Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Figure 5. ESI spectrum showing the derivatized protein resulting from reacting the reduced �-lactoglobulin A with ebselen. The superscript in the charge number shows the number of selenium tags added to the protein after derivatization. High selectivity and efficiency of the selenium chemistry investigated in this study makes it useful in identification of the number of free and bound thiol groups in proteins, which is of importance in the protein structural analysis. The reaction of intact protein �-lactoglobulin A with ebselen as described above shows that the protein has only one free cysteine residue. We further examined the derivatization reaction with reduced protein. In the experiment, the �-lactoglobulin A was first reduced by TCEP, which is known to be more stable and effective to reduce disulfide bonds than DTT. 46 After reduction and removal of the excess amount of TECP, the reagent ebselen was added to the protein solution for thiol derivatization. As shown in Figure 5, the reduced �-lactoglobulin A containing three selenium tags has a high relative abundance. It is likely that the reduction of disulfide bond of Cys 66 -Cys 160 is easier than the other Cys 106 -Cys 119 bond leading to reduced protein mainly with three free thiols. 47 Another possible reason is that the reduced protein is partially folded so that it is not easy for the relatively large ebselen reagent to access all of the free thiols. Nevertheless, in Figure 5, the multiply charged ions of fully modified proteins with five selenium tags were clearly observed, suggesting that the reduced protein has a maximum of five free cysteine residues. This indicates that the four additional free cysteine residues result from reduction and the protein has two disulfide bonds prior to reduction, which is exactly in agreement with the known structure (46) Han, J. C.; Han, G. Y. Anal. Biochem. 1994, 220, 5–10. (47) Sakai, K.; Sakurai, K.; Sakai, M.; Hoshino, M.; Goto, Y. Protein Sci. 2000, 9, 1719–1729. 6932 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 of �-lactoglobulin A as mentioned before. Thus, it is possible to identify the number of free cysteine, total cysteine residues, and disulfide linkages in proteins using a simple approach based on the selenium chemistry. CONCLUSIONS In summary, ebselen and N-(phenylseleno)phthalimide, as Se-N bond containing compounds, are excellent labeling reagents for characterization of thiol-containing compounds by mass spectrometry. In this study we examined a series of reactions of these two selenium reagents with amino acids, peptides, and proteins. Our study reveals that the thiol derivatization reaction is highly selective, rapid, reversible, and efficient (quantitative in the case of �-lactoglobulin A derivatization by ebselen), which is of high value in proteomics research. In comparison to the wellknown Michael-addition reactions used for thiol tagging, selenium reagents appear to be more efficient and faster. Analytical applications stemming from this investigation include (i) fast screening of peptides/proteins containing free cysteine residues from complex mixtures and (ii) identification of the number of free and bound thiols of proteins and their locations using MS/ MS experiments. Given the significance of thiols in life and the important reaction features uncovered, it is expected that there will be many novel MS applications based on the powerful selenium chemistry reported in this study. ACKNOWLEDGMENT This work was supported by U.S. NSF (Grant CHE-0911160), National Basic Research Program of China (973 Program, Grant 2007CB936000), National Natural Science Funds for Distinguished Young Scholar (Grant No. 20725518), National Natural Science Foundation of China (Grant No. 20875057), and Natural Science Foundation of Shandong Province in China (Grant No. Y2007B02). Part of the research described in this manuscript was performed at the W. R. Wiley Environmental Molecular Sciences Laboratory (EMSL), a national scientific user facility sponsored by the U.S. Department of Energy’s Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory (PNNL). PNNL is operated by Battelle for the U.S. Department of Energy. We also thank Mr. Xiaoyong Lu for his help. SUPPORTING INFORMATION AVAILABLE Additional supporting mass spectra. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review May 4, 2010. Accepted July 6, 2010. AC1011602
Anal. Chem. 2010, 82, 6933–6939 Difference between Ultramicroelectrodes and Microelectrodes: Influence of Natural Convection Christian Amatore,* Cécile Pebay, Laurent Thouin,* Aifang Wang, and J-S. Warkocz Ecole Normale Supérieure, Département de Chimie, UMR CNRS-ENS-UPMC 8640 “Pasteur”, 24 rue Lhomond, F-75231 Paris Cedex 05, France Natural convection in macroscopically immobile solutions may still alter electrochemical experiments performed with electrodes of micrometric dimensions. A model accounting for the influence of natural convection allowed delineating conditions under which it interferes with mass transport. Several electrochemical behaviors may be observed according to the time scale of the experiment, electrode dimensions, and intensity of natural convection. The range of parameters in which ultramicrelectrodes behave under a true diffusional steady state was identified. Mapping of concentration profiles was performed experimentally by scanning electrochemical microscopy in the vicinity of microelectrodes of various radii. The results validated remarkably the predictions of the model, evidencing in particular the alteration of the diffusional steady state by natural convection. Microelectrodes are versatile tools for the study of electrochemical processes of mechanistic and/or analytical interest. Their advantageous properties stem from their small size. Microelectrodes may be used in highly resistive environments and in very small sample volumes. They enable the detection of very small amounts of material and allow short time responses. 