various nanoparticles (NPs,mely SiO2, ZnS, TiO2, Fe3O4, Fe3O4/TiO2, and Au)also usefulwithout the addition of glycerolforLDI-MS [412]. Because of their unique chemical andysical properties, NPs can also act as selective probesd/or efficient ionization nanomaterials. For example,and TiO2 NPs are suitable for the concentration andization of aminothiols and catechins, respectively, inLDI-MS [8, 11]. One other advantage of using NPs ist fewer sweet spots are formed, thereby maximiz-reproducibility. Although NPs have been used

ccessfully for the determination of a range of analytesom small analytes to proteins), a review of therature reveals that the various NPs provide quiteferent results in terms of sensitivity, reproducibility,d mass range. Thus, our aim in this study was to

L) was deposited into one of the wells of the MS plateand dried under ambient conditions for at least 1 h.Next, one of the sample solutions (1 L) was added intothe well. Each sample solution contained one of thefollowing analytes: glutathione (MW 307.32 Da),angiotensin I (1296.48 Da), insulin (5734.52 Da), cyto-chrome c (ca. 12 kDa), trypsinogen (ca. 24 kDa), andchymotrypsin (ca. 25 kDa). The aqueous sample solu-tions were premixed with ammonium citrate buffer(0.550 mM; pH 4.0); the ammonium ions acted asproton sources for the SALDI process [810]. Afterdrying under ambient conditions, the samples weresubjected to SALDI-MS analysis.

Mass spectrometry experiments were performed inthe positive or negative ion mode using a MicroflexMALDI-TOF mass spectrometer (Bruker Daltonics, Bre-men, Germany). A pulsed nitrogen laser (337 nm; 3-nspulses at 10.0 Hz; fluence 4066.25 J) was used in theMS system, which was operated in the reflectron (orlinear) mode with an ion source voltage of 19.00 (20.00)

dress reprint requests to Dr. H.-T. Chang, Department of Chemistry,tional Taiwan University, 1, Section 4, Roosevelt Road, Taipei 106,wan. E-mail: changht@ntu.edu.tw

Published online March 30, 2010010 American Society for Mass Spectrometry. Published by Elsevier Inc. Received October 8, 20094-0305/10/$32.00 Revised February 24, 2010esorption/Ionization Mass Septides and Proteins

eng-Kang Chiang, Ni-Chen Chiang, Zong-ng-Wei Lin, and Huan-Tsung Changpartment of Chemistry, National Taiwan University, Taipei, Taiw

e have investigated six nanomaterials for their applicabptides and proteins using surface-assisted laser desorLDI-MS). Gold nanoparticles (NPs) were useful nantathione); Pt nanosponges and Fe3O4 NPs were efficientper detectable mass limit of ca. 25 kDa. Nanomaterials htrices, including lower limits of detection for small analytewer problems associated with sweet spots), when usedectrometry. (J Am Soc Mass Spectrom 2010, 21, 1204120ectrometry

urface-assisted laser desorption/ionization massspectrometry (SALDI-MS) was developed recentlyusing nanomaterials rather than organic com-

unds as matrices, for the determination of analytes oferest. For example, laser desorption/ionization (LDI)intact proteins and protein aggregates in the presenceglycerol has been demonstrated using cobalt particles. 30 nm) [1]. Similar to the role played by organictrices, the particles absorb energy from the laseradiation and transfer it efficiently to the analytes,reby inducing desorption and ionization. Mixturesgraphite particles (2150 m) and glycerol have beeni:10.1016/j.jasms.2010.02.028ctrometry of

ng Lin, Guo-Yu Lan,

as surfaces for the analyses of/ionization mass spectrometryterials for small analytes (e.g.,omaterials for proteins, with anseveral advantages over organicd lower batch-to-batch variationsaser desorption/ionization mass2010 American Society for Mass

aluate the performance of several types of NPs for thealysis of peptides and proteins.

perimental

nanomaterialsAu NPs, TiO2 NPs, Se NPs, CdTeantum dots (QDs), Fe3O4 NPs, and Pt nanospongesSPs)were tested for the SALDI-MSbased analysespeptides and proteins; they were prepared in aque-s solutions and characterized according to proce-res described in the literature [8, 11, 1316]. A two-er preparation method was applied to deposit theAccepted February 24, 2010

