Analytical Chemistry Chemical Cytometry Quantitates Superoxide

More documents

Recommendations

Info

performed in linear and positive ionization mode on an Ultraflex III instrument (Bruker Daltonik) equipped with a Smartbeam laser using sinapinic acid (SA) as the matrix. A 5 pmol portion of protein was applied to a ground steel target (Bruker Daltonik) using the double layer method as described in the manufacturer’s manual of the Ultraflex III mass spectrometer. Nano-HPLC/MALDI-TOF/TOF Mass Spectrometry. Peptide mixtures derived from enzymatic digestions of Munc13-1 and PPARR were analyzed by offline coupling of a nano-HPLC system (Ultimate 3000, Dionex Corp., Idstein, Germany) to a MALDI- TOF/TOF mass spectrometer (Ultraflex III, Bruker Daltonik). Samples were injected via the autosampler to a precolumn (Acclaim PepMap, C18, 300 µm × 5 mm, 5 µm, 100 Å, Dionex) and desalted by washing the precolumn for 15 min with 0.1% TFA before the peptides were eluted onto the separation column (Acclaim PepMap, C18, 75 µm × 250 mm, 5 µm, 100 Å, Dionex), which had been equilibrated with 95% solvent A (5% ACN, 0.05% TFA). For PPARR, peptides were separated with a 60 min gradient (0-60 min, 5-50% B; 60-62 min, 50-100% B; 62-67 min, 100% B; 67-72 min, 5% B; solvent B ) 80% ACN, 0.04% TFA) at a flow rate of 300 nL/min with UV detection at 214 and 280 nm. Eluates were fractionated into 18 s fractions using the fraction collector Proteineer fc (Bruker Daltonik), mixed with 1.1 µL of matrix solution (0.8 mg/mL HCCA in 90% ACN, 0.1% TFA, 1 mM NH4H2PO4) and directly prepared on a 384 MTP 800 µm AnchorChip target (Bruker Daltonik). For separation of Munc13-1 peptides, slightly different nano-HPLC conditions were employed. Tryptic peptide mixtures were separated by nano-HPLC (30 min, 5-50% B), and fractions were collected on the AnchorChip 384/800 target in 30 s steps. MALDI-TOF/TOF-MS/MS analyses were conducted in the positive ionization and reflectron mode by accumulating 2000 laser shots in the range m/z 700-5000 to one mass spectrum. Mass spectra were externally calibrated using Peptide Calibration Standard II (Bruker Daltonik). Signals with a signal-to-noise ratio (S/N) > 15 were selected and subjected to laser-induced fragmentation (LID). In-source decay (ISD) was performed manually on isolated cross-linking product candidates by increasing the laser energy by ca. 10% relative to the usual laser energy in LID-MS/ MS. Data acquisition was done automatically by the WarpLC 1.1 software (Bruker Daltonik) coordinating MS data acquisition (FlexControl 1.3) and data processing (FlexAnalysis 3.0). Nano-HPLC/Nano-ESI-LTQ-Orbitrap Mass Spectrometry. Fractionation of tryptic peptide mixtures (Munc13-1 and PPARR) was carried out on an Ultimate nano-HPLC system (Dionex) using reversed-phase C18 columns (precolumn, Acclaim PepMap, 300 µm × 5 mm, 5 µm, 100 Å; separation column, Acclaim PepMap, 75 µm × 250 mm, 5 µm, 100 Å; Dionex). After being washed on the precolumn for 15 min with water containing 0.1% TFA, the peptides were eluted and separated using gradients from 0% to 50% B (90 min) and from 50% to 100% B (1 min) and 100% B (5 min), with solvent A being 5% ACN containing 0.1% FA and solvent B being 80% ACN containing 0.1% FA. The nano-HPLC system was directly coupled to the nano-ESI source (Proxeon, Odense, Denmark) of an LTQ-orbitrap XL hybrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). 31 MS data were (31) Hu, Q.; Noll, R. J.; Li, H.; Makarov, A.; Hardman, M.; Graham Cooks, R. J. Mass Spectrom. 2005, 40, 430–443. 6960 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 acquired over 122 min in data-dependent MS 2 mode: Each highresolution full scan (m/z 300-2000, R ) 60 000) in the orbitrap was followed by three product ion scans in the orbitrap (R ) 7500) on the three most intense signals in the full-scan mass spectrum (isolation window 1.5 u). MS 3 was performed on the three most intense signals in CID tandem mass spectra, and fragment ions were either analyzed in the linear quadrupole ion trap (LTQ) or in the orbitrap (R ) 7500). Dynamic exclusion (exclusion duration 180 s, exclusion window -1 to +1 Th) was enabled to allow detection of less abundant ions. Data acquisition was controlled via XCalibur 2.0.7 (Thermo Fisher) in combination with DCMS link 2.0 (Dionex). Analysis of Cross-Linked Products. Cross-linked products were identified by analyzing the MS data using General Protein Mass Analysis for Windows (GPMAW), 32 version 8.10 (Lighthouse Data, Odense, Denmark, http://www.gpmaw.com) and the Cool- ToolBox software program, an upgrade of the VIRTUALMSLAB software program. 