1-9 However, the definition of a microelectrode is still nowadays ambiguous. Actually, the notion of a microelectrode differs greatly according to the particular origin of electrochemists, i.e., electroanalytical chemists or molecular electrochemists. The term microelectrode may then encompass electrodes of either millimetric or micrometric dimensions. Electrodes of smaller sizes are referred to as ultramicroelectrodes. Such definitions, based mainly on historical * To whom correspondence should be addressed. E-mail: christian.amatore@ ens.fr (C.A.); laurent.thouin@ens.fr. (1) Fleischmann, M.; Pons, S.; Rolison, D. R. Ultramicroelectrodes; Datatech Systems, Inc.: Morgantown, NC, 1987. (2) Bond, A. M.; Oldham, K. B.; Zoski, C. G. Anal. Chim. Acta 1989, 216, 177–230. (3) Wightman, R. M.; Wipf, D. O. Electroanalytical Chemistry; Marcel Dekker: New York, 1989; Vol. 15, pp 267-353. (4) Montenegro, M. I.; Queiros, M. A.; Daschbach, J. L. Microelectrodes: Theory and Applications; Kluwer Academic Press: Dordrecht, The Netherlands, 1991; Vol. 197. (5) Aoki, K. Electroanalysis 1993, 5, 627–639. (6) Heinze, J. Angew. Chem., Int. Ed. 1993, 32, 1268–1288. (7) Amatore, C. Electrochemistry at ultramicroelectrodes. In Physical Electrochemistry; Rubinstein, I., Ed.; Marcel Dekker: New York, 1995. (8) Stulik, K.; Amatore, C.; Holub, K.; Marecek, V.; Kutner, W. Pure Appl. Chem. 2000, 72, 1483–1492. (9) Forster, R. J. Encyclopedia of Electrochemistry; John Wiley & Sons: New York, 2003; Vol. 3, pp 160-195. criteria, may appear useless since they better define the origin of the users than the object itself. A better classification of these electrodes would be obtained if it were based on their particular properties. Since electrochemical reactions are interfacial reactions, mass transport is one of the key processes to consider. 10 In a liquid, elementary contributions in the mass transport are diffusion, migration, and convection. Under most circumstances, migration is suppressed by adding a large excess of dissociated inert salt or supporting electrolyte. Convection is often neglected at electrodes of micrometric dimensions in macroscopically still solutions. Indeed, convection originates from movement of fluid packets of micrometric size. 11 It necessarily vanishes close to the electrode interface over distances where concentrations differ significantly from their bulk values. 12,13 In such a case, only diffusion is assumed to govern the final approach of an electroactive molecule toward the electrode. However, according to the size of these electrodes and time scale of the experiments, convective fluxes due to natural convection may still compete with diffusional fluxes in motionless solutions. This may occur even in the absence of any density gradients 14 or effects induced by a magnetic field. 15 These situations arise as soon as the thickness of the diffusion layer becomes comparable to the thickness of the convection-free domain. 7 Under such conditions, the responses do not follow the classical relationships given for currents in dynamic and steady-state regimes. Therefore, under given experimental conditions, it is of importance to decide the largest size of an electrode for eliminating any influence of natural convection. 16,17 Such a criterion may then allow distinguishing properties of ultramicroelectrodes from those of other electrodes of micrometric sizes. To assess the conditions of convection-free regimes at electrodes of micrometric dimensions, we investigated in some previous studies the current responses of micrometric disk (10) Bard, A. J.; Faulkner, L. R. Electrochemical Methods, 2nd ed.; John Wiley & Sons: New York, 2001. (11) Moreau, M.; Turq, P. Chemical Reactivity in Liquids: Fundamental Aspects; Kluwer Academic/Plenum Press: New York, 1988; pp 561-606. (12) Levich, V. G. Physicochemical Hydrodynamics; Prentice Hall: Englewoods Cliffs, NJ, 1962. (13) Davies, J. E. Turbulence Phenomena; Academic Press: New York, 1972. (14) Li, Q. G.; White, H. S. Anal. Chem. 1995, 67, 561–569. (15) Grant, K. M.; Hemmert, J. W.; White, H. S. J. Electroanal. Chem. 2001, 500, 95–99. (16) Hapiot, P.; Lagrost, C. Chem. Rev. 2008, 108, 2238–2264. (17) Molina, A.; Gonzalez, J.; Martinez-Ortiz, F.; Compton, R. G. J. Phys. Chem. C 2010, 114, 4093–4099. 10.1021/ac101210r © 2010 American Chemical Society 6933 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Published on Web 07/26/2010
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Anal. Chem. 2010, 82, 6745-6750 Let
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purchased from Invitrogen-Molecular
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Figure 3. Electropherograms of TPP-
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Anal. Chem. 2010, 82, 6751-6755 Res
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(pH 8.0) with cysteine and cystamin
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Figure 4. Ion mobility mass spectra
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GOx glucose + O298 gluconic acid +
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coater at 2000 rpm for 20 s, and th
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Figure 5. Comparison of cyclic volt
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in channels with either no grooves
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indicators of atmospheric processin
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Figure 1. GC/MS total ion chromatog
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Table 2. Concentrations and Stable
