Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Scheme 3. Fragmentation Mechanism of Protonated 2 upon CID, Delivering Two Complementary Doublets of 26 u Mass Shifted Product Ions a a Product ions of peptide 1 are 6a and 7a, and product ions of peptide 2 are 6b and 7b. Scheme 4. Fragmentation Mechanism of a Protonated Type 1 Modified Peptide (3) upon CID, Delivering a Product Ion That Is Modified with BuUr [M + H + BuUr] + (7) by a CNL of Pyrolidinone (85 u) Table 1. Summary of the Characteristic CNLs and Fragment Ion Mass Shifts Used for Discrimination of the Different Cross-Linking Types (ESI-Ion Trap-MS/MS and MALDI-TOF/TOF-MS/MS) cross-link type hydrolyzed, type 0 aminolyzed, type 0 intrapeptide, type 1 interpeptide, type 2 CNL 103 and 129 u 102 and 128 u 85 u no. of 26 u doublets 1 1 2 the latter presents the respective product ion spectrum of [M + H + BuUr] + at m/z 969.53 (7). In both experiments the product ion analysis was conducted in the orbitrap at a mass accuracy of 1-2 ppm employing a resolving power of 15 000. In the MS 3 product ion spectra presented in Figure 1B,C, the precursor ions exhibit frequent losses of water and ammonia, and more importantly, extensive backbone cleavages allow an efficient sequencing of the peptide. In the present case, b-type ions containing the cross-linker dominate the spectra. From the CID fragmentation pattern (Figure 1A) it is immediately obvious that fragmentation of the urea cross-linker is strongly preferred, while the peptide backbone remains virtually intact. The effective fragmentation of the partially hydrolyzed cross-linker 4 leads to the formation of prominent “26 u doublet” product ions 6 and 7 (Table 1). As highlighted above, these product ions are indicative of a selective and sensitive detection of the respective cross-linked 6962 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 species as they cannot be confused with b- and corresponding a-type ions giving a mass difference of 28 u (CO). Peptides that are modified by an amidated cross-linker (5), resulting from the quenching reaction with ammonium bicarbonate, are also observed in the product ion mass spectra, 37 accordingly exhibiting CNLs of 128 and 102 u (Scheme 2, Table 1). It should be noted that peptides modified by hydrolyzed (4) and amidated (5) linkers were readily separated by our nano-HPLC method with ca. 30 s elution time difference for a 1000 Da peptide. An analogous fragmentation behavior was observed for a modified tryptic peptide of PPARR comprising amino acids 259-272. ESI-CID-MS 2 experiments resulted in characteristic CNLs of 129 and 103 u with the indicative 26 u pattern due to (37) Kalkhof, S.; Sinz, A. Anal. Bioanal. Chem. 2008, 392, 305–312.
Figure 1. (A) ESI-LTQ-CID-MS 2 product ion spectrum of the type 0 modified tryptic peptide AKANWLR ion [M + H + BuUrBu-OH] + (structure 4 (Scheme S2, Supporting Information) at m/z 1072.60) of Munc13-1. ESI-LTQ-MS 3 product ion spectra of [M + H + Bu] + at m/z 943.55 (B) and of [M + H + BuUr] + at m/z 969.53 (C). cleavage of 4 (Figure S1 of the Supporting Information). In subsequent MS 3 product ion experiments, the peptide backbone was sequenced, clearly evidencing the modification of Lys-266 in the flexible ω loop of PPARR (Figure S1B,C). Intrapeptide Cross-Linking Products (Type 1). As the next step, we aimed to evaluate the behavior of compound 1 in intrapeptide cross-linked products (3, Scheme 2S, Supporting Information). In Figure 2, ESI-CID-MS 2 and -MS 3 data of a derivatized PPARR peptide (aa 196-210) are exemplarily presented. In contrast to peptides that were modified by a partially hydrolyzed cross-linker (type 0), intrapeptide crosslinks (type 1, structure 3, Scheme 2S) exhibit a characteristic neutral loss of 85 u upon CID (Table 1), resulting from the elimination of a pyrolidinone as illustrated in Scheme 3. This property renders our novel cross-linker highly advantageous for distinguishing different types of cross-links from each other and thus makes it an extremely valuable tool for a rapid and automated screening of cross-linking products. ESI-MS 2 and -MS 3 product ion experiments enabled us to readily identify Lys-204 to be connected with Lys-208 in the N-terminal helix of PPARR. The efficient fragmentation of cross-linked species compared to conventional NHS ester cross-linkers, such as bis(sulfosuccin- imyl) suberate (BS 3 ), 38 makes it clearly superior to these noncleavable derivatives. Interpeptide Cross-linking Product (Type 2). Finally, we examined the fragmentation behavior of 1 for the analysis of interpeptide cross-links (type 2, structure 2), which present the most informative distance constraints with respect to analyzing 3D structures of proteins or to mapping protein-protein interaction sites. Modified Munc13-1 (Figure 3) and PPARR (273 aa; Figure S2, Supporting Information) peptides deliver the two indicative 26 u doublets of fragment ions 6a, 7a and 6b, 7b (Scheme 3, Table 1), which allowed an unambiguous identification of cross-linked lysines. For the Munc13-1 peptide, amino acids 3-9 were found to be connected with amino acids 10-15, clearly pointing to a cross-link between Lys-4 and Lys-13 (Figure 3). For the PPARR peptide, Lys-208 was demonstrated to be cross-linked to Lys-216 (Figure S2), a cross-linking product that had not been identified in previous cross-linking studies of PPARR with noncleavable cross-linkers. 39 Interestingly, the most abundant signals in the MS 3 product ion spectra (Figure S2B,C) correspond to both unmodified peptides comprising the cross-linking product (38) Dihazi, G. H.; Sinz, A. Rapid Commun. Mass Spectrom. 2003, 17, 2005– 2014. Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6963
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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
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RESULTS AND DISCUSSION Sulfur Detec
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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 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 and 214: m/z 1172.935 was observed for Ser14
Page 215 and 216: linked products via affinity tags.
Page 217: Scheme 2. Fragmentation Mechanism o
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
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
Page 265 and 266: electroactive-species loaded liposo
Page 267 and 268: 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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