33 The length of the cross-linker was calculated using the ViewerLight 5.0 software (Accelrys). 34 RESULTS AND DISCUSSION Cross-Linker Design. The dissociative cross-linker specifically designed for CID tandem MS is a simple and symmetric molecule with a central urea moiety (Scheme 1), similar to the previously introduced thiourea derivative. 14 The practical strategy of inducing a preferred and defined dissociation of the chemical cross-linker upon CID was adapted from the Edman degradation mechanism, which was exemplified for a cross-linker containing a thiourea moiety that is connected to proline via a glycine residue. 13 The refined analytical concept of the herein presented novel cleavable cross-linker relies on an intramolecular nucleophilic attack at the central urea carbonyl, resulting in an efficient cleavage of the cross-linker and giving rise to characteristic fragment ions and constant neutral losses (CNLs). The symmetric compound is simple as it consists of a central urea moiety that is flanked by two aminobutyric acids connected to activated NHS esters, which react with lysines or free N-termini in proteins. 4 An abbreviated notation, BuUrBu, is used throughout this paper for the novel cross-linker 1, which is derived from urea (Ur) and aminobutyric acid (Bu) (Scheme 1). The protein derivatization reactions of 1 are illustrated in Scheme 2S of the Supporting Information. After one reactive site has reacted with a primary amine in a protein, the second reactive site has at least two options to react with proteins: (a) it might form an intermolecular cross-link (2) with another protein (type 2 cross-link 35 ) or (b) it might react with another amine group of the same protein, leading to an intramolecular cross-link (type 1, structure 3). Alternatively, the second NHS functionality is hydrolyzedsin case no amine group is within the correct distance to be crosslinkedsor it is aminolyzed during reaction quenching with ammonium salts (type 0 cross-link linker, 35 structures 4 and 5, respectively). (32) Peri, S.; Steen, H.; Pandey, A. Trends Biochem. Sci. 2001, 26, 687–689. (33) de Koning, L. J.; Kasper, P. T.; Back, J. W.; Nessen, M. A.; Vanrobaeys, F.; Van Beeumen, J.; Gherardi, E.; de Koster, C. G.; de Jong, L. FEBS J. 2006, 273, 281–291. (34) ViewerLight 5.0, Accelrys, San Diego, CA. (35) Schilling, B.; Row, R. H.; Gibson, B. W.; Guo, X.; Young, M. M. J. Am. Soc. Mass Spectrom. 2003, 14, 834–850.
Scheme 2. Fragmentation Mechanism of Protonated 4 (Scheme S2, Supporting Information) upon CID, Delivering a Doublet of 26 u Mass Shifted Product Ions [M + H + Bu] + (6) and [M + H + BuUr] + (7) by CNLs of 129 and 103 u Reactivity and CID Fragmentation. The novel symmetric urea-based cross-linker is unique in that it allows a discrimination of different types of cross-linked products, namely, type 0 (a peptide that is modified by a partially hydrolyzed cross-linker 35 ), type 1 (intrapeptide cross-link), and type 2 (interpeptide crosslink), based on characteristic fragmentation reactions. As such, the cross-linker allows a screening for cross-linked species in a highly automated fashion. The CID fragmentation mechanism of peptides modified with hydrolyzed linkers (the type 0 cross-link linker 4 35 is depicted in Scheme 2). A nucleophilic attack at the carbonyl carbon of the urea moiety leads to a cleavage of the crosslinker at either urea amide bond. Depending on which oxygen attacks the urea carbonyl carbon, either the peptide modified with 4-aminobutyric acid (6) ([M + H + 85 u] + ) corresponding to a CNL of 129 u (1,3-oxazepan-2-one) or the peptide that is decorated with 1,3-oxazepan-2-one (7) ([M + H + 111 u] + ) corresponding to a CNL of 103 u (4-aminobutyric acid) is observed. Intriguingly, the two product ions 6 and 7 formed are mass shifted by 26 u, which is a unique indicator of a “deadend” (type 0) cross-link, i.e., a peptide that is modified by a partially hydrolyzed cross-linker (Scheme 2). In analogy to the fragmentation behavior of the type 0 crosslink (Scheme 2), an interpeptide (type 2) cross-link (2) results in the cleavage of the urea amide bond, yielding a pair of complementary product ions that originate from the symmetric structure of the cross-linker (Scheme 3). Either peptide ions are generated that are modified with 4-aminobutyric acid (ions 6a and 6b) or, alternatively, peptide product ions are formed carrying a 1,3oxazepan-2-one (7a and 7b) modification. This specific fragmentation behavior of an intermolecular cross-linked precursor ion (2) gives rise to two doublets of peptide ions, which exhibit the characteristic mass difference of 26 u. This unique pattern of a “doublet of 26 u doublets” in CID mass spectra is highly indicative in evidencing the presence of an interpeptide cross-link (type 2), while a dead-end (type 0) cross-link only generates a single 26 u doublet as discussed above. Strikingly different from the characteristic fragment ions created for type 0 and type 2 cross-links is the fragmentation pattern of a type 1 intramolecular cross-link (3). As shown in Scheme 4, CID of intramolecular cross-links leads to the effective formation of pyrolidinone, detectable as a CNL for 85 u. A summary of the characteristic fragment ions and CNLs created for the different cross-linked products that allow for a rapid screening is shown in Table 1. We also observed mixed species, i.e., peptides containing more than one cross-linker molecule, which were not considered in this study. Nano-HPLC/Nano-ESI-LTQ-Orbitrap Mass Spectrometry. To assess the behavior of the NHS-BuUrBu-NHS cross-linker (1) in CID experiments, two model substances, the 22-amino acid Munc13-1 peptide (CRAKANWLRAFNKVRMQLQEAR) and the 28 kDa ligand binding domain of PPARR, were selected. Both were cross-linked with 1, enzymatically digested and subjected to nano- HPLC/nano-ESI-LTQ-orbitrap-MS n . The Munc13-1 peptide is currently under investigation in the Sinz lab 36 and was chosen as a model peptide for evaluating the properties of 1 as it contains three primary amines (N-terminus, Lys-4 and Lys-13) that are separated by four arginines, allowing a tryptic cleavage. Peptides Modified with a Partially Hydrolyzed Cross- Linker (Dead-End Cross-Links, Type 0). In Figure 1A, the ESI- CID-MS/MS product ion spectrum of a modified tryptic peptide (AKANWLR) of Munc13-1 is presented. The abundant precursor ion at m/z 1072.60 was selected and completely dissociated by effective collision activation in the linear quadrupole ion trap. The ion at m/z 1072.60 was assigned as a type 0 cross-linked ion [M + H + BuUrBu-OH] + that is modified with a partially hydrolyzed cross-linker (4) at the lysine residue. This assignment is consistent with the fragmentation scheme shown in Scheme 2. The expected CNLs of 129 and 103 u consequently lead to the observation of specific fragment ions with an indicative mass difference of 26 u, in this case found at m/z 943.55 and at m/z 969.53 (Figure 1A). Structure identification of these ions was achieved by interpretation of the LTQ-MS 3 product ion spectra shown in Figure 1B,C. The former spectrum shows the respective product ion spectra of [M + H + Bu] + at m/z 943.55 (6), while (36) Dimova, K.; Kalkhof, S.; Pottratz, I.; Ihling, C.; Rodriguez-Castaneda, F.; Liepold, T.; Griesinger, C.; Brose, N.; Sinz, A.; Jahn, O. Biochemistry 2009, 48, 5908–5921. Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6961
Page 1 and 2:
Anal. Chem. 2010, 82, 6745-6750 Let
Page 3 and 4:
purchased from Invitrogen-Molecular
Page 5 and 6:
Figure 3. Electropherograms of TPP-
Page 7 and 8:
Anal. Chem. 2010, 82, 6751-6755 Res
Page 9 and 10:
(pH 8.0) with cysteine and cystamin
Page 11 and 12:
Figure 4. Ion mobility mass spectra
Page 13 and 14:
GOx glucose + O298 gluconic acid +
Page 15 and 16:
coater at 2000 rpm for 20 s, and th
Page 17 and 18:
Figure 5. Comparison of cyclic volt
Page 19 and 20:
in channels with either no grooves
Page 21 and 22:
indicators of atmospheric processin
Page 23 and 24:
Figure 1. GC/MS total ion chromatog
Page 25 and 26:
Table 2. Concentrations and Stable
