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(MALDI)1,2 and electrospray ionization (ESI),3 had MS been widely applied to ...... phase cleavage of peptide bonds or the ready interpretation of tan...
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Anal. Chem. 2008, 80, 6995–7003

Reactive-Electrospray-Assisted Laser Desorption/ Ionization for Characterization of Peptides and Proteins Ivory X. Peng,† Rachel R. Ogorzalek Loo,‡ Jentaie Shiea,§ and Joseph A. Loo*,†,‡ Department of Chemistry and Biochemistry and Department of Biological Chemistry, David Geffen School of Medicine, University of CaliforniasLos Angeles, Los Angeles, California 90095, and Department of Chemistry, National Sun Yat-Sen University and National Sun Yat-Sen UniversitysKaohsiung Medical University Joint Center, Kaohsiung, Taiwan Electrospray-assisted laser desorption/ionization (ELDI) is a soft ionization method for mass spectrometry (MS) and combines features of both electrospray ionization (ESI) and matrix-assisted laser desorption/ionization to generate ESI-like multiply charged molecules. The ELDI process is based on merging ESI-generated, charged droplets with particles UV laser desorbed from dried or wet sample deposits. We previously reported that ELDI is amenable for MS-based protein identification of large peptides and small proteins using top-down and bottomup techniques (Peng, I. X.; Shiea, J.; Ogorzalek Loo, R. R.; Loo, J. A. Rapid Commun. Mass Spectrom. 2007, 21, 2541-2546). We have extended our studies by applying collisionally activated dissociation and electron-transfer dissociation MSn to protein analysis and show that ELDI is capable of multistage MS to MS4 for top-down characterization of large proteins such as 29 kDa carbonic anhydrase. Multiply charged proteins generated by the ELDI mechanism can be shifted to higher charge by increasing the organic content in the ESI solvent to denature the protein molecules, or by adding m-nitrobenzyl alcohol to the ESI solvent. Furthermore, we introduce “reactive-ELDI”, which supports chemical reactions during the ELDI process. Preliminary data for online disulfide bond reduction using dithiothreitol on oxidized glutathione and insulin show reactive-ELDI to be effective. These data provide evidence that the laser-desorbed particles merge with the ESI-generated charge droplets to effect chemical reactions prior to online MS detection. This capability should allow other chemical and enzymatic reactions to be exploited as online protein characterization tools, as well as extending them to flexible, spatially resolved tissue screening and imaging. Also, these reactive-ELDI disulfide reduction experiments enable direct top-down protein identification for proteomic study, side * To whom correspondence should be addressed. E-mail: JLoo@ chem.ucla.edu. † Department of Chemistry and Biochemistry, University of CaliforniasLos Angeles. ‡ Department of Biological Chemistry, University of CaliforniasLos Angeles. § National Sun Yat-Sen University and National Sun Yat-Sen Universitys Kaohsiung Medical University Joint Center. 10.1021/ac800870c CCC: $40.75  2008 American Chemical Society Published on Web 08/07/2008

stepping laborious, time-consuming sample preparation steps such as in-solution reduction and alkylation. Mass spectrometry (MS) is a widely used analytical technique for the detection and structural characterization of a wide variety of molecules. However, not until the development of two soft ionization methods, matrix-assisted laser desorption/ionization (MALDI)1,2 and electrospray ionization (ESI),3 had MS been widely applied to the study of proteins and other large biomolecules, culminating in the introduction of a new research field, proteomics. Both MALDI and ESI play important roles in proteomic studies.4 MALDI typically generates singly charged molecules and is able to measure peptides and proteins beyond 100 kDa. For proteomics applications, a MALDI peptide mass fingerprint spectrum, coupled with tandem mass spectrometry (MS/MS) can be effective for the identification of proteins.5 ESI, on the other hand, often generates multiply charged ions. It is useful in protein structure and folding studies,6 in the measurement of protein noncovalent complexes,7–10 and for highthroughput protein identifications through bottom-up and topdown MS techniques.4,11–13 Recent efforts have been made toward developing new ionization methods that promise high throughput and/or convenience for measurements performed under ambient conditions. Some of these methods are variations of ESI and MALDI, and they offer potential enhancements in sensitivity and ease of sample introduc(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. (3) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64–71. (4) Aebersold, R.; Mann, M. Nature 2003, 422, 198–207. (5) Henzel, W. J.; Watanabe, C.; Stults, J. T. J. Am. Soc. Mass Spectrom. 2003, 14, 931–942. (6) Kaltashov, I. A.; Eyles, S. J. Applications of Mass Spectrometry in Biophysics: Conformation and Dynamics of Biomolecules; John Wiley & Sons; Chichester, UK, 2005. (7) Loo, J. A. Mass Spectrom. Rev. 1997, 16, 1–23. (8) Loo, J. A. Int. J. Mass Spectrom. 2000, 200, 175–186. (9) Ruotolo, B. T.; Giles, K.; Campuzano, I.; Sandercock, A. M.; Bateman, R. H.; Robinson, C. V. Science 2005, 310, 1658–1661. (10) Sharon, M.; Robinson, C. V. Annu. Rev. Biochem. 2007, 76, 167–193. (11) Aebersold, R.; Goodlett, D. R. Chem. Rev. 2001, 101, 269–295. (12) Kelleher, N. L. Anal. Chem. 2004, 76, 197A–203A. (13) Han, X.; Jin, M.; Breuker, K.; McLafferty, F. W. Science 2006, 314, 109– 112.

