Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Anal. Chem. 2010, 82, 6958–6968 Cleavable Cross-Linker for Protein Structure Analysis: Reliable Identification of Cross-Linking Products by Tandem MS Mathias Q. Müller, † Frank Dreiocker, ‡ Christian H. Ihling, † Mathias Schäfer,* ,‡ and Andrea Sinz* ,† Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther-Universität Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, D-06120 Halle (Saale), and Institute for Organic Chemistry, Department of Chemistry, Universität zu Köln, Greinstrasse 4, D-50939 Cologne, Germany Chemical cross-linking combined with a subsequent enzymatic cleavage of the created cross-linked complex and a mass spectrometric analysis of the resulting crosslinked peptide mixture presents an alternative approach to high-resolution analysis, such as NMR spectroscopy or X-ray crystallography, to obtain low-resolution protein structures and to gain insight into protein interfaces. Here, we describe a novel urea-based cross-linker, which allows distinguishing different cross-linking products by collision-induced dissociation (CID) tandem MS experiments based on characteristic product ions and constant neutral losses. The novel cross-linker is part of our ongoing efforts in developing collision-induced dissociative reagents that allow an efficient analysis of cross-linked proteins and protein complexes. Our innovative analytical concept is exemplified for the Munc13-1 peptide and the recombinantly expressed ligand binding domain of the peroxisome proliferator-activated receptor r, for which cross-linking reaction mixtures were analyzed both by offline nano-HPLC/MALDI-TOF/TOF mass spectrometry and by online nano-HPLC/nano-ESI-LTQ-orbitrap mass spectrometry. The characteristic fragment ion patterns of the novel cross-linker greatly simplify the identification of different cross-linked species, namely, modified peptides as well as intrapeptide and interpeptide cross-links, from complex mixtures and drastically reduce the potential of identifying false-positive cross-links. Our novel ureabased CID cleavable cross-linker is expected to be highly advantageous for analyzing protein 3D structures and protein-protein complexes in an automated manner. Chemical cross-linking 1 combined with mass spectrometry presents an alternative approach to study the tertiary and quaternary structure of proteins and protein complexes. 2-4 However, an unambiguous, sensitive, reliable, and fast identifica- * To whom correspondence should be addressed. (A.S.) Phone: +49-345- 5525170. Fax: +49-345-5527026. E-mail: andrea.sinz@pharmazie.uni-halle.de. (M.S.) Phone: +49-221-4703086. Fax: +49-221-4703064. E-mail: mathias.schaefer@ uni-koeln.de. † Martin-Luther-Universität Halle-Wittenberg. ‡ Universität zu Köln. (1) Sinz, A. Angew. Chem., Int. Ed. 2007, 46, 660–662. (2) Sinz, A. J. Mass Spectrom. 2003, 38, 1225–1237. (3) Trakselis, M. A.; Alley, S. C.; Ishmael, F. T. Bioconjugate Chem. 2005, 16, 741–750. 6958 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 tion of the amino acids involved in the covalent derivatization by chemical cross-linking remains challenging. A mass spectrometric identification of the chemically modified amino acids in the respective protein is performed after proteolytic digestion of the cross-linking reaction mixtures. This is often hampered by the complexity of the created peptide mixtures, wherein only a relatively small percentage of cross-linked products are present besides a majority of unmodified peptides. To safeguard for a selective identification of cross-linked peptides using electrospray ionization (ESI) 5-7 combined with collision-induced dissociation (CID) 8,9 tandem MS, 10 several approaches have been suggested. 11-14 They include cross-linking reagents that incorporate the use of marker ions resulting from low-energy CID 15,16 or metastable decay, 17 isotope-coding strategies, such as proteolytic digestion in 18 O water, 18 isotope coding of the cross-linking reagents 19-22 or of the proteins, 23 and an enrichment of cross- (4) Sinz, A. Mass Spectrom. Rev. 2006, 25, 663–682. (5) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64–71. (6) Cole, R. Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation and Applications; 1997. (7) Kebarle, P.; Verkerk, U. H. Mass Spectrom. Rev. 2009, 28, 898–917. (8) Wells, J. M.; McLuckey, S. A. The Encyclopedia of Mass Spectrometry; 2003; p1. (9) Shukla, A. K.; Futrell, J. H. J. Mass Spectrom. 2000, 35, 1069–1090. (10) Hunt, D. F.; Yates, J. R., III; Shabanowitz, J.; Winston, S.; Hauer, C. R. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 6233–6237. (11) Soderblom, E. J.; Goshe, M. B. Anal. Chem. 2006, 78, 8059–8068. (12) Soderblom, E. J.; Bobay, B. G.; Cavanagh, J.; Goshe, M. B. Rapid Commun. Mass Spectrom. 2007, 21, 3395–3408. (13) Dreiocker, F.; Müller, M. Q.; Sinz, A.; Schäfer, M. J. Mass Spectrom. 2010, 45, 178–189. (14) Müller, M. Q.; Dreiocker, F.; Ihling, C. H.; Sinz, A.; Schäfer, M. J. Mass Spectrom., in press. (15) Back, J. W.; Hartog, A. F.; Dekker, H. L.; Muijsers, A. O.; de Koning, L. J.; de Jong, L. J. Am. Soc. Mass Spectrom. 2001, 12, 222–227. (16) Tang, X.; Munske, G. R.; Siems, W. F.; Bruce, J. E. Anal. Chem. 2005, 77, 311–318. (17) Young, M. M.; Tang, N.; Hempel, J. C.; Oshiro, C. M.; Taylor, E. W.; Kuntz, I. D.; Gibson, B. W.; Dollinger, G. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5802–5806. (18) Back, J. W.; Notenboom, V.; de Koning, L. J.; Muijsers, A. O.; Sixma, T. K.; de Koster, C. G.; de Jong, L. Anal. Chem. 2002, 74, 4417–4422. (19) Petrotchenko, E. V.; Olkhovik, V. K.; Borchers, C. H. Mol. Cell. Proteomics 2005, 4, 1167–1179. (20) Schulz, D. M.; Kalkhof, S.; Schmidt, A.; Ihling, C.; Stingl, C.; Mechtler, K.; Zschörnig, O.; Sinz, A. Proteins 2007, 69, 254–269. (21) Ihling, C.; Schmidt, A.; Kalkhof, S.; Schulz, D. M.; Stingl, C.; Mechtler, K.; Haack, M.; Beck-Sickinger, A. G.; Cooper, D. M.; Sinz, A. J. Am. Soc. Mass Spectrom. 2006, 17, 1100–1113. 10.1021/ac101241t © 2010 American Chemical Society Published on Web 07/22/2010
linked products via affinity tags. 1,24,25 Identification of cross-linked peptide ions by CID tandem MS or MS n is not trivial as product ion spectra of these contain a number of product ions, i.e., band y-type ions, 26,27 originating from both peptides involved in the cross-linking product. To enable a more effective analysis of cross-linked peptides by CID tandem MS analysis, we have developed a novel concept for collision-induced dissociative crosslinking reagents. As such, the novel cross-linker contains a labile covalent bond located within the linker region, which is selective and preferably cleaved by collision activation in the gas phase. Highly efficient and reproducible cleavage of our novel urea-based cross-linker is mediated by a nucleophilic attack of a carbonyl oxygen in the cross-linker’s spacer chain at the urea carbonyl carbon. The synthesis and application of the predecessor of this cross-linker, a thiourea derivative, has been studied in detail previously. 14 However, that thiourea-based cross-linker exhibited a number of properties which were unfavorable for analyzing cross-linked products by CID in an automated fashion. Most importantly, we were unable to differentiate between the different types of cross-linked species, namely, peptides that are modified by a partially hydrolyzed cross-linker or intramolecular type 1 cross-linked products. The urea-based cross-linker presented herein overcomes the deficiencies of the thiourea-based reagent. It leads to the formation of indicative fingerprint mass shifted product ions and neutral losses in the product ion mass spectra, which allow unambiguous identification of the different types of cross-linking products. The properties of the novel cross-linker were evaluated for the Munc 13-1 peptide and the 28 kDa ligand binding domain of the peroxisome proliferator-activated receptor R (PPARR), impressively indicating its power for efficiently analyzing cross-linked products in a highly automated fashion. EXPERIMENTAL SECTION Materials. All chemicals and solvents used for synthesis of the cross-linker were used as purchased without further purification (Acros Organics, Geel, Belgium; ABCR, Karlsruhe, Germany). Dichloromethane and pyridine were dried by distillation from calcium hydride. All other solvents were distilled over a column prior to use. Buffer reagents and chemicals were obtained from Sigma (Taufkirchen, Germany). Proteases (trypsin and GluC, sequencing grade) were obtained from Roche Diagnostics (Mannheim, Germany). Nano-HPLC solvents were spectroscopic grade (Uvasol, VWR, Darmstadt, Germany). MALDI matrixes and calibration standards were purchased from Bruker Daltonik (Bremen, Germany). Water was purified with a Direct-Q5 water purification system (Millipore, Eschborn, Germany). The ligand binding domain of PPARR was expressed in Escherichia coli and purified (22) Schmidt, A.; Kalkhof, S.; Ihling, C.; Cooper, D. M.; Sinz, A. Eur. J. Mass Spectrom. 2005, 11, 525–534. (23) Taverner, T.; Hall, N. E.; O’Hair, R. A.; Simpson, R. J. J. Biol. Chem. 2002, 277, 46487–46492. (24) Hurst, G. B.; Lankford, T. K.; Kennel, S. J. J. Am. Soc. Mass Spectrom. 2004, 15, 832–839. (25) Sinz, A.; Kalkhof, S.; Ihling, C. J. Am. Soc. Mass Spectrom. 