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1205J Am Soc Mass Spectrom 2010, 21, 12041207 SALDI-MS OF PEPTIDES AND PROTEINS, a second ion source voltage of 16.25 (18.40) kV, as voltage of 9.70 (7.00) kV, a reflector voltage of 20.0.0) kV, and an extraction delay time of 200 (450) ns.ALDI mass spectra resulting from 300 laser pulsesre collected and averaged for each acquisition.

sults and Discussion

ble S1, which can be found in the electronic version ofs article summarizes the pseudomolecular ions ob-ved from the analytes when using these six nanoma-ials in SALDI-MS. Although the Au NPs were selec-e and efficient nanomaterials for glutathione, theyre not useful for the analysis of analytes havinglues of m/z greater than 1300. When using the Ses, TiO2 NPs, and CdTe QDs for SALDI-MS analyses,mass range reached as high as the mass of cyto-

rome c. The SALDI-MS approach allowed the detec-n of chymotrypsin by using the Pt NSPs and Fe3O4s. Interestingly, we detected the [M H] ion oftathione when using the TiO2 NPs for SALDI-MSasurement, but the [M Na] ion when using theer five nanomaterials (Table S1). For angiotensin I,detected the [MH] and [MNa] ions in all six

ses; in addition, we also detected the [M K] ionen using the Fe3O4 NPs. When using Fe3O4 NPs andTe QDs in the analysis of insulin, we detectedducts of Fe and Cd, respectively. Presumably, themation of metalanalyte adducts was due to strongeractions between the Fe3/Cd2 ions and the pro-ns through metalligand (carboxylate and amino)

Figure 1. Surface-assisted laser desorption ionizapmol) recorded at 51.25 J using Fe3O4 nanopar

ammonium citrate (pH 4.0). Inset: SALDI mass spe11,80012,800.ordination [9]. We detected a greater number ofducts for the larger protein cytochrome c when usingFe3O4 NPs and CdTe QDs, mainly because therere more Fe ions desorbed and ionized from the NPsn those from the mixtures of the metal ions (lowncentrations) and sinapinic acid (SA). For compari-n, we also recorded the mass spectra of cytochrome cing SA as the matrix in the absence and presence of3 or Cd2 ions. Figure S1a, which can be found inelectronic version of this article, presents the mass

ectrum of cytochrome c obtained using SA (10 mg/) containing Fe3 ions (1 mM) as the matrix. Al-ugh we observed the formation of Feanalyte ad-cts, the most abundant peak corresponds to the [M ion; in contrast, Figure 1 (Fe3O4 NPs) reveals thatmost abundant peaks were those of Feanalyte

ducts. In the absence of Fe3 (Figure S1b), we de-ted no Feanalyte adducts. Figure S1a and b botheal the formation of SAanalyte adducts. We alsoserved Cdcytochrome c adducts when analyzingtochrome c using either CdTe QDs as the nanomate-ls or SA (10 mg/mL) and added Cd2 ions (1 mM) asmatrices, and the [M H] ion when using SA asmatrix (data not shown). The limits of detection

ODs; signal-to-noise ratio 3) for cytochrome c was. 5.6 fmol when using the Fe3O4 NPs in SALDI-MS,ch lower than that (2.5 104 fmol) obtained whening the CdTe QDs. When using the Fe3O4 NPs andTe QDs in SALDI-MS measurements, the intensitiesthe signals for the metal adducts varied by 16 and%, respectively, over 50 sample spots; in contrast, the

(SALDI) mass spectrum of cytochrome c (50(NPs) (800 nM, 1 L), prepared in 50 mMcoadthewethacosousFethespmLthoduH]theadtecrevobcyriathethectrum of cytochrome c in the range m/z

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1206 CHIANG ET AL. J Am Soc Mass Spectrom 2010, 21, 12041207riations were greater than 50% when using SA as thetrix, either in the absence or presence of Fe3 or Cd2

s.Figure S2 presents the SALDI mass spectra of cyto-rome c, trypsinogen, and chymotrypsin that we ob-ned using the Pt NSPs. Unlike the situation for thenanomaterials, which produced many Au clusters,did not detect any Pt clusters when using the PtPs as the nanomaterials, primarily because of theatively low thermal conductivity of Pt (71.6 W m11 at 300 K; cf. 318 m1 K1 for Au) [17]. For smallalytes (e.g., glutathione), the Au NPs provided muchtter sensitivity than did the Pt NSPs, mainly due tore-efficient desorption and ionization. The massit reached only 5735.49 Da (insulin) when using theNPs, primarily because of strong interactions

