Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Figure 4. MALDI-TOF/TOF product ion spectrum of the type 0 modified Munc13-1 peptide (peptide amino acids 3-9) [M + H + BuUrBu-OH] + at m/z 1072.6. Intrapeptide Cross-Linking Products (Type 1). Also for intrapeptide cross-links, MALDI-TOF/TOF data were comparable to ESI-CID-MS 2 data for Munc13-1 (data not shown) and a PPARR peptide (Supporting Information, Figure S4). Interpeptide Cross-Linking Products (Type 2). For interpeptide cross-links 2 within the Munc13-1 peptide, MALDI-TOF/ TOF experiments impressively demonstrated the fragility of the urea cross-linker upon high-energy CID, which is the reason for the increased abundance of the two 26 u doublets of product ions dominating the product ion spectrum of the type 2 modified precursor ion at m/z 1789.2 (Figure 5, Table 1). According to this spectrum, the fragmentation characteristics of 1 seem to be perfectly suited for MALDI-TOF/TOF applications, being even better than for low-energy MS 2 experiments in a quadrupole ion trap (Figure 3). The two pairs of 26 u mass shifted product ions reveal that the R-peptide and �-peptide comprising the cross-linked product are modified either with 4-aminobutyric acid or with 1,3oxazepan-2-one. Additionally conducted MALDI-ISD-MS/MS experiments confirmed the amino acid sequences of both peptides connected via the BuUrBu linker. This fingerprint feature of two 26 u doublets evidencing the interpeptide cross-link was also found in the MALDI-TOF/TOF fragment ion spectrum of an interpeptide (type 2) modified PPARR peptide (Lys-208 connected with Lys- 216) as the most abundant signals in the mass spectrum (Figure S5, Supporting Information; Table 1). A full list of cross-linking products from PPARR, which were identified by nano-HPLC/ MALDI-TOF/TOF-MS/MS, is presented in the Supporting Information (Table S1). CONCLUSIONS In this paper, we describe the synthesis and application of a novel dissociative amine-reactive cross-linker that shows enhanced 6966 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 fragmentation capabilities in both ESI-CID-ion trap-MS n and MALDI-TOF/TOF-CID-MS/MS and ISD-MS/MS and as such possesses a versatile applicability. The reactivity and the kinetics of our novel cross-linker were found to be comparable with that of other NHS esters, such as BS 3 . The cross-linker 1 contains a urea moiety allowing for a facilitated cleavage upon CID. The key step in the fragmentation mechanism of the cross-linker is a nucleophilic attack at the urea carbonyl group, enabling a highly effective cleavage of the urea moiety. The synthesis of the cross-linker is simple compared to that of the previously published “Edman” cross-linker 13 and is achieved in two steps with inexpensive starting materials. The BuUrBu structure is symmetric, and hence, assignment of cross-linked product ions is facilitated compared to the rather complicated product ion spectra obtained with the previously published unsymmetric tandem MS cleavable linker. 13 The effective fragmentation properties of the CID cleavable cross-linker safeguarding for a selective identification of modified peptides by tandem MS improves the sensitivity of chemical crosslinking for protein structure analysis. This is highly advantageous for discriminating cross-linked species compared to isotope-labeled, i.e., deuterated linkers, which are employed as 1:1 mixtures of nondeuterated and deuterated derivatives, and in constrast to those, no decrease in MS signal intensity is observed for CID labile cross-linkers. Especially during MALDI-TOF/TOF experiments, higher quality MS/MS data were obtained compared to those for conventional noncleavable NHS esters, such as BS 3 . From CID MS 2 product ion mass spectra, the type of cross-linking present is readily visible: Peptides that are modified with a hydrolyzed linker (dead-end or type 0 cross-links) deliver a single pair of product ions mass shifted by 26 u due to the loss of 103 and 129 u
Figure 5. (A) MALDI-TOF/TOF product ion spectrum of the type 2 modified Munc13-1 peptide at m/z 1789.2. (B) Upper panel: ISD-MS 2 product ion spectrum of the �-peptide [� + H + Bu] + at m/z 819.4. Lower panel: ISD-MS/MS product ion spectrum of the �-peptide [� + H + BuUr] + at m/z 845.5. (C) Upper panel: ISD-MS 2 product ion spectrum of the R-peptide [R +H + Bu] + at m/z 943.5. (C) Lower panel: ISD-MS/MS product ion spectrum of the R-peptide [R +H + BuUr] + at m/z 969.5. (Table 1). The resulting 26 u product ion pair (“doublet”) greatly simplifies identification of the respective cross-linking type and therefore allows for an automated analysis of tandem MS data. A CNL of 85 u (pyrolidinone) indicates an intrapeptide cross-link (type 1), whereas an interpeptide (type 2) cross-link delivers two sets of 26 u doublets originating from both R- and �-peptides, each Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6967
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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
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Anal. Chem. 2010, 82, 6911-6918 Dir
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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 and 218: Scheme 2. Fragmentation Mechanism o
Page 219 and 220: Figure 1. (A) ESI-LTQ-CID-MS 2 prod
Page 221: Figure 3. (A) ESI-LTQ-CID-MS 2 prod
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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
Page 269 and 270: Figure 6. Fluorescence imaging of Q
Page 271 and 272: 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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