A. Kononikhin et al., Eur. J. Mass Spectrom. 19, 247–252 (2013) Received: 25 September 2013 n Accepted: 14 October 2013 n Publication: 16 October 2013 247 EUROPEAN JOURNAL OF MASS SPECTROMETRY Signal enhancement in electrospray laser desorption/ionization mass spectrometry by using a black oxide-coated metal target and a relatively low laser fluence Alexey Kononikhin,a,d Min-Zong Huang,b Igor Popov,c,d Yury Kostyukevich,a,d Evgeny Kukaev,a,d Alexey Boldyrev,a Alexander Spasskiy,a Ilya Leypunskiy,a Jentaie Shieab and Eugene Nikolaeva,c,e* a Institute for Energy Problems of Chemical Physics, 119334 Moscow, Russia. E-mail: ennikolaev@rambler.ru b Department of Chemistry, National Sun Yat-Sen University, Kaohsiung, Taiwan c Emanuel Institute of Biochemical Physics, Moscow, Russia d Moscow Institute of Physics and Technology, Dolgoprudny, Russia e Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, ul. Pogodinskaya 10, 119121 Moscow, Russia The electrospray laser desorption/ionization (ELDI) method is actively used for direct sample analysis and ambient mass spectrometry imaging. The optimizing of laser desorption conditions is essential for this technology. In this work, we propose using a metal target with a black oxide (Fe3O4) coating to increase the signal in ELDI-MS for peptides and small proteins. The experiments were performed on an LTQ-FT mass spectrometer equipped with a home-made ELDI ion source. A cutter blade with black oxide coating was used as a target. A nitrogen laser was used with the following parameters: 337 nm, pulse duration 4 ns, repetition rate 10 Hz, fluence ~ 700 J m–2. More than a five times signal increase was observed for a substance P peptide when a coated and a non-coated metal target were compared. No ion signal was observed for proteins if the same fluence and the standard stainless steel target were used. With the assistance of the Fe3O4 coated metal target and a relatively low laser fluence (≤ 700 J m–2), proteins such as insulin, ubiquitin and myoglobin were successfully ionized. It was demonstrated that the Fe3O4-coated metal target can be used efficiently to assist laser desorption and thus significantly increase the analyte signal in ELDI-MS. A relatively low laser fluence (≤ 700 J m–2) was enough to desorb peptides and proteins (up to 17 kDa) with the assistance of the Fe3O4-coated metal target under ambient conditions. Keywords: electrospray laser desorption/ionization (ELDI), assistance of laser desorption, ambient mass spectrometry, high-resolution mass spectrometry (FT-ICR), metal oxide (Fe3O4) Introduction A combination of laser desorption (LD) with electrospray ionization (ESI) has resulted in the development of the electrospray laser desorption/ionization (ELDI)1 method for mass spectrometry and its different modifications such as matrix-assisted laser desorption electrospray ionization ISSN: 1469-0667 doi: 10.1255/ejms.1229 (MALDESI)2 and laser ablation electrospray ionization (LAESI).3 Generally in these techniques, laser desorbed materials from the solid substrate are post-ionized by electrospray at atmospheric pressure and finally ESI-like multiply charge ions are generated in contrast to matrix-assisted laser desorption © IM Publications LLP 2013 All rights reserved 248 Signal Enhancement in Electrospray Laser Desorption/Ionization Mass Spectrometry (MALDI) and surface-assisted laser desorption/ionization (SALDI) methods.1 Today, this technology stands in line with methods for direct sample analysis at ambient conditions such as desorption electrospray ionization (DESI), direct analysis in real time (DART), desorption atmospheric pressure chemical ionization (DAPCI) and others.4–9 Optimizing laser desorption conditions is essential for ELDIMS. The influences of organic and inorganic matrices, the laser energy, the laser wavelength and the sample holder material on desorption of protein molecules from sample plates were demonstrated10 and, in particular, it was shown that carbon powder and nanoparticles can influence desorption/ ionization processes. 11 Assistance with the desorption/ ionization processes by inorganic materials was first realised by Tanaka et al. during LDI-MS analysis of the proteins.12 They successfully employed cobalt powder (~30 nm) mixed with glycerol for the desorption/ionization of large proteins (up to 25 kDa). Later, Sunner et al. used micro-sized graphite powder mixed with glycerol for laser desorption/ionization of peptides and proteins from liquid solutions and termed this approach as surface-assisted laser desorption/ionization (SALDI).13 The type, form and size of SALDI substrates are the critical parameters affecting ion generation efficiency. Numerous materials were used for assisting laser desorption/ionization based on silicon, carbon and metal.14–16 It was shown that oxide nanoparticles [(NPs), namely SiO 2, TiO 2, Fe 3O 4 and Fe3O4 /TiO2] can be used efficiently to assist laser desorption and ionization processes for peptides and proteins.9,10 Metal oxide films such as TiO2, ZnO, SnO2 and ZrO2 were also used successfully as assisting materials for LDI-MS analysis.17,18 Authors have shown that the best sensitivity can be obtained for peptides and proteins using TiO2 specially fabricated film with citric buffer as the proton source. In this work, we propose to use a metal target with a black oxide (Fe3O4) coating to increase the signal in electrospray laser desorption/ionization (ELDI)-MSfor peptides and proteins. One advantage of black oxide over other coatings is the cheapness and cost effectiveness of the technology.19 Black oxide or blackening is a conversion coating for ferrous materials, copper and copper-based alloys, zinc, powdered metals and silver solder.19,20 It is used to add mild corrosion resistance and for appearance. During blackening of ferrous alloys, oxidizing salts react with the iron to form magnetite (Fe3O4)—a black oxide layer 1–10 µm thick. We have found that a metal target with an Fe3O4 coating