rough AuS bonds) between the Au NPs and thealytes. Compared with the Fe3O4, TiO2, and CdTenomaterials, the Pt NSPs required less laser energy. 4043.75 J) to induce the desorption and ioniza-n of either cytochrome c or chymotrypsin, mainlycause of the relatively higher thermal conductivity of

ble 1. Experimental parameters, linear ranges (LR), limits of deges for the SALDI-MS analyses of the various analytes using the

anomaterial/iameter (nm)b Analyte

Concentrationof NPs

Lase

NPs/14 (2) Glutathionec 15 nM 4Angiotensin I 15 nM 4Insulin 1.5 nM 4Cytochrome c Chymotrypsin

2 NPs/5 (1) Glutathione 24 nM 5Angiotensin I 24 nM 5Insulin 24 nM 5Cytochrome c 24 M 6Chymotrypsin

NPs/100 (10) Glutathione 50 pM 4Angiotensin I 1 nM 4Insulin 1 nM 4Cytochrome c 1 nM 5Chymotrypsin

Te QDs/3 (0.5) Glutathione 500 nM 4Angiotensin Ic 500 nM 4Insulin 500 nM 5Cytochrome c 25 M 6Chymotrypsin

3O4 NPs/13 (3) Glutathione 8 nM 4Angiotensin I 80 nM 4Insulinc 800 nM 5Cytochrome cc 800 nM 5Chymotrypsin 4 M 5

NSPs/37 (6) Glutathione 10 pM 4Angiotensin I 50 pM 4Insulin 50 pM 4Cytochrome c 4 nM 4Chymotrypsinc 8 nM 4

Not detected in the mass spectrum.l experiments were performed in linear positive (for m/z 6000) or reflde used for the detection of glutathione using TiO2.zes of the nanomaterials were calculated from five different batches b

alyte exhibiting the best sensitivity among the six nanomaterials.timal MS range of analytes for a given nanomaterial.Larger molecules have more adsorption sites forir binding to the surfaces of inorganic materials,king it much more difficult to detach them. There-e, macromolecules require a higher laser power thansmall molecules for detection using SALDI-MS.

cause we employed lower laser energies when usingPt NSPs, fewer fragment ions were formed. Further-re, the Pt NSPs featured surface projections thatted as tiny antennas, producing significant field en-ncements in their vicinity, leading to greater desorp-n and ionization efficiency. Indeed, relative to the3O4 NPs, the Pt NSPs provided very clean masstterns for the analysis of proteins. We detected mul-ly charged ions for cytochrome c, trypsinogen, andymotrypsin when using the Fe3O4 NPs for SALDI-MSalyses. In our experiments, NH4

ions acted as thein proton donors during the desorption and ioniza-n processes. Notably, we had prepared the Fe3O4s from an ammonia solution under basic conditions.e observed similar results when analyzing cyto-rome c using the TiO2 NPs as the added nanomate-

