Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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of Ni(II)- nor Mg(II)-adducted arachidonic acid (Figure 3a,b). We hypothesize that Ni(II) or Mg(II) adduction may yield a “less fixed” charge compared to Mn(II) adduction, thereby failing to provide all double bond locations in EID. EID of Mn(II)-adducted arachidonic acid was compared to IRMPD of the same species. In contrast to EID (believed to occur via both electronic and vibrational activation), dissociation in IRMPD occurs solely via vibrational excitation. The mixture of mostly [C xHy + Mn] + and [CxHyO2 + Mn] + species observed in EID (Figure 4a) implies competition between charge-driven and charge-remote processes. However, IRMPD of the same precursor ions mainly yielded product ions from charge-driven fragmentation, [CxHy + Mn] + , as expected from vibrational activation (Figure 4b). In contrast, high energy CAD (known to involve electronic excitation) provides mainly charge-remote product ions. 9 The internal energy required for charge-remote fragmentation is estimated to be ∼1.4-2.9 eV for protonated fatty acids. 39 In 70 eV EI, molecular ions of small alkenes were found to be isomerized to a mixture of interconverting structures. 40 We propose that the 25-50 eV electron energies used in EID are sufficient to promote charge-remote fragmentation but not high enough to cause isomerization of Mn(II)-adducted fatty acids, which would result in double bond migration. CONCLUSION Mn(II)-adducted fatty acids were analyzed to investigate the utility of EID for determining double bond locations. Charge- (39) Deterding, L.; Gross, M. L. Org. Mass. Spectrom. 1988, 23, 169–177. (40) Borchers, F.; Levsen, K.; Schwartz, H.; Wesdemiotis, C.; Winkler, H. C. J. Am. Chem. Soc. 1977, 99, 6359–6365. 6946 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 remote product ion abundances of [CxHyO2 + Mn] + -type fragments generated by EID are significantly reduced at double bond positions. Analysis of [CxHyO2 + Mn] + -type product ion abundances from EID of Mn(II)-adducted fatty acids allowed determination of all double bond positions. However, other metal adducts did not generally provide characteristic product ion abundances at all double bond locations. The resulting structural information on double bond locations for Mn(II)adducted fatty acids may be explained by dominant electronic excitation processes in EID and efficient generation of a fixed charge at the carboxylate end due to strong interaction between Mn(II) cation and carboxylate anion. EID of Mn(II)-adducted arachidonic acid was compared with IRMPD of the same species. As expected, mostly charge-driven fragmentation was observed in IRMPD whereas both charge-remote and chargedriven product ions were observed in EID. In contrast, high energy CAD is known to occur mainly via electronic excitation and results in dominant charge-remote product ions. ACKNOWLEDGMENT This work was supported by the University of Michigan and an NSF CAREER award to K.H. (CHE-05-47699). SUPPORTING INFORMATION AVAILABLE Peak assignments and calculations for Figures 1a and 2a. EID spectra of Li-, Zn-, Co-, and Ni-adducted arachidonic acid. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review May 8, 2010. Accepted July 14, 2010. AC101217X
Anal. Chem. 2010, 82, 6947–6957 Identification of Metallothionein Subisoforms in HPLC Using Accurate Mass and Online Sequencing by Electrospray Hybrid Linear Ion Trap-Orbital Ion Trap Mass Spectrometry Sandra Mounicou,* ,† Laurent Ouerdane, † BéatriceL’Azou, ‡ Isabelle Passagne, ‡ CélineOhayon-Courtès, ‡ Joanna Szpunar, † and Ryszard Lobinski † CNRS/UPPA, Laboratoire de Chimie Analytique Bio-Inorganique et Environnement, UMR 5254, 2, av. Pr. Angot, 64053 Pau, France, and EA 3672 Santé-Travail-Environnement, Université Victor Segalen, 146, rue Léo Saignat, 33076 Bordeaux A comprehensive approach to the characterization of metallothionein (MT) isoforms based on microbore HPLC with multimodal detection was developed. MTs were separated as Cd7 complexes, detected by ICP MS and tentatively identified by molecular mass measured with 1-2 ppm accuracy using Orbital ion trap mass spectrometry. The identification was validated by accurate mass of the corresponding apo-MTs after postcolumn acidification and by their sequences acquired online by higher-energy collision dissociation MS/MS. The detection limits down to 10 fmol and 45 fmol could be obtained by ESI MS for apo- and Cd7-isoforms, respectively, and were lower than those obtained by ICP MS (100 fmol). The individual MT isoforms could be sequenced at levels as low as 200 fmol with the sequence coverage exceeding 90%. The approach was successfully applied to the identification of MT isoforms induced in a pig kidney cell line (LLC-PK1) exposed to CdS nanoparticles. Mammalian metallothioneins (MT) are low molecular weight proteins (60-62 amino acid with molecular mass of ca. 6000-7000 Da) characterized by high cysteine content (up to 30% residues) enabling to bind a wide range of transition and heavy metal ions. 1-3 They are involved in a variety of biochemical processes essential for life, such as cellular growth, stress response, copper and zinc homeostasis, and detoxification of heavy metals. Hence, MTs can be valuable biomarkers of stress conditions and several pathologies which require the development of methods for the determination and identification of the individual MT isoforms and products of their post-translational modifications. 