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tion. These methods include desorption electrospray ionization (DESI),14 electrospray-assisted laser desorption/ionization (ELDI),15–17 ambient liquid mass spectrometry (ALMS),18 matrixassisted laser desorption electrospray ionization (MALDESI),19 laser ablation electrospray ionization,20 direct analysis in real time,21 atmospheric pressure solids analysis probe,22 desorption atmospheric pressure chemical ionization,23 easy ambient sonic spray ionization (EASI, previously called desorption sonic spray ionization),24 and a new version of EASI, easy ambient sonic spray ionization-membrane interface mass spectrometry (EASI-MIMS).25 The DESI approach is carried out by electrospraying solvent toward a surface coated with a dried sample film; the ESIgenerated droplets desorb and ionize the materials present on the surface.14,26 DESI mass spectra are similar to ESI mass spectra, with multiply charged molecules observed for peptides and proteins. However, most of the DESI applications are limited to small organic and inorganic molecules, as the sensitivity of DESI is relatively low for large biomolecules. EASI is a desorption/ ionization method similar to DESI, except that it uses high-voltagefree sonic spray,27 rather than electrospray, to generate the charged droplets for the desorption and ionization of the dried sample molecules.24 EASI-MIMS applies the EASI source for solution constituent analysis by using a membrane as an interface; charged droplets pick up the analyte molecules from the external surface of the membrane across which the analyte had selectively permeated.25 Recently, a new method termed “reactive DESI” has been introduced.28–36 By electrospraying reagent solution, reactive DESI allows heterogeneous ion/molecule reactions to take place at the interface between ESI charged microdroplets and the solid surface bearing the condensed-phase analytes. Reactive DESI reactions reported so far include ion-transfer and adduct formation to enhance the detection sensitivity of explosives28–30,33 and counterfeit antimalarial drugs,35 oxidation reactions of copper complexes,32 and reactions of phenylboronic acid with sugars and estriol,31 boric acid with alkylmethylphosphonic acids,36 and hydroxylamine with the carbonyl group of steroids.34 Most (14) Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Science 2004, 306, 471–473. (15) Shiea, J.; Huang, M. Z.; HSu, H. J.; Lee, C. Y.; Yuan, C. H.; Beech, I.; Sunner, J. Rapid Commun. Mass Spectrom. 2005, 19, 3701–3704. (16) Huang, M. Z.; Hsu, H. J.; Lee, L. Y.; Jeng, J. Y.; Shiea, J. J. Proteome Res. 2006, 5, 1107–1116. (17) Peng, I. X.; Shiea, J.; Ogorzalek Loo, R. R.; Loo, J. A. Rapid Commun. Mass Spectrom. 2007, 21, 2541–2546. (18) Shiea, J.; Yuan, C. H.; Huang, M. Z. Mol. Cell. Proteomics 2006, 5, S351– S351. (19) Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2006, 17, 1712–1716. (20) Nemes, P.; Barton, A. A.; Li, Y.; Vertes, A. Anal. Chem. 2008, 80, 4575– 4582. (21) Cody, R. B.; Laramee, J. A.; Durst, H. D. Anal. Chem. 2005, 77, 2297– 2302. (22) McEwen, C. N.; McKay, R. G.; Larsen, B. S. Anal. Chem. 2005, 77, 7826– 7831. (23) Williams, J. P.; Patel, V. J.; Holland, R.; Scrivens, J. H. Rapid Commun. Mass Spectrom. 2006, 20, 1447–1456. (24) Haddad, R.; Sparrapan, R.; Eberlin, M. N. Rapid Commun. Mass Spectrom. 2006, 20, 2901–2905. (25) Haddad, R.; Sparrapan, R.; Kotiaho, T.; Eberlin, M. N. Anal. Chem. 2008, 80, 898–903. (26) Cooks, R. G.; Ouyang, Z.; Takats, Z.; Wiseman, J. M. Science 2006, 311, 1566–1570. (27) Hirabayashi, A.; Sakairi, M.; Koizumi, H. Anal. Chem. 1995, 67, 2878– 2882.

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reactive DESI reactions reported to date are limited to adduct formation and reactions of small organic or inorganic molecules. ELDI, as well as its related ALMS and MALDESI, combines features of both ESI and MALDI.15–19 In these methods, desorption of analytes and ionization of those desorbed particles are performed as separate, discrete steps. ELDI desorbs analytes from dried samples deposited on a sample plate using 337-nm ultraviolet radiation from a N2 laser. Neutral particles are postionized by fusing with charged, ESI-generated solvent droplets. Droplet evaporation and ion desorption/desolvation proceed as in normal ESI and generate multiply charged ions.15 ALMS is very similar to ELDI, except that it desorbs analytes from a sample solution drop containing matrix carbon powder, rather than from a dried, pure sample, deposited on the sample plate.18 MALDESI uses the organic matrixes commonly employed for MALDI and atmospheric pressure MALDI to assist the desorption event,19 although the original ELDI reports found matrix to be detrimental.15,16 ELDI is a soft ionization method. More importantly, as in ESI, it forms multiply charged molecules offering MS and tandem MS measurements of large peptides and proteins. The potential enhancement in MS/MS efficiency offered by the multiple charging capabilities is a distinguishing feature of ELDI over MALDI. We have previously shown that (1) ELDI ionizes proteins up to 29 kDa bovine carbonic anhydrase and 66 kDa bovine serum albumin and (2) ELDI-MS/MS can be applied for bottom-up identification of horse cytochrome c (12 kDa) and top-down identification of bee venom melittin (2.8 kDa) and bovine ubiquitin (8.6 kDa).17 In this report, we extend our previous studies coupling ELDI to top-down proteomic studies and characterization of larger proteins.17 We demonstrate that ELDI is amenable for MS and top-down MS/MS of large proteins up to 29 kDa. The applicability of electron-transfer dissociation (ETD) to ELDI-MS/MS peptide sequencing is presented. We also show the effect of the ESI solution’s organic solvent content on protein conformation during the ELDI ionization process, as ELDI is based on the merging of ESI solvent droplets and the laser-desorbed particles. Finally, gasphase reactive-ELDI experiments are demonstrated for the first time. Chemical reactions can be performed during the ELDI ionization process. Preliminary reactive-ELDI experiments show gas-phase disulfide bond reduction using dithiothreitol (DTT, Cleland’s reagent). MS and MS/MS data show that reactive-ELDI with DTT successfully reduces insulin and oxidized glutathione in the gas phase. (28) Chen, H.; Chen, H.; Cooks, R. G. J. Am. Soc. Mass Spectrom. 2004, 15, 998–1004. (29) Cotte-Rodriguez, I.; Takats, Z.; Talaty, N.; Chen, H. W.; Cooks, R. G. Anal. Chem. 2005, 77, 6755–6764. (30) Takats, Z.; Cotte-Rodriguez, I.; Talaty, N.; Chen, H.; Cooks, R. G. Chem. Commun. 2005, 1950–1952. (31) Chen, H.; Cotte-Rodriguez, I.; Cooks, R. G. Chem. Commun. 2006, 597– 599. (32) Nefliu, M.; Cooks, R. G.; Moore, C. J. Am. Soc. Mass Spectrom. 2006, 17, 1091–1095. (33) Song, Y.; Cooks, R. G. Rapid Commun. Mass Spectrom. 2006, 20, 3130– 3138. (34) Huang, G.; Chen, H.; Zhang, X.; Cooks, R. G.; Ouyang, Z. Anal. Chem. 2007, 79, 8327–8332. (35) Nyadong, L.; Green, M. D.; De Jesus, V. R.; Newton, P. N.; Fernandez, F. M. Anal. Chem. 2007, 79, 2150–2157. (36) Song, Y.; Cooks, R. G. J. Mass Spectrom. 2007, 42, 1086–1092.

