Analytical Chemistry Chemical Cytometry Quantitates Superoxide

More documents

Recommendations

Info

Figure 3. Comparison of Macro performance with contemporary database searching algorithms. The same data set in Figure 2b was researched with other commonly used database searching algorithms, including Mascot, OMSSA, and X!Tandem. All algorithms were run in single-threaded mode to provide an accurate comparison. legacy SEQUEST and performs the fast Xcorr preprocessing math on the resultant sparse array. Given the mass precision and resolution of ion traps, MS/MS data from such instruments normally defines these bin widths as 1 m/z wide, and the spectral preprocessing logic of SEQUEST determines which ions fall into which m/z bin(s). Figure 4a depicts an MS/MS spectrum collected in an LTQ Orbitrap with a resolution setting of 15 000. Figure 4b describes this spectrum preprocessed for fast Xcorr analysis by Macro with a bin width of 1 m/z, while Figure 4c depicts an array from the same spectrum but with bin widths of 0.01 m/z. Note that, in the insets, binning logic in Macro reduced the number of available ions for matching across a 2 m/z-wide window in the original spectrum from 6 to 2 when 1 m/z-wide bins were used versus 0.01 m/z-wide bins. Clearly, the higher “resolution” of the array in Figure 4c allows for more accurate ion-to-bin assignments and consequently a more robust discrimination between ion fragments of similar m/z. This is also apparent in the significant reduction in the average magnitude of negative values in the high-resolution preprocessed array (Figure 4c), which spreads these anticorrelations out over a much larger bin space. Similar to the preprocessing steps that were performed for experimental spectra, theoretical spectra for each candidate peptide were created using the same bin width scaling factor, and Xcorr values were then calculated from the dot product of the two arrays. We then tested the utility of these scalable MS/MS arrays by collecting data from our LTQ Orbitrap at relatively high resolution and mass accuracy (R ) 15 000). We analyzed our HeLa cell 80-120 kDa protein lysate digest over a 90 min LTQ Orbitrap gradient, during which a total of 5 725 MS/MS spectra were collected. We then used Macro to iteratively search this run with fragment ion bin widths of 1.0, 0.75, 0.5, 0.25, 0.1, 0.075, 0.05, 0.025, and 0.01 m/z. Because the size of the arrays scales inversely with bin width, there is a modest but significant speed penalty 6826 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 associated with high-resolution scoring. On our Intel Conroe test platform fitted with 8Gb of RAM, we observed a 3× increase in search time using bin widths of 0.025 m/z relative to unit resolution and a 6× increase in search time when using bin widths of 0.01 m/z. However, we also observed a significant increase in the total number of true positive (TP) PSMs at a 1% false discovery rate (FDR). Figure 5a depicts the total number of TP PSMs for this run as a function of search bin width. We observed a 24% increase in the number of TP PSMs when using bin widths of 0.025 m/z versus 1.0 m/z bin widths. We considered the possibility that simply limiting the number of candidate PSMs by using a very narrow precursor ion tolerance may allow these “rescuable” PSMs to rise in rank. However, consistent with previous reports, 24,25 we only observed a very slight increase in TP assignments in a search limited to ±10 ppm precursor ion mass measurement accuracy (Figure 5b). This suggests that the sensitivity gains we observe by high-resolution fragment ion searching represent PSMs that are not accessible by simply limiting the number of candidate PSMs in a search. Although these mediocre yet ”rescuable” MS/MS spectra are being considered in the context of fewer competing candidates during a narrow window precursor ion search, their inherently poor correlation characteristics do not sufficiently enhance their differential Xcorr rank to allow for their rescue in this search mode. This increase in true positive assignments can be ascribed to a combination of features of merit associated with the higher resolution versions of Xcorr and dCn. Although we did not see an appreciable change in Xcorr for TP PSMs when searching at higher fragment ion resolution, we noted that false positive (FP) PSMs exhibited a decrease in their median Xcorr values (from 1.6 to 1.2 for 1.0 and 0.025 m/z-wide bins, respectively; Figure 5c). This allows for significantly lower Xcorr cutoff values to be used when establishing a 1% FDR. From Figure 5c, clearly many of the “rescued” TP PSMs come from low Xcorr scores