4-7 The primary * To whom correspondence should be addressed. † Laboratoire de Chimie Analytique Bio-Inorganique et Environnement. ‡ Université Victor Segalen. (1) Hamer, D. H. Annu. Rev. Biochem. 1986, 55, 913–951. (2) Kagi, J. H. R. Methods Enzymol. 1991, 205, 613–626. (3) Metallothioneins; Stillman, M. J.; Shaw, C. F. I.; Suzuki, K. T., Eds.; VCh: New York, 1992. (4) Kagi, J. H. R.; Schaffer, A. Biochemistry 1988, 27, 8509–8515. (5) Cousins, R. J. Physiol. Rev. 1985, 65, 238–309. (6) Klaassen, C. D.; Liu, J.; Choudhuri, S. Ann. Rev. Pharmacol. Toxicol. 1999, 39, 267–294. structure of metallothioneins exhibits modifications up to 15 amino acids leading to the occurrence of several subisoforms. 1-3 Electrospray ionization (ESI) MS was first proposed by Fenselau group for in vitro studies of the complexation of metalions by MTs. 8,9 These seminal works have been followed by a large number of studies using ESI MS to study the stoichiometry of metal binding to MTs and its domains, 10-14 reactivity and kinetics of metal exchange, 15-18 and reaction of MT with metallodrugs 19 which have been competently reviewed. 15,20,21 Recombinant and highly purified MTs were used in these studies because of the very low purity of the commercial MT which are mixtures of different isoforms. Electrospray MS, coupled with a high-resolution separation techniques, such as reversed-phase chromatography 22 or capillary electrophoresis, 23-25 was shown to be an attractive technique to (7) Coyle, P.; Philcox, J. C.; Carey, L. C.; Rofe, A. M. Cell. Mol. Life Sci. 2002, 59, 627–647. (8) Yu, X.; Wojciechowski, M.; Fenselau, C. Anal. Chem. 1993, 65, 1355– 1359. (9) Afonso, C.; Hathout, Y.; Fenselau, C. J. Mass Spectrom. 2002, 37, 755– 759. (10) Gehrig, P. M.; You, C.; Dallinger, R.; Gruber, C.; Brouwer, M.; KÃ´gi, J. H. R.; Hunziker, P. E. Protein Sci. 2000, 9, 395–402. (11) Merrifield, M. E.; Huang, Z.; Kille, P.; Stillman, M. J. J. Inorg. Biochem. 2002, 88, 153–172. (12) Ngu, T. T.; Krecisz, S.; Stillman, M. J. Biochem. Biophys. Res. Commun. 2010, 396, 206–212. (13) Orihuela, R.; Domènech, J.; Bofill, R.; You, C.; Mackay, E. A.; Kägi, J. H. R.; Capdevila, M.; Atrian, S. J. Biol. Inorg. Chem. 2008, 13, 801–812. (14) Palumaa, P.; Eriste, E.; Kruusel, K.; Kangur, L.; Jörnvall, H.; Sillard, R. Cell. Mol. Biol. (Noisy-le-Grand, Fr.) 2003, 49, 763–768. (15) Duncan, K. E. R.; Ngu, T. T.; Chan, J.; Salgado, M. T.; Merrifield, M. E.; Stillman, M. J. Exper. Biol. Med. 2006, 231, 1488–1499. (16) Palumaa, P.; Eriste, E.; Njunkova, O.; Pokras, L.; Jörnvall, H.; Sillard, R. Biochemistry 2002, 41, 6158–6163. (17) Vaher, M.; Romero-Isart, N.; Vasak, M.; Palumaa, P. J. Inorg. Biochem. 2001, 83, 1–6. (18) Zeitoun-Ghandour, S.; Charnock, J. M.; Hodson, M. E.; Leszczyszyn, O. I.; Blindauer, C. A.; Stürzenbaum, S. R. FEBS J. 2010, 277, 2531–2542. (19) Karotki, A. V.; Vasak, M. J. Biol. Inorg. Chem. 2009, 14, 1129–1138. (20) Chan, J.; Huang, Z.; Merrifield, M. E.; Salgado, M. T.; Stillman, M. J. Coord. Chem. Rev. 2002, 233-234, 319–339. (21) Ngu, T. T.; Stillman, M. J. Dalton Trans. 2009, 5425–5433. (22) Chassaigne, H.; Lobinski, R. J. Chromatogr., A 1998, 829, 127–136. (23) Mounicou, S.; Polec, K.; Chassaigne, H.; Potin-Gautier, M.; Lobinski, R. J. Anal. At. Spectrom. 2000, 15, 635–642. (24) Andon, B.; Barbosa, J.; Sanz-Nebot, V. Electrophoresis 2006, 27, 3661– 3670. 10.1021/ac101245h © 2010 American Chemical Society 6947 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Published on Web 07/29/2010
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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)
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Figure 2. Product ion mass spectra
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-70 °C resolved the problem, givin
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Table 1. Intraday Precision and Acc
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Anal. Chem. 2010, 82, 6887-6894 Fer
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Figure 1. Infrared spectra of (A) u
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Figure 3. Cyclic voltammograms obta
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Figure 6. Calculated charge from ch
Page 151 and 152: Anal. Chem. 2010, 82, 6895-6903 Ele
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 and 188: Figure 4. (a) ESI-MS spectrum showi
Page 189 and 190: Anal. Chem. 2010, 82, 6933-6939 Dif
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: Figure 4. EID (a) and IRMPD (b) of
Page 205 and 206: Figure 1. Schematic flowchart showi
Page 207 and 208: difference, ppm compound Table 1. I
Page 209 and 210: isoforms, its successful use, in th
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
Page 237 and 238: quent ligation of the aptamers afte
Page 239 and 240: Anal. Chem. 2010, 82, 6983-6990 Imp
Page 241 and 242: Figure 2. Configuration editor wind
Page 243 and 244: Figure 4. Dependencies between volu
Page 245 and 246: Since the time for liquid expulsion
Page 247 and 248: Anal. Chem. 2010, 82, 6991-6999 Int
Page 249 and 250: Figure 1. Representation of the Car
Page 251 and 252: 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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