EXPERIMENTAL SECTION Samples and Reagents. All peptides and proteins, DTT, 2,5dihydroxybenzoic acid (DHB), m-nitrobenzyl alcohol (m-NBA), and formic acid (FA) were purchased from Sigma-Aldrich (St. Louis, MO). Peptide-1 (Phe-Arg-Glu-Asp-Trp-Met-Cys-Leu-Ala-PhePhe-Glu-Arg-Thr- Tyr, MW 2014.3) was synthesized and HPLCpurified by AnaSpec (San Jose, CA). HPLC grade acetonitrile (ACN) was from EMD Chemicals (Gibbstown, NJ). All materials were used as received without further purification. ELDI Ion Source and Mass Spectrometers. The ELDI source, similar to that previously reported,17 includes a pulsed 10-Hz, 337-nm nitrogen laser to desorb sample molecules from a dried sample or liquid drop, an ESI source to generate charged carrier solvent droplets, and an x-y-z sample translation stage. No nebulizing gas was used to assist the electrospray. (Caution must be exercised in the laboratory, as the current platform has exposed high voltage on the ESI needle without a safety enclosure, and the laser radiation is not enclosed. Proper eye protection should be required under such circumstances.) A few modifications have been incorporated to increase the stability of the electrospray and to accommodate a larger sample plate. These changes include the following: (1) a high positive voltage (3.5 kV) is applied to the ESI tip, rather than a grounded source, and -3.5 kV applied to the MS inlet, to generate electrospray for positive ion mass spectra; (2) a home-built stainless steel tube ion guide (5-cm length, 1-mm internal diameter) is adapted to the MS inlet of the quadrupole ion trap system, to allow the ESI tip and sample translation stage to withdraw further to accommodate a larger sample plate, such as a regular 2-in. × 2-in., 100-well MALDI plate; (3) the stainless steel sample plate is connected to ground potential instead of to -3.0 kV; (4) the angle of incidence of the laser increases from 30° to 45°; and (5) the distance between the point where the laser irradiates the target to the MS inlet increases from 3 to 6 mm, while the distance of the ESI tip to the MS inlet remains the same. ELDI mass spectra were acquired using either an Agilent 1100 series MSD quadrupole ion trap mass spectrometer (Agilent Technologies, Santa Clara, CA) or a Thermo LTQ linear ion trap mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Tandem MS spectra were acquired by fragmenting isolated precursor ions with collisionally activated dissociation (CAD)37 using helium or with ETD (LTQ instrument)38 using fluoranthene anions generated by negative chemical ionization. Sample Preparation for ELDI-MS and MSn. For peptide/ protein measurements using ELDI-MS, ESI solvent was prepared by mixing 1:1 v/v of ACN/deionized water with 0.2% FA and spraying at a flow rate of 0.5-2.0 µL min-1. Samples either were deposited as a liquid drop or were dried on the sample plate. Samples in liquid drops were prepared by mixing 1:1 v/v of protein aqueous solution (typically 10-200 µM) and matrix solution (10 mg mL-1 in 50% ACN), as an organic matrix commonly used in MALDI-MS is required for laser desorption of polypeptides from liquids. Four microliters of the mixture was deposited onto the sample plate. For the analysis of samples dried onto the sample plate, 10 µL of 10-100 µM protein aqueous solutions was (37) Loo, J. A.; Edmonds, C. G.; Smith, R. D. Science 1990, 248, 201–204. (38) Syka, J. E.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 9528–9533.