n (LOD), coefficients of variation (CV), and mass spectralanomaterialsa

werLR (pmol) LOD (fmol) CV

MS range(Da)d

2.5100 1.4 102 18% 3001,300250 8.1 102 29% 7.7 103 25% ND ND

20100 2.2 103 9% 1,20012,0000.420 8.3 101 23%

4.7 101 25% 1.5 104 20% ND 3.3 104 19% 1,20012,000

0.430 1.6 102 29% 1.8 101 25% 2.3 103 27% ND 4100 1.9 102 25% 30012,000

0.450 1.1 101 16% 1.4 101 23% 2.5 104 26% ND

30200 8.3 103 14% 1,20025,0000.13 9.8 101 7%

1.2 101 15% 5.6 16% 4.4 104 20% 2.3 104 17% 1,20025,000

0.170 4.3 101 16% 3.6 101 17% 1.5 104 35% 1.1 104 32%

positive (for m/z 6000) modes, except for the reflectron negative

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1207J Am Soc Mass Spectrom 2010, 21, 12041207 SALDI-MS OF PEPTIDES AND PROTEINSl; notably, the TiO2 NPs had been prepared underidic conditions.Table 1 summarizes the SALDI-MS analyses of thee analytes using the six nanomaterials throughLDI-MS measurement. For small analytes (e.g., glu-hione), the Au NPs provided the lowest LOD (140ol). Upon increasing the mass of the analytes, theDs increased as a result of their stronger interactionsth Au NP surfaces and greater ionization energies.cause we required high laser powers for the largerlecules (MW:12 kDa), Au clusters caused suppres-n of the sample ion peaks and difficulty in peakignment. CdTe QDs provided good sensitivity foralytes in the mass range from 330.07 Da to 12 kDa.ong the six nanomaterials, the Fe3O4 NPs and PtPs provided the greatest sensitivity for larger pro-ns (1225 kDa). Relative to the Pt NSPs, the Fe3O4s provided better sensitivity (LOD 5.6 fmol versuspmol) for cytochrome c, but poorer sensitivity forymotrypsin (LOD 44 pmol versus 11 pmol). Tablelso provides a quick reference guide for the NPs mostitable for certain molecular-weight ranges of analytes.e note that the mass limits obtained using Fe3O4 NPsd Pt NSPs are the highest ever reported for SALDI-S; indeed, they are higher than those reported whening silane-immobilized Fe3O4 NPs (16 kDa) or Fe3O4/2 core/shell NPs (24 kDa) [9, 10]. When using the PtPs for SALDI-MS analyses, the batch-to-batch varia-ns for the smaller (12 kDa) and larger (1225 kDa)oteins were 16% and 35%, respectively.

nclusions

e have tested six nanomaterials for their suitability inLDI-MS for the detection of small peptides andoteins (307.3 Da25 kDa). Au NPs provided the bestsitivity for small analytes; Pt NSPs and Fe3O4 NPsovided the highest mass limit (25 kDa) for proteins.the best of our knowledge, this paper is the first toscribe the use of Pt NSPs for SALDI-MS analyses. Foricient desorption and ionization, the nanomaterialst absorb laser energy efficiently and transfer it toanalytes efficiently, but its interactions with the

alytes must not be too strong. For high sensitivity, thenomaterial must be stable under laser irradiation andly minimally form clusters (e.g., Au clusters). Toovide selectivity for certain analytes, aptamers andtibodies can be used to prepare functionalized Pt NSPsough strong Pt-S or Pt-N bonding [18]. The bondergies for Pt-S and Pt-N are 174 and 137 kJ/mol,pectively, which are higher that that of Au-S bonding4 kJ/mol). For example, we can prepare 11-mercapto-,9-trioxaundecyl-r-D-mannopyranoside and aptamernctionalized Pt NSPs for the detections of concanavalind platelet-derived growth factors, respectively, through

LDI-MS [19, 20].cknowledgmentse authors acknowledge support for this study by the Nationalence Council of Taiwan under contracts NSC 98-2113-M-002--MY3, 98-2627-M-002-013, and 98-2627-M-002-014.

ppendix Applementary Material

pplementary data associated with this article can bend, in the online version, at doi:10.1016/j.jasms.2010..028.

ferencesTanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T.; Matsuo,T. Protein and Polymer Analyses up to m/z 100 000 by Laser IonizationTime-of-Flight Mass Spectrometry. Rapid Commun. Mass Spectrom. 1988,2, 151153.Sunner, J.; Dratz, E.; Chen, Y.-C. Graphite Surface Assisted LaserDesorption/Ionization Time-of-Flight Mass-Spectrometry of Peptidesand Proteins from Liquid Solutions. Anal. Chem. 1995, 67, 43354342.Dale, M.; Knochenmuss, R.; Zenobi, R. Graphite/Liquid Mixed Matricesfor Laser Desorption/Ionization Mass Spectrometry. Anal. Chem. 1996,68, 33213329.Ugarov, M. V.; Egan, T.; Khabashesku, D. V.; Schultz, J. A.; Peng, H.;Khabashesku, V. N.; Furutani, H.; Prather, K. S.; Wang, H.-W. J.; Jackson,S. N.; Woods, A. S. MALDI Matrices for Biomolecular Analysis Based onFunctionalized Carbon Nanomaterials. Anal. Chem. 2004, 76, 67346742.Najam-ul-Haq, M.; Rainer, M.; Huck, C. W.; Hausberger, P.; Kraushaar,H.; Bonn, G. K. Nanostructured Diamond-Like Carbon on DigitalVersatile Disc as a Matrix-Free Target for Laser Desorption/IonizationMass Spectrometry. Anal. Chem. 2008, 80, 74677472.Wen, X.; Dagan, S.; Wysocki, V. H. Small-Molecule Analysis withSilicon-Nanoparticle-Assisted Laser Desorption/Ionization Mass Spec-trometry. Anal. Chem. 2007, 79, 434444.Kailasa, S. K.; Kiran, K.; Wu, H.-F. Comparison of ZnS SemiconductorNanoparticles Capped with Various Functional Groups as the Matrixand Affinity Probes for Rapid Analysis of Cyclodextrins and Proteins inSurface-Assisted Laser Desorption/Ionization Time-of-Flight MassSpectrometry. Anal. Chem. 2008, 80, 96819688.Huang, Y.-F.; Chang, H.-T. Nile Red-Adsorbed Gold Nanoparticle Ma-trixes for Determining Aminothiols Through Surface-Assisted Laser De-sorption/Ionization Mass Spectrometry. Anal. Chem. 2006, 78, 14851493.Chen, W.-Y.; Chen, Y.-C. Affinity-Based Mass Spectrometry UsingMagnetic Iron Oxide Particles as the Matrix and Concentrating Probesfor SALDI MS Analysis of Peptides and Proteins. Anal. Bioanal. Chem.2006, 386, 699704.Chen, C.-T.; Chen, Y.-C. Fe3O4/TiO2 Core/Shell Nanoparticles as AffinityProbes for the Analysis of Phosphopeptides Using TiO2 Surface-AssistedLaser Desorption/Ionization Mass Spectrometry. Anal. Chem. 2005, 77,59125919.Lee, K.-H.; Chiang, C.-K.; Lin, Z.-H.; Chang, H.-T. Determining EnediolCompounds in Tea Using Surface-Assisted Laser Desorption/Ioniza-tion Mass Spectrometry with Titanium Dioxide Nanoparticle Matrices.Rapid Commun. Mass Spectrom. 2007, 21, 20232030.McLean, J. A.; Stumpo, K. A.; Russell, D. H. Size-Selected (210 nm)Gold Nanoparticles for Matrix Assisted Laser Desorption Ionization ofPeptides. J. Am. Chem. Soc. 2005, 127, 53045305.Lin, Z.-H.; Wang, C. R. C. Evidence on the Size-Dependent AbsorptionSpectral Evolution of Selenium Nanoparticles. Mater. Chem. Phys. 2005,92, 591594.Gao, M.; Kirstein, S.; Mhwald, H.; Rogach, A. L.; Kornowski, A.; Eych-mller, A.; Weller, H. Strongly Photoluminescent CdTe Nanocrystals byProper Surface Modification. J. Phys. Chem. B 1998, 102, 83608363.Smith, J. E.; Medley, C. D.; Tang, Z.; Shangguan, D.; Lofton, C.; Tan, W.Aptamer-Conjugated Nanoparticles for Selective Collection and Detec-tion of Cancer Cells. Anal. Chem. 2006, 78, 29182924.Lin, Z.-H.; Lin, M.-H.; Chang, H.-T. Facile Synthesis of CatalyticallyActive Platinum Nanosponges, Nanonetworks, and Nanodendrites.Chem. Eur. J. 2009, 15, 46564662.Kawasaki, H.; Yonezawa, T.; Watanabe, T.; Arakawa, R. PlatinumNanoflowers for Surface-Assisted Laser Desorption/Ionization MassSpectrometry of Biomolecules. J. Phys. Chem. C 2007, 111, 1627816283.Kiguchi, M.; Miura, S.; Takahashi, T.; Hara, K.; Sawamura, M.; Mura-koshi, K. Conductance of Single 1,4-Benzenediamine Molecule Bridgingbetween Au and Pt Electrodes. J. Phys. Chem. C 2008, 112, 1334913352.Huang, C.-C.; Chen, C.-T.; Shiang, Y.-C.; Lin, Z.-H.; Chang, H.-T. Synthesisof Fluorescent Carbohydrate-Protected Au Nanodots for Detection ofConcanavalin A and Escherichia coli. Anal. Chem. 2009, 81, 875882.Huang, C.-C.; Chiang, C.-K.; Lin, Z.-H.; Lee, K.-H.; Chang, H.-T. Biocon-

jugated Gold Nanodots and Nanoparticles for Protein Assays Based onPhotoluminescence Quenching. Anal. Chem. 2008, 80, 14971504.

Nanomaterial-Based Surface-Assisted Laser Desorption/Ionization Mass Spectrometry of Peptides and ProteinsExperimentalResults and DiscussionConclusionsAppendix ASupplementary Material

AcknowledgmentsReferences