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on a substrate are preferred. 20-24
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Figure 4. SERS analysis of NAADP co
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Anal. Chem. 2010, 82, 6775-6781 Hig
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tion, 2 µL of proprionaldehyde wer
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Figure 4. Analysis of 2a by HPLC-MS
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eaction of 1a with PBH can be condu
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Numerous references had demonstrate
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Figure 1. TEM images of the prepare
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Figure 4. Schematic representation
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esult in a big SPR signal change wi
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also reduces chemical noise, which
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Table 1. Extraction Yields, Liquid
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scanning of AMPP amides of the anal
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Anal. Chem. 2010, 82, 6797-6806 δ
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Figure 1. Schematic view of the pre
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from the specimen and enclosed in a
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Figure 4. (A) IRMS mass-44 chromato
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Table 3. Ambient Measurement Result
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Anal. Chem. 2010, 82, 6807-6813 Dir
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Polymerase (1 U per sample). Reacti
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of which was constant for all ampli
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analytically useful signals at less
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carbon black and RP-C18 for the ext
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solution in an equal volume, and 1
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Table 2. Concentrations and Ratios
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Anal. Chem. 2010, 82, 6821-6829 Mac
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mg/mL protein, followed by separati
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Figure 2. Productivity of SEQUST an
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Figure 4. High-resolution MS/MS spe
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Figure 6. Characterization of PSMs
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containing T-T mismatches. 23 Based
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Figure 1. Extinction spectra of sol
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Figure 3. (A) The value of Ex650 nm
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Figure 5. Extinction spectra and co
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wished to explore the dehydration o
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constant medium for separation; we
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Figure 2. Standards of (Pi)n, n ) 1
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Figure 5. Quantitative calibration
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Anal. Chem. 2010, 82, 6847-6853 Met
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Figure 1. 226 Ra spectrum by liquid
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Table 1. Counting Properties and De
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Table 3. Analysis of 226 Ra in Sedi
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mercial microarray scanner and fabr
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Figure 2. Optical transmission meas
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Figure 5. Volcano plots detailing t
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data as well. The 41 genes in Table
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corresponding compound if its chemi
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Figure 1. Schematic illustration of
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Table 1. Absolute Quantification Re
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ment. Therefore, the long-time drea
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Figure 1. Chemically actuated micro
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Figure 2. Influence of a surfactant
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Figure 5. Device to eject and mix s
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Anal. Chem. 2010, 82, 6877-6886 Imm
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NaCl, phosphate buffer saline (PBS)
Page 137 and 138: Figure 2. Product ion mass spectra
Page 139 and 140: -70 °C resolved the problem, givin
Page 141 and 142: Table 1. Intraday Precision and Acc
Page 143 and 144: Anal. Chem. 2010, 82, 6887-6894 Fer
Page 145 and 146: Figure 1. Infrared spectra of (A) u
Page 147 and 148: Figure 3. Cyclic voltammograms obta
Page 149 and 150: Figure 6. Calculated charge from ch
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Page 153 and 154: Table 1. Chemical Structure, pKa Va
Page 155 and 156: pH with a tilted baseline (Figure 1
Page 157 and 158: Table 2. Linearity and Detection Li
Page 159 and 160: ascorbic acid (AA), uric acid (UA),
Page 161 and 162: the multielement capabilities, the
Page 163 and 164: RESULTS AND DISCUSSION Sulfur Detec
Page 165 and 166: Table 2. Molecular Properties and C
Page 167 and 168: Anal. Chem. 2010, 82, 6911-6918 Dir
Page 169 and 170: dilution and hybridization buffer.