Figure 1. Reactive-ELDI experiment. The reactant is introduced either by laser desorption or by ESI infusion. (A) For the normal ELDI process, a nitrogen laser desorbs the protein analyte, which mixes with the multiply charged droplets generated by an ESI source. (B) For the reactive-ELDI process, the chemical reactant is desorbed either by laser ablation while the protein analyte is infused by ESI (reactive-ELDILD) or (C) the reactant is introduced by ESI and the protein analyte is introduced by laser desorption (reactive-ELDIESI).

deposited onto the sample plate, spread over a 1-cm × 1-cm square area, and allowed to dry without the presence of a matrix. For experiments designed to monitor the ability of organic solvents to induce conformational changes on ubiquitin during the ELDI process, the ESI solvents used were 0.1% FA with ACN concentrations ranging from 20 to 80%. Ubiquitin (10 µL of 100 µM protein solution) dissolved in 20, 50, and 80% ACN solutions with 10 mg mL-1 DHB were deposited on the sample plate and laser desorbed directly for ELDI-MS. Reactive-ELDI for Online Disulfide Bond Reduction. To couple the reactants for the reactive-ELDI experiments, DTT was introduced by either laser desorption or ESI infusion, as shown in Figure 1. For DTT (5 mM in 50% ACN) introduction by ESI (1 µL min-1), the protein sample (10 µL of a 100 µM aqueous solution, dried on the sample plate) is desorbed by laser irradiation. For DTT introduction by laser desorption, DTT (saturated in 50% ACN, with 10 mg mL-1 DHB, deposited onto the sample plate) is desorbed by the laser. Accordingly, the protein sample (1 µM in 50% ACN) is continuously infused by ESI (1 µL min-1). RESULTS AND DISCUSSION ELDI-MS and MSn of Peptides and Proteins. We have previously reported ELDI-MS and CAD MS/MS data for top-down protein characterization of 2.8 kDa melittin and 8.6 kDa bovine ubiquitin, as well as the bottom-up identification (i.e., via trypsin digestion) of 12 kDa cytochrome c.17 All of the previous experiments were performed by laser desorbing protein analyte from a matrix-containing solution deposited on the sample plate and MS measurement employed a quadrupole ion trap mass spectrometer. Different from our previous studies, many of the ELDI-MS and MSn studies described herein are based on laser desorption of the dried protein analyte from a sample plate and, perhaps more Analytical Chemistry, Vol. 80, No. 18, September 15, 2008

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Figure 2. ELDI-MS and MS/MS of peptide-1. The peptide is laser-desorbed from 10 µL of a 100 µM sample solution spread over a 1-cm × 1-cm square area without the presence of a matrix and allowed to dry completely. (A) ELDI mass spectrum of peptide-1 showing 2+ and 3+ charged peptide molecules. (B) CAD-MS/MS of the 2+-charged peptide (m/z 1007), and (C) ETD-MS/MS of the 3+-charged peptide (m/z 672) resulted in nearly complete sequence coverage. Mass spectra were collected on the LTQ linear ion trap instrument. Data from ∼300 laser shots were averaged to obtain each spectrum. The sequence depicted in the inset shows b-/y-products from CAD and c-/z•-products from ETD.

Figure 3. ELDI-MS and ETD-MS/MS spectra of 2.8 kDa melittin. Melittin is laser-desorbed from 10 µL of a 50 µM sample solution spread over a 1-cm × 1-cm square area without the presence of a matrix and allowed to dry completely. (A) ELDI-MS spectrum of melittin (from 100 laser shots). (B) ELDI-ETD-MS/MS (from 300 laser shots) of the 5+ melittin ion (m/z 570) generates a series of c- and z-product ions, including zn2+ (labeled with b; n ) 5-9, 11-25), zn3+ (labeled with 9; n ) 13-23, 25), cn2+ (labeled with O; n ) 9, 10, 14, 17, 20-25), and cn3+ (labeled with 0; n ) 22, 23, 25). Smaller contributions from singly charged y8 and z8-10 and doubly charged y18-20, 24 were also observed. Mass spectra were acquired with an LTQ linear ion trap instrument.

importantly, without the presence of a matrix. In addition, signal stability and, to some extent, sensitivity have improved by adding an ion-transfer tube onto the quadrupole ion trap, modifying the source as described in the Experimental Section or by using the linear ion trap instrument. 6998

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Figures 2–4 show the ELDI-MS and MSn spectra of a 2 kDa peptide (peptide 1), 2.8 kDa melittin, and 29 kDa carbonic anhydrase, as an illustration of the ELDI-MS experiment depicted in Figure 1A. The ELDI-MS spectrum of 15-amino acid residue peptide-1 shows mainly doubly and triply charged molecules

Figure 4. ELDI-MS and CAD-MSn of 29 kDa bovine carbonic anhydrase. The protein was laser-desorbed from 10 µL of a 500 µM sample solution spread over a 1-cm × 1-cm square area without the presence of a matrix and allowed to dry completely. (A) ELDI mass spectrum of carbonic anhydrase, and CAD top-down MSn spectra: (B) MS2 of the (M + 29H)29+ ion (m/z 1001), generating series of y-products, including yn3+ (labeled with 2; n ) 18-23), yn4+ (labeled with ∆; n ) 25), yn5+ (labeled with b; n ) 60-63, 67), yn6+ (labeled with 0; n ) 50, 52, 59-63, 66-69), yn7+ (labeled with 9; n ) 54, 61, 62, 66, 67), and yn8+ (labeled with O; n ) 61, 62, 65-67, 76); (C) MS3 of y677+ (m/z 1086), from MS2 of (M + 29H)29+, generating yn3+ (labeled with 2; n ) 25, 27), yn4+ (labeled with ∆; n ) 36, 39, 43-45, 47-49), yn5+ (labeled with b; n ) 47-49, 60, 61), and yn6+ (labeled with 0; n ) 61-63); (D) MS4 of y616+ (m/z 1174), generating yn3+ (labeled with 2; n ) 25, 27), yn4+ (labeled with ∆; n ) 36, 42-45, 48, 49), yn5+ (labeled with b; n ) 47-49) and b46,474+. ELDI mass spectra were acquired with the LTQ linear ion trap instrument. Data from ∼100 laser shots were averaged for the ELDI-MS spectrum, and ∼600 laser shots for each of the MSn spectra.

