Analytical Chemistry Chemical Cytometry Quantitates Superoxide

More documents

Recommendations

Info

Figure 5. Behavior of primary scoring metrics for high-resolution correlation analysis. (a) Histogram depicting the sensitivity of an LC-Orbitrap- MS/MS analysis as a function of fragment ion bin widths at FDR < 1%. (b) High-resolution correlation analysis rescues false negatives that are inaccessible by high-mass accuracy precursor ion searching alone. (c) ROC plot using XCorr filtering during 1.0, 0.25, and 0.025 m/z-wide bin searches. Insets show histograms of the behavior of Xcorr during 1.0 and 0.025 m/z-wide bin searches. The 1.0 m/z TP distribution is blue, while the 1.0 m/z FP distribution is red. The 0.025 m/z TP distribution is green, while the 0.025 m/z FP distribution is orange. Note that while the primary score for TPs remains largely unchanged, FP scores are significantly reduced. (d) ROC plot using dCn filtering during 1.0, 0.25, and 0.025 m/z-wide bin searches. Insets show histograms of the behavior of dCn during 1.0 and 0.025 m/z-wide bin searches; the distributions are colored the same as in part c. DISCUSSION Soon, proteomics researchers will be swimming in a sea of mass spectra. During the later stages of development of Macro, we were asked to search a5hLTQVelos run with Macro and were surprised to find that it consisted of ∼170 000 tandem mass spectra. Clearly, the relative processing speed of computer-assisted spectral matching algorithms will be a critical feature for these algorithms in the coming years. Our intent with developing Macro was to refresh a scoring metric (Xcorr) that adds significant value to proteomics research by reorganizing the way it conducted searches. The fact that Macro is also very competitive with other search engines reinforces our view that candidate-centric searching represents a practical approach to search algorithm design in general. The core Macro process is also scalable: the MS/MS spectral array may be accessed in main CPU memory by multiple independent processing cores, each of which could digest a different portion of the FASTA database or perform scoring functions, or both; design modifications such as this are planned for future versions of Macro. Although the SEQUEST-specific version of Macro contains proprietary information that precludes its pubic release, we offer the source code for MacroParser to the proteomics community as a general framework for candidate-centric database parsing into which any peptide scorer could be inserted. Details regarding the Macro project can be found on our lab Web server at http:// proteomics.dartmouth.edu. We feel that the performance gains associated with Macro can allow for a host of features and functions to be added to the classical scoring function Xcorr, including post-translational modi- 6828 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 fication scanning and, as we show here, high-resolution correlation analysis. Although a parameter does exist in legacy SEQUEST that appears to allow users to scale fragment ion tolerances, closer inspection of the SEQUEST source code and search output when using this parameter reveals that it is not performing these searches as the user likely intends. Our inclusion of a variable fragment ion bin width parameter that accurately bins fragment ions into a scalable sparse array enables users with a mass spectrometer capable of better than unit resolution MS/MS spectra to search their data with the corresponding fragment ion mass tolerance. This has the added benefit of improved overall search accuracy relative to unit resolution searching. Although this does come as a speed penalty, we note that even when running at 0.025 m/z bin width, Macro is still 20× faster than SEQUEST on our platform. We anticipate that these highresolution correlation searches will result in a significant decrease in the minimum Xcorr threshold required to achieve a desired FDR: we observed the median Xcorr value of rescued PSMs, across all charge states, to be 1.52, with the lowest rescued PSM yielding an Xcorr of 0.67, albeit with a dCn of 0.08. Legacy SEQUEST users will likely need some adjusting to these unusually low score results, but with careful application of strategies to estimate data set false discovery rates 21 and calculate PSM posterior probability assignments, 26 we hope they will also ultimately appreciate the benefit of high mass precision to the improved accuracy of their data sets. (26) Kall, L.; Storey, J. D.; MacCoss, M. J.; Noble, W. S. J. Proteome Res. 2008, 7, 29–34.
Figure 6. Characterization of PSMs “rescued” by high-resolution correlation analysis. (a) Top-ranked PSMs and scores for a given MS/MS search result using 1.0 and 0.025 m/z-wide bins. Note that these sequences are identical, except for a K/Q substitution in the middle of the peptide, marked by a red box. While the 1.0 m/z-wide bin search rank 1 PSM satisfies a precursor ion tolerance and Xcorr cutoff, the dCn (0.005) precludes unambiguous TP assignment. The top-ranked PSMs and scores from the 0.025 m/z-wide bin search are shown below. The resolving power of much narrower fragment ion bins clearly allows for discrimination of the K-containing ion fragments, resulting in a dCn of 0.36 and an unambiguous TP PSM assignment. (b) Graphical representation of the distribution of candidate PSMs by Xcorr for the 1.0 (blue) and 0.025 (red) m/z-wide bin searches of the PSM from part a. (c) Histogram depicting the relative ranks of rescued PSMs in the lower resolution search. The lowest ranked PSM was rescued from the 4 715th position. (d) Reciprocal MS/MS plots, sequence, and matched fragment ions for the PSM rescued from the 4 715th rank position. The upper MS/MS spectrum is from the endogenous peptide from the HeLa cell lysate, and the lower MS/MS spectrum is from the synthetic peptide “AASVHTVGEDTEETPHR”. ACKNOWLEDGMENT We thank Mike Senko, Justin Blethrow, and colleagues at ThermoFisher Scientific for facilitating transfer of the SEQUST source code, additional platform testing, and general discussions. We thank Arminja Kettenbach for preparing the HeLa cell lysate digest and phosphopeptide-enriched sample and also thank Jason Gilmore for critical reading of the manuscript. This work was supported by the National Institutes of Health Grant P20-RR018787 from the IDeA Program of the National Center for Research Resources (to S.A.G.) and a predoctoral fellowship from the National Institute of General Medical Sciences Grant T32- GM008704 (to B.K.F.). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review March 26, 2010. Accepted July 8, 2010. AC100783X Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6829
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: Figure 4. High-resolution MS/MS spe
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 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
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?