Page 171 and 172: solution under appropriate incubati
Page 173 and 174: Figure 4. Standardization curve for
Page 175 and 176: Anal. Chem. 2010, 82, 6919-6925 Ele
Page 177 and 178: the ×10 objective, to have a large
Page 179 and 180: Figure 2. With a suitable removal o
Page 181 and 182: the NB signal in a much better foot
Page 183 and 184: Scheme 1. Reactions of Selenium Rea
Page 185 and 186: Figure 2. (a) ESI-MS spectrum showi
Page 187: Figure 4. (a) ESI-MS spectrum showi
Page 191 and 192: trode 28 by a finite element using
Page 193 and 194: Figure 4. Comparison between simula
Page 195 and 196: Figure 6. Comparison between simula
Page 197 and 198: educed in the vicinity of double bo
Page 199 and 200: Figure 2. Normalized product ion ab
Page 201 and 202: Figure 4. EID (a) and IRMPD (b) of
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Page 205 and 206: Figure 1. Schematic flowchart showi
Page 207 and 208: difference, ppm compound Table 1. I
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Page 211 and 212: Figure 5. Extracted ion current ESI
Page 213 and 214: m/z 1172.935 was observed for Ser14
Page 215 and 216: linked products via affinity tags.
Page 217 and 218: Scheme 2. Fragmentation Mechanism o
Page 219 and 220: Figure 1. (A) ESI-LTQ-CID-MS 2 prod
Page 221 and 222: Figure 3. (A) ESI-LTQ-CID-MS 2 prod
Page 223 and 224: Figure 5. (A) MALDI-TOF/TOF product
Page 225 and 226: Anal. Chem. 2010, 82, 6969-6975 Ana
Page 227 and 228: Figure 3. Equilibrium response as a
Page 229 and 230: Figure 6. The average measured resp
Page 231 and 232: for the fill time, and we find that
Page 233 and 234: nucleotide tails. 3-5 Thus, the amo
Page 235 and 236: allow the use of higher aptamer con
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Anal. Chem. 2010, 82, 6983-6990 Imp
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Figure 2. Configuration editor wind
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Figure 4. Dependencies between volu
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Since the time for liquid expulsion
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Anal. Chem. 2010, 82, 6991-6999 Int
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Figure 1. Representation of the Car
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Table 2. Capillary Electrophoresis
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Figure 4. (A) Typical raw electroph
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field. DNA profiles were delivered
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Another approach to handle this lim
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(principal components, PCs) in whic
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Figure 5. Relevant score plots and
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CONCLUSIONS In this paper we have i
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electroactive-species loaded liposo
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Figure 2. Qdot-based FLFTS response
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Figure 6. Fluorescence imaging of Q
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Anal. Chem. 2010, 82, 7015-7020 Sel
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Table 1. Effect of 1 D-LC Condition
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Figure 3. Peak-production rate vers
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Anal. Chem. 2010, 82, 7021-7026 Cel
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luciferase (T7 control vector) was
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Figure 3. Inhibitory effects of luc
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Anal. Chem. 2010, 82, 7027-7034 Pat
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Electrochemistry. Electrochemical m
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Figure 2. SEM images of Au substrat
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Table 3. Film Thickness Measurement
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Anal. Chem. 2010, 82, 7035-7043 Qua
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Figure 1. (A) FL spectra of BSPOTPE
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Figure 4. (A) Variation in the FL i
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Figure 6. (A) FL spectra of BSPOTPE
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Scheme 1. Proposed Mechanism for Fl
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one or more drawbacks including poo
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Figure 2. Free fluorophores do not
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Anal. Chem. 2010, 82, 7049-7052 Dev
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Figure 2. Comparative analysis of d
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