(Figure 2A). CAD-MS/MS of the doubly charged ion shows mostly y- and b- product ions (Figure 2B), whereas ETD analysis of the triply charged ion shows mainly c- and z · -product ions (Figure 2C). Both tandem mass spectra give detailed, unambiguous information to sequence characterize the peptide, and the combined measurements yield complete sequence coverage, with 14 out of 14 inter-residue linkages cleaved. The ELDI-MS spectrum of 26-residue melittin shows 3+, 4+, and 5+ charged melittin molecules, with the m/z 712 4+ ion as the most abundant (Figure 3). ELDI-ETD-MS/MS experiments of the 5+ melittin molecule show mostly z- and c-product ions (Figure 3B), while ELDI-CAD-MS/MS data for melittin 4+ ions (m/z 712) show mainly y- and b-product ions, with y132 ion the most abundant product (data not shown). An MS3 experiment by further dissociating the y132 ion (m/z 812) provides more detailed sequence information for melittin. Enhanced sequence coverage was observed for the ETD experiments compared to CAD for melittin, consistent for electron capture dissociation (ECD) spectra of melittin and other biomolecules.39–41 Cleavage of all 25 interresidue linkages was measured from a single ETD spectrum (of the 5+ charged precursor), compared to 18 inter-residue cleavages from CAD-MS2 of the 4+ charged precursor combined with CADMS3 of the y132 product. For 29 kDa carbonic anhydrase, the ELDI-MS spectrum shows a multiple charge distribution similar to that measured by conventional infusion ESI-MS (Figure 4A). Although the sensitivity of the current ELDI technique needs further improvement, the (39) Lin, C.; Cournoyer, J. J.; O’Connor, P. B. J. Am. Soc. Mass Spectrom. 2006, 17, 1605–1615. (40) Liu, H.; Håkansson, K. J. Am. Soc. Mass Spectrom. 2007, 18, 2007–2013. (41) Zubarev, R. A.; Horn, D. M.; Fridriksson, E. K.; Kelleher, N. L.; Kruger, N. A.; Lewis, M. A.; Carpenter, B. K.; McLafferty, F. W. Anal. Chem. 2000, 72, 563–573.

quality of the ELDI-MS spectra of peptides and proteins is comparable to that generated by conventional ESI. Top-down MS of carbonic anhydrase (MW 29024.6) is carried out by CAD, and tandem MS data were collected up to MS4. MS2 of the 29+ charged intact protein shows mostly multiply charged y-product ions, with the most abundant product ions y67 and y61 from the cleavage of the N-terminal amide bonds to Pro-193 and Pro-199, respectively (Figure 4B), which is consistent with the previously reported ESI-MSn data for carbonic anhydrase.42 MS3 of the 7598 Da y677+ ion shows y-products not observed from CAD of the intact protein, in addition to an abundant y616+ ion (Figure 4C). Further dissociation of the 7042 Da y616+ ion in an MS4 experiment shows again mainly y-ions, including an abundant y474+ from cleavage of the Glu-212/Pro-213 amide bond (Figure 4D). These data demonstrate that ELDI generates multiply charged molecules, which can be effectively and efficiently dissociated by tandem MS techniques for top-down proteomic studies. The laser desorption process produces primarily neutral and singly charged molecules for peptides and proteins, but the conversion of the neutral, and possibly charged, protein particles to multiply charged species by the ESI-generated droplets allows the proteins to be detected at lower m/z values and allows for more efficient CAD and ETD processes. Sequence-informative product ions for protein identification can be generated for large multiply charged protein molecules by both CAD and ETD techniques, and potentially by ECD.41 In our previous work in which the protein molecules were laser-desorbed from a sample solution, the presence of a matrix seemed to aid the desorption process.17 In our current experiments where ELDI mass spectra were obtained from dried protein (42) Loo, J. A.; Ogorzalek Loo, R. R.; Andrews, P. C. Org. Mass Spectrom. 1993, 28, 1640–1649.

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Figure 5. Effect of ESI solvent and protein solution composition on the structural conformers of ubiquitin measured by ELDI-MS. Droplets formed by electrospraying solutions composed of 0.1% FA with 20, 50, and 80% ACN are mixed with laser-desorbed ubiquitin (10 µL of 100 µM concentration) dissolved in 20, 50, and 80% ACN solution with DHB. Data were acquired with an Agilent quadrupole ion trap mass spectrometer.

samples, the presence of the matrix seemed to decrease ELDI sensitivity, especially for larger proteins such as carbonic anhydrase. More work is necessary to fully understand the role of the matrix in ELDI and MALDESI experiments. ELDI-MS sensitivity for intact protein analysis appears to be comparable or even slightly higher compared to DESI and MALDESI. For a medium-sized protein such as 25.6 kDa chymotrypsinogen, Basile and co-workers reported a DESI detection limit (with a signal-to-noise ratio of 3 or better) of 100 ng mm-2.43 Muddiman and co-workers reported acquiring MALDESI mass spectra from 0.8 µL of a 250 µM solution of 8.6 kDa ubiquitin deposited on the laser desorption surface.44 With our ELDI platform, we are able to measure a mass spectrum with a signalto-noise ratio of 3 for 29 kDa carbonic anhydrase from 10 µL of a 1 µM solution dried to a spot area of 13 mm2, corresponding to 23 ng mm-2. Protein Multiple Charging and the ELDI Process. In an ELDI-MS experiment, laser-desorbed particles merge and mix with ESI-generated charged solvent droplets. To investigate this mixing process, the effect of organic solvent on the multiple charge distribution of ubiquitin was measured by ELDI-MS. It is known that, in the ESI process, the conformation of the polypeptide is an important factor affecting its charge distribution. A protein in its native, globular state exhibits a charge distribution envelope at higher m/z, representing lower charge-state molecules. A more extended, denatured state shows a multiple charge envelope at lower m/z, representing higher charge states.45 A change in a protein’s conformation can be induced by a variety of factors, including decreasing the pH, or increasing the organic content, in the ESI solvent.46 For ubiquitin, previous work (43) Shin, Y. S.; Drolet, B.; Mayer, R.; Dolence, K.; Basile, F. Anal. Chem. 2007, 79, 3514–3518. (44) Dixon, R. B.; Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C. Anal. Chem. 2008, 80, 5266-5271. (45) Loo, J. A.; Ogorzalek Loo, R. R.; Udseth, H. R.; Edmonds, C. G.; Smith, R. D. Rapid Commun. Mass Spectrom. 1991, 5, 101–105. (46) Loo, J. A.; Udseth, H. R.; Smith, R. D. Biomed. Environ. Mass Spectrom. 1988, 17, 411–414.