Page 27 and 28:
on a substrate are preferred. 20-24
Page 29 and 30:
Figure 4. SERS analysis of NAADP co
Page 31 and 32:
Anal. Chem. 2010, 82, 6775-6781 Hig
Page 33 and 34:
tion, 2 µL of proprionaldehyde wer
Page 35 and 36:
Figure 4. Analysis of 2a by HPLC-MS
Page 37 and 38:
eaction of 1a with PBH can be condu
Page 39 and 40:
Numerous references had demonstrate
Page 41 and 42:
Figure 1. TEM images of the prepare
Page 43 and 44:
Figure 4. Schematic representation
Page 45 and 46:
esult in a big SPR signal change wi
Page 47 and 48:
also reduces chemical noise, which
Page 49 and 50:
Table 1. Extraction Yields, Liquid
Page 51 and 52:
scanning of AMPP amides of the anal
Page 53 and 54:
Anal. Chem. 2010, 82, 6797-6806 δ
Page 55 and 56:
Figure 1. Schematic view of the pre
Page 57 and 58:
from the specimen and enclosed in a
Page 59 and 60:
Figure 4. (A) IRMS mass-44 chromato
Page 61 and 62:
Table 3. Ambient Measurement Result
Page 63 and 64:
Anal. Chem. 2010, 82, 6807-6813 Dir
Page 65 and 66:
Polymerase (1 U per sample). Reacti
Page 67 and 68:
of which was constant for all ampli
Page 69 and 70:
analytically useful signals at less
Page 71 and 72:
carbon black and RP-C18 for the ext
Page 73 and 74:
solution in an equal volume, and 1
Page 75 and 76:
Table 2. Concentrations and Ratios
Page 77 and 78:
Anal. Chem. 2010, 82, 6821-6829 Mac
Page 79 and 80:
mg/mL protein, followed by separati
Page 81 and 82:
Figure 2. Productivity of SEQUST an
Page 83 and 84:
Figure 4. High-resolution MS/MS spe
Page 85 and 86:
Figure 6. Characterization of PSMs
Page 87 and 88:
containing T-T mismatches. 23 Based
Page 89 and 90:
Figure 1. Extinction spectra of sol
Page 91 and 92:
Figure 3. (A) The value of Ex650 nm
Page 93 and 94:
Figure 5. Extinction spectra and co
Page 95 and 96:
wished to explore the dehydration o
Page 97 and 98:
constant medium for separation; we
Page 99 and 100:
Figure 2. Standards of (Pi)n, n ) 1
Page 101 and 102:
Figure 5. Quantitative calibration
Page 103 and 104:
Anal. Chem. 2010, 82, 6847-6853 Met
Page 105 and 106:
Figure 1. 226 Ra spectrum by liquid
Page 107 and 108:
Table 1. Counting Properties and De
Page 109 and 110:
Table 3. Analysis of 226 Ra in Sedi
Page 111 and 112:
mercial microarray scanner and fabr
Page 113 and 114:
Figure 2. Optical transmission meas
Page 115 and 116:
Figure 5. Volcano plots detailing t
Page 117 and 118:
data as well. The 41 genes in Table
Page 119 and 120:
corresponding compound if its chemi
Page 121 and 122:
Figure 1. Schematic illustration of
Page 123 and 124:
Table 1. Absolute Quantification Re
Page 125 and 126:
ment. Therefore, the long-time drea
Page 127 and 128:
Figure 1. Chemically actuated micro
Page 129 and 130:
Figure 2. Influence of a surfactant
Page 131 and 132:
Figure 5. Device to eject and mix s
Page 133 and 134:
Anal. Chem. 2010, 82, 6877-6886 Imm
Page 135 and 136:
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
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 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: linked products via affinity tags.
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
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
Page 273 and 274:
Table 1. Effect of 1 D-LC Condition
Page 275 and 276:
Figure 3. Peak-production rate vers
Page 277 and 278:
Anal. Chem. 2010, 82, 7021-7026 Cel
Page 279 and 280:
luciferase (T7 control vector) was
Page 281 and 282:
Figure 3. Inhibitory effects of luc
Page 283 and 284:
Anal. Chem. 2010, 82, 7027-7034 Pat
Page 285 and 286:
Electrochemistry. Electrochemical m
Page 287 and 288:
Figure 2. SEM images of Au substrat
Page 289 and 290:
Table 3. Film Thickness Measurement
Page 291 and 292:
Anal. Chem. 2010, 82, 7035-7043 Qua
Page 293 and 294:
Figure 1. (A) FL spectra of BSPOTPE
Page 295 and 296:
Figure 4. (A) Variation in the FL i
Page 297 and 298:
Figure 6. (A) FL spectra of BSPOTPE
Page 299 and 300:
Scheme 1. Proposed Mechanism for Fl
Page 301 and 302:
one or more drawbacks including poo
Page 303 and 304:
Figure 2. Free fluorophores do not
Page 305 and 306:
Anal. Chem. 2010, 82, 7049-7052 Dev
Page 307 and 308:
Figure 2. Comparative analysis of d
show all

Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Create successful ePaper yourself

Delete template?

Save as template?