significantly increases analyte signal intensity during ELDI-MS. Experimental In the study, for a stainless steel target with an Fe3O4 coating, we used a common cutter blade (Sparta Company, Russia). The experiments were performed on a 7-Tesla LTQ-FT Ultra (Thermo Electron, Bremen, Germany) mass spectrometer equipped with a home-made ELDI ion source [Figure 1(a)]. The following conditions were used for electrospray: flow rate 3 µL min−1, positive ion mode; needle voltage 3.5 kV; no sheath and auxiliary gas flow; MS inlet voltage 10 V; heated capillary temperature 250°C. The nitrogen laser (SESI, USA) parameters were as follows: wavelength 337 nm, pulse duration 4 ns, repetition rate 10 Hz; fluence ~ 700 J m–2. Fourier transform ion cyclotron resonance (FT-ICR) mass spectra were acquired with a resolution of R = 100,000 at m/z 400. For the analysis of ELDI-MS, we used standard peptide and proteins purchased commercially (Sigma, St Louis, MO, USA): substance P (1347.6 Da), insulin (5.7 kDa), ubiquitin (8.5 kDa) and myoglobin (16.9 kDa). The sample preparation was as follows: the analyte was dissolved in water and then 2 µL of solution was dropped on the target. Results and discussion A relatively low laser fluence (≤ 700 J m–2) was used to investigate the effect of the laser desorption assistance while usually for an ELDI-like method more than 10 times higher fluence is used.4,11 As shown in Figure 2, the effect of the black oxide coating is demonstrated for the doubly charged molecular ion of the substance P peptide [M + 2H]2+ with m/z 674.5 [Figures 1(b) and 1(c)]. A signal increase of more than five times is observed if the coated and non-coated metal targets are compared [Figures 1(b) and 1(c)]. The ion signals were also obtained from a wet spot of substance P. The same signal intensity was observed in the case of wet and dry spot analysis [Figures 1(c) and 1(d)]. No ion signal was observed for proteins if the same fluence (≤ 700 J m–2) and the standard stainless steel target were used. It was demonstrated that proteins from the aqueous solution can be detected by ELDI-MS under ambient conditions. 4,11 The presence of fine particles of carbon powder or Au nanoparticles (NPs) in the sample solution was helpful for the desorption of the protein molecules by UV laser with the fluence (~15 kJ m –2 ). 11 Herein, we demonstrate that proteins up to 17 kDa can be detected by ELDI in combination with FT-ICR-MS. With the assistance of the Fe3O4-coated metal target, a relatively low laser fluence (≤ 700 J m–2) was enough to desorb proteins such as insulin, ubiquitin and myoglobin (Figure 2). As shown in Figure 2(a), the multiply charged ions from +4 to +6 were detected from the dry insulin spot. The signals were high enough to observe isotopic distribution for each of the protein charge states [Figure 3(a)—inserts]. As with the results of the substance P peptide, the ion signals from the insulin wet spot were also successfully detected. The mass spectra in both dry and wet insulin spots showed that the ion signal intensity was equally high and charge distribution from +4 to +6 was the same [Figures 3(a) and 3(b)]. Further, the wet spots containing ubiquitin and myoglobin proteins were analyzed via ELDI-MS. The multiply charged distribution from +6 to +8 and +14 to +25 for ubiquitin and myoglobin were successfully detected, respectively [Figures 2(c) and 2(d)]. The results are in accord with previous observations A. Kononikhin et al., Eur. J. Mass Spectrom. 19, 247–252 (2013) 249 Figure 1. ELDI ion trap mass spectra of substance P (10 pmol µL−1), laser fluence (≤ 700 J m–2): (a)—Schematic view of homemade ELDI ion source; (b)—stainless steel target, dry spot; (c)—metal target with black oxide coating, dry spot; (d)—metal target with black oxide coating, wet spot. of Chang et al.15 which showed that Fe3O4 nanoparticles can be efficiently used for assistance in laser desorption and thus provides the mass limit up to 25 kDa for proteins in SALDI-MS. Analysis of the black oxide surface morphology before and after laser shots was performed using a scanning electronic microscope, Philips SEM 515, with computer registration of images. Electron-microscopic analysis of the surface was performed at different magnifications in the range from 156× up to 10,000× (Figure 3). Vertical strips observed on images are caused by a relief of rolling which is partially smoothed out by a thick multilayer of oxide film [Figure 3(c)]. It can be seen from the image that, after a dozen laser shots, the oxide film (layer) is seriously damaged [Figure 3(d)]. It was also noted during ELDI-MS analysis that the signal decreased with the laser shots and one needed to move to another spot inside the sample. Conclusion It was demonstrated that an Fe3O4-coated target can significantly increase analyte signal in ELDI MS. As a metal oxide layer we propose to use Fe3O4 as a cheap and cost-effective material. A more than five times signal increase was observed for peptides desorbed from the black oxide-coated target. We demonstrate the possibilities of using the ELDI method in combination with high-resolution mass spectrometry (FT-ICR) for small proteins analysis. With the assistance of the Fe3O4 coated metal target, proteins up to 17 kDa were analyzed using 250 Signal Enhancement in Electrospray Laser Desorption/Ionization Mass Spectrometry Figure 2. ELDI FT-ICR mass spectra of the proteins, black oxide-coated metal target, laser fluence (≤ 700 J m–2): (a)—insulin (10–4 M), dry spot; (b)—insulin (10–4 M), wet spot; (c)—Ubiqutin (10–4 M), wet spot; (d)—myoglobin (10–4 M), wet spot. a relatively low laser fluence (≤ 700 J m–2). The signal intensity was high enough to observe isotopic distribution for each of the protein charge states, which is essential for accurate mass measurements. At the same laser fluence, no signal was observed for proteins deposited on the standard stainless steel target. 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