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demonstrated that high organic solvent content (e.g., ACN) can effectively denature the protein’s structure, resulting in a shift to higher ESI charging.45 To study the mixing process of the laserdesorbed particles with the ESI-generated droplets, we monitored the ubiquitin charge distribution as a function of ACN content in both the ESI solvent and the analyte droplet on the sample stage. According to previous ESI studies, the most abundant peaks for ubiquitin under native solution conditions are m/z 1071 (8+) and 1224 (7+). For denatured ubiquitin, the most abundant peaks are around m/z 780 (11+).45 Figure 5 shows ubiquitin ELDI mass spectra collected by ESI spraying 20, 50, and 80% ACN solution (with 0.1% formic acid) and laser-desorbing ubiquitin from a liquid droplet containing various concentrations of ACN. With a high ACN concentration (80%) in the ESI solvent, ubiquitin is primarily denatured (higher charged states) (Figure 5H and I). Low ACN concentration (20%) in the protein solution mixed with the ESI 80% ACN droplets tends to partially retain the native protein conformation originally present on the plate, as a bimodal charge distribution is observed (Figure 5G). These experiments demonstrate effective mixing between the laser-desorbed ubiquitin molecules and the ESI-generated solvent droplets. However, the mixing may not be complete, as there is still a significant amount of native ubiquitin molecules, when the protein is originally in 20% ACN native condition, with ESI spraying 80% ACN solution (Figure 5G). Likewise, with 80% ACN in the protein solution and 20% ACN in the ESI solution to promote an unfolded-to-folded transition, the resulting mass spectrum shows a mixture of folded and unfolded conformations (Figure 5C). For ubiquitin, rates for the folding process range from 1556 to 400 s-1, or 0.6-2.5 ms.47,48 For a 2-µm-diameter droplet produced by ESI, the axial mean velocity is ∼5-10 m/s,49–51 corresponding to a transit time of roughly 1-2 ms. Thus, although the folding/unfolding rates for (47) Colley, C. S.; Clark, I. P.; Griffiths-Jones, S. R.; George, M. W.; Searle, M. S. Chem. Commun. 2000, 1493–1494. (48) Mia, D.; Liub, G. R.; Wang, J.-S.; Li, Z. R. J. Theor. Biol. 2006, 241, 152– 157. (49) Olumee, Z.; Callahan, J. H.; Vertes, A. J. Phys. Chem. A 1998, 102, 9154– 9160.

Figure 6. Addition of m-NBA into the ESI solvent to increase the charge state of human insulin and bovine carbonic anhydrase during the ELDI process. Aqueous solutions of insulin (10 µL, 100 µM) and carbonic anhydrase (10 µL, 500 µM) were spread and dried over a 1-cm × 1-cm square onto the sample plate. A solution composed of 50% ACN/0.1% FA with (A and C, for insulin and carbonic anhydrase, respectively) and without (B and D, insulin and carbonic anhydrase, respectively) m-NBA was electrosprayed and the droplets were mixed with the laserdesorbed proteins. Mass spectra were acquired with the LTQ linear ion trap instrument.

ubiquitin correspond well to the droplet velocity, this does not consider variables, such as the efficiency of mixing of the desorbed protein particles and the ESI-generated droplets, and solvent composition from evaporation as the droplet traverses toward the mass spectrometer inlet. Another test of the mixing and its effect on protein charging is to incorporate a chemical compound that affects charging. It has been reported that the addition of m-NBA in the ESI solvent increases the surface tension of ESI-generated charged droplets and therefore promotes the positive “supercharging” of protein ions produced by ESI.52–54 The addition of m-NBA can be useful to promote increased charging for tryptic peptides characterized by ESI-ETD-MS/MS,54 as ETD efficiency is increased for higher charged peptides. Supercharging of ELDI-generated ions can be induced as well. The small 5.8 kDa human insulin protein and the larger 29 kDa carbonic anhydrase were tested. Panels A and B of Figure 6 show the ELDI mass spectra of laser-desorbed insulin postioned by the ESI droplets with and without m-NBA in the ESI solution. With 0.25% m-NBA in the ESI solution, the highest charge state of ELDI insulin ions is 6+ (m/z 969), while the highest charge state is 5+ (m/z 1162) without addition of m-NBA to the ESI solvent. The average charge state of the ELDIgenerated insulin molecules increased from 4.0 to 4.8 with m-NBA in the ESI solution. For carbonic anhydrase (Figure 6C and D), the presence of m-NBA shifts the maximum charge state from 36+ to 41+, and the average charge state is increased similarly from +27.7 to +30.0. (50) Olumee, Z.; Callahan, J. H.; Vertes, A. Anal. Chem. 1999, 71, 4111–4113. (51) Wortmann, A.; Kistler-Momotova, A.; Zenobi, R.; Heine, M. C.; Wilhelm, O.; Pratsinis, S. E. J. Am. Soc. Mass Spectrom. 2007, 18, 385–393. (52) Iavarone, A. T.; Jurchen, J. C.; Williams, E. R. Anal. Chem. 2001, 73, 1455– 1460. (53) Iavarone, A. T.; Williams, E. R. J. Am. Chem. Soc. 2003, 125, 2319–2327. (54) Kjeldsen, F.; Giessing, A. M. B.; Ingrell, C. R.; Jensen, O. N. Anal. Chem. 2007, 79, 9243–9252.