2005, 16, 1921– 1931. (26) Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 11, 601. (27) Papayannopoulos, I. A.; Biemann, K. Protein Sci. 1992, 1, 278–288. Scheme 1. Structure of the Symmetric NHS-BuUrBu-NHS Compound (1) for Chemical Cross-Linking according to a previously published protocol. 28 The Munc13-1 peptide was synthesized by Dr. O. Jahn (MPI for Experimental Medicine, Göttingen, Germany). Synthesis of the Cross-Linker. Aminobutanoic acid was dissolved in 4 M NaOH, and 0.175 equiv of triphosgen in dioxane was added at 0 °C (see Scheme S1 of the Supporting Information). The reaction mixture was brought to room temperature overnight, and the solid was removed by filtration. The solvent was removed under reduced pressure, and the residue was recrystallized from 6 M hydrochloric acid. The product was isolated by filtration, washed with acetone, and dried in vacuum. HO-BuUrBu-OH was isolated as a colorless solid in a yield of 26%. 29 An abbreviated notation, BuUrBu, is used throughout this paper for the backbone of cross-linker 1, which is derived from urea (Ur) and aminobutyric acid (Bu) (Scheme 1). HO-BuUrBu-OH was dissolved in pyridine and treated with N-(trifluoroacetoxy)succinimidesprepared from N-hydroxysuccinimide (NHS) and trifluoroacetic anhydridesat 0 °C. 30 The reaction mixture was brought to room temperature within 2 h. After addition of ethyl acetate the raw product was isolated by filtration and dissolved in a mixture of dichloromethane and methanol. Insoluble components were removed by filtration, and the filtrate was evaporated. NHS-BuUrBu-NHS (1) was isolated as a colorless solid in a yield of 82%. Cross-Linking Reactions. For cross-linking experiments, aqueous stock solutions of Munc13-1 peptide (100 µg/mL) or PPARR ligand binding domain (2 mg/mL) were diluted with 20 mM HEPES, 150 mM NaCl, 1 mM TCEP, 10% (v/v) glycerol, pH 8.0, to a volume of 1 mL, giving a final protein/peptide concentration of 10 µM. The cross-linker (200 mM stock solution in DMSO) was added in 50, 100, and 200 M excess to the protein/peptide solution, and the reactions were allowed to proceed for 5, 15, 30, 60, and 120 min. Reactions were quenched with ammonium bicarbonate (20 mM final concentration). One 200 µL aliquot was taken from each sample and stored at -20 °C before MS analysis. Cross-linked Munc13-1 peptide was digested with trypsin, whereas PPARR was digested either with trypsin or with trypsin/GluC (each 1:100 (w/w) enzyme to substrate ratio) overnight at 25 °C according to an existing protocol. 20 The resulting digests were stored at -20 °C before MS analysis. Linear Mode MALDI-TOF Mass Spectrometry. MALDI- TOF mass spectrometry of intact cross-linked PPARR was (28) Müller, M. Q.; Roth, C.; Sträter, N.; Sinz, A. Protein Expression Purif. 2008, 62, 185–189. (29) Coe, S.; Kane, J. J.; Nguyen, T. L.; Toledo, L. M.; Wininger, E.; Fowler, F. W.; Lauher, J. W. J. Am. Chem. Soc. 1997, 119, 86–93. (30) Rao, T. S.; Nampalli, S.; Sekher, P.; Kumar, S. Tetrahedron Lett. 2002, 43, 7793–7795. Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6959
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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
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Anal. Chem. 2010, 82, 6895-6903 Ele
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Table 1. Chemical Structure, pKa Va
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pH with a tilted baseline (Figure 1
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Table 2. Linearity and Detection Li
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ascorbic acid (AA), uric acid (UA),
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the multielement capabilities, the
Page 163 and 164: RESULTS AND DISCUSSION Sulfur Detec
Page 165 and 166: Table 2. Molecular Properties and C
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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 and 202: Figure 4. EID (a) and IRMPD (b) of
Page 203 and 204: Anal. Chem. 2010, 82, 6947-6957 Ide
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: m/z 1172.935 was observed for Ser14
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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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
Page 253 and 254: Figure 4. (A) Typical raw electroph
Page 255 and 256: field. DNA profiles were delivered
Page 257 and 258: Another approach to handle this lim
Page 259 and 260: (principal components, PCs) in whic
Page 261 and 262: Figure 5. Relevant score plots and
Page 263 and 264: 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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