Although the small increase in charge may have limited practical applications, the experiments with m-NBA experiments were performed to test the capabilities of the ELDI process for incorporating additives to the droplets. On the otherhand, Jensen’s laboratory recently reported the application of m-NBA for increasing the charge state of tryptic peptides.54 Although the chargestate distributions of the tryptic peptides increase slightly, increasing the abundance of the 3+ charged peptides dramatically enhances the efficiency for sequencing peptides using ETD compared to 2+ charged peptides. More importantly, these experiments suggest that the laser-desorbed protein particles can merge into the ESI solvent droplets and undergo a transformation as they travel toward the MS inlet of the mass spectrometer. Reduction of Peptide Disulfide Bonds by Reactive-ELDI. A goal of the development of reactive-ELDI is to provide additional analytical capabilities by the incorporation of gas-phase reactions prior to the ELDI-MS measurements. Many proteins contain intramolecular disulfide bonds formed between cysteine residues. The existence of disulfide bonds often hinders appropriate gasphase cleavage of peptide bonds or the ready interpretation of tandem MS spectra from such experiments, and potentially it is more difficult to characterize proteins with disulfide linkages by top-down MS. Usually, one needs to perform time-consuming pretreatments, such as disulfide bond reduction and sulfhydryl alkylation, prior to MS or MS/MS analysis. A commonly used disulfide reducing agent for protein pretreatment is DTT.55 DTT is used to reduce the disulfide bonds of proteins and to prevent intramolecular and intermolecular disulfide bonds from forming between cysteine residues. The reduced form of DTT is a molecule with two free thiol groups connected by four carbon atoms, while the oxidized form is a stable cyclized six-membered ring formed by bringing the two sulfur atoms (55) Cleland, W. W. Biochemistry 1964, 3, 480–482.

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Figure 7. Reactive-ELDIESI experiments for gas-phase disulfide reduction of oxidized glutathione with DTT. A DTT solution (5 mM in 50% ACN) was introduced by ESI infusion at a flow rate of 1 µL min-1. Oxidized glutathione (10 µL, 100 µM in water) was deposited, spread, and dried on the sample plate for laser desorption. ELDI mass spectra were acquired using a quadrupole ion trap mass spectrometer. (A) ELDI-MS of oxidized glutathione, showing a singly charged ion m/z 613. (B) Reactive-ELDI-MS for disulfide reduction of oxidized glutathione with DTT in the ESI solution.

together. The DTT reduction of a disulfide bond is usually accomplished by two sequential thiol-disulfide exchange reactions, resulting in polypeptides with reduced free cysteines residues, and the formation of the stable ring of oxidized DTT with an internal disulfide bond. The reducing power of DTT is limited to pH values above ∼7, since only the negatively charged thiolate S- is reactive and the protonated sulfur form SH has lower nucleophilicity.56 The efficiency of protein disulfide reduction via DTT and reactive-ELDI was tested. DTT is introduced into the system either by ESI (reactive-ELDIESI) or by laser desorption (reactiveELDILD). When DTT is introduced by ESI, the protein sample is desorbed by laser ablation. When introduced by laser desorption, DTT is deposited on the sample plate with DHB and desorbed while still in solution. Accordingly, the protein sample is continuously infused by ESI. Preliminary data show that reactive-ELDI with DTT successfully reduces the disulfide bonds of insulin and oxidized glutathione in the vapor/droplet phase online with MS detection, and the resulting signals can persist for more than 1 h. Figures 7 and 8 show the reactive-ELDI experiments for online DTT reduction of oxidized glutathione and human insulin, respectively. In these experiments, the reactions are carried out by ESI infusing DTT and laser-desorbed polypeptide molecules from dried samples without matrix. Oxidized glutathione is composed of two identical tripeptides (γGlu-Cys-Gly) linked by a disulfide bond. ELDI-MS spectra of the gas-phase DTT reduction of oxidized glutathione showed, besides the unreacted singly charged (M + H)+ of oxidized glutathione (m/z 613), an additional peak at m/z 307 (Figure 7B) for the singly charged reduced glutathione molecule. The disulfide reduction reaction is believed to take place in the gas phase or in the microdroplets from the merging and mixing of DTT and peptide molecules during the ELDI ionization process. Conventional ELDI-MS measurements for oxidized glutathione (Figure 7A) showed that the laser desorption process is not capable of cleaving the disulfide bond. (56) Ruegg, U. T.; Rudinger, J. Methods Enzymol. 1977, 47, 111–116.

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Online disulfide reduction of insulin showed two new peaks at m/z 858 and 1144 (Figure 8A) besides the remaining original intact insulin peaks at m/z 969 (6+) and 1162 (5+). The new peaks at m/z 858 and 1144 observed upon introduction of DTT by ESI are identified to be insulin B-chain with 4+ and 3+ charges, respectively. (The sensitivity for positively charged A-chain insulin is lower compared to the B-chain polypeptide by conventional ESIMS.) The two disulfide bonds joining A-chain and B-chain together were reduced by online DTT-induced reactions. CAD tandem MS analysis of the m/z 1144 ion showed y21-28 and b23-27 product ions, further confirming the insulin B-chain identity (Figure 8B). Disulfide bond reduction by online reactive-ELDI can be carried out also by electrospraying the protein solution and laserdesorbing DTT from a saturated DTT solution mixed with DHB (reactive-ELDILD). Figure 8C shows the disulfide reduction of human insulin using this method. When the laser is off and no DTT is desorbed, ESI analysis of insulin showed mainly the 4+ and 5+ intact insulin ions (data not shown). Turning on the laser to desorb DTT generates three additional peaks at m/z 858 (insulin B-chain, 4+), 1144 (insulin B-chain, 3+), and 1193 (insulin A-chain, 2+) and a reduction in relative abundance of the 4+- and 5+-charged intact insulin molecules (Figure 8C). CAD tandem MS data of m/z 1144 further confirmed the B-chain identity (data not shown). Although both modes of reactive-ELDI, i.e., laser desorbing or ESI-introduced reagent, are effective for disulfide reduction, electrospraying DTT is more reliable and reproducible in our preliminary experiments. From these experiments, it is not clear how the laser-desorbed particles (and droplets from desorbing liquids) are mixing with the electrospray-produced charged droplets or gas-phase molecules. From the solvent composition/charge-state experiments for ubiquitin (Figure 5) and the increased charging from the incorporation of m-NBA (Figure 6), mixing is sufficiently efficient to affect the resulting mass spectra. Furthermore, the reduction of disulfide bonds of glutathione (Figure 7) and insulin (Figure 8) by DTT demonstrate that chemical reactions can be promoted by the online reactive-ELDI process. Complete disulfide bond reduction was not observed, as a significant amount of the nonreduced polypeptide was measured and reduction was estimated to be no more than 30% efficient. This raises questions on its applicability to larger proteins. However, it is somewhat surprising that any disulfide bond reduction was observed. Mixing of the reactants and the chemical reaction must be completed in the submillisecond time frame as the microdroplets travel at atmospheric pressure toward the mass spectrometer orifice. Reduction of disulfide bonds in solution for standard proteins such as insulin and ribonuclease is reported to occur in the hundreds of seconds to several minutes.57–59 No disulfide reduction was observed to occur for the reaction between 10 µM insulin and 10 mM DTT mixed in solution for 10 min and monitored by ESI-MS and MALDI-TOF-MS (data not shown). The reactive-ELDI conditions are far from standard solution conditions. ESI-generated droplets are on the order of tens of micrometers in diameter (and smaller), and they are highly charged. It is possible that the local concentration of the reductant is in far greater excess to the protein analyte in the evaporating droplet, thereby accelerating (57) Fukada, H.; Takahashi, K. J. Biochem. 1980, 87, 1111–1117. (58) Garel, J. R. FEBS Lett. 1977, 79, 135–138. (59) Singh, R.; Whitesides, G. M. J. Org. Chem. 1991, 56, 2332–2337.

Figure 8. Reactive-ELDI experiments for online disulfide reduction of insulin with DTT. A DTT solution (5 mM in 50% ACN) was introduced by ESI infusion at a flow rate of 1 µL min-1. Human insulin (10 µL, 100 µM in water) was deposited, spread, and dried on the sample plate for laser desorption. ELDI mass spectra were acquired using a quadrupole ion trap mass spectrometer. (A) Reactive-ELDIESI-MS for disulfide reduction of insulin with DTT in the ESI solution yield new peaks at m/z 858 (insulin B-chain, 4+) and 1144 (insulin B-chain, 3+). (B) Subsequent CADMS/MS of m/z 1144 confirmed the B-chain identity. (C) A solution of DTT (saturated in 50% ACN, with 10 mg mL-1 DHB) was deposited onto the sample plate and desorbed by laser irradiation (reactive-ELDILD). Insulin (1 µM in 50% ACN) was infused by ESI at 1 µL min-1. Only intact insulin was observed with the laser turned off. With the laser turned on to desorb DTT, insulin and the products of disulfide reduction, A-chain and B-chain insulin, were measured.

the reaction. The 5 mM concentration of DTT (in the ESI solution) required and described in this report was determined empirically; DTT concentrations less than 1 mM did not successfully display reactivity by the technique. The concentration requirements of the reactants and the effects of other experimental variables, such as temperature, need to be studied further. Whether small, highly charged surfaces and droplets can promote and accelerate such chemical reactions remains to be studied. However, it is clear that laser-desorbed particles can merge and mix with ESI-generated charged droplets. This should allow other chemical and enzymatic reactions to be exploited online as tools for protein characterization, as well as for tissue screening and imaging. Also, these reactive-ELDI disulfide reduction experiments provide a possibility for direct top-down protein identification for proteomic study, without the necessity of in-solution reduction and alkylation. CONCLUSIONS We demonstrate that ELDI is a soft ionization method for MS measurements of peptides and proteins from both dried and wet samples under ambient conditions. ELDI generates multiply charged ions, which allows laser-desorbed species to readily access the advantages of tandem mass spectrometry for producing sequence-informative product ions by top-down mass spectrometry. The ELDI ionization process separates the desorption and ionization steps, and it is based on the merging and mixing of ESI-generated solvent droplets and laser-desorbed particles. This should allow for online chemical reactions between the ESI solution and the laser-desorbed sample. Reactive-ELDI data show that disulfide bonds can be reduced online by either ESI infusion or laser-desorbing the reducing agent, DTT. Reactive-ELDI

provides a possibility for direct top-down protein identification of disulfide bond-containing proteins for proteomic study, without the necessity of the additional steps of in-solution reduction and alkylation. Reactive-ELDI with laser desorption of a disulfide reducing agent could be combined with LC-MS/MS for online top-down protein identification. Additional chemical and enzymatic reactions will be exploited in the future with reactive-ELDI experiments to achieve more convenient and high throughput proteomics analysis. The primary intention of our report is to discuss progress toward improvements in ELDI performance and to introduce the concept of “reactive” ELDI. We envision that ELDI-MS and ELDIMSn would have unique advantages for analysis of intact protein samples embedded in complex matrixes, such as tissues and fluids, as demonstrated already by Shiea and co-workers,16,18 and even 1D- and 2D-gels without the laborious procedure required for intact protein extraction. ACKNOWLEDGMENT The authors thank Dr. Minzong Huang and Wenhan Zhan (National Sun Yat-Sen University) for helpful experimental advice. The W. M. Keck Foundation is acknowledged for the establishment of the UCLA Functional Proteomics Center. The project is financially supported by a grant from the National Institutes of Health (National Cancer Institute, NCI; R21CA126106 to J.A.L.).

Received for review April 29, 2008. Accepted June 25, 2008. AC800870C Analytical Chemistry, Vol. 80, No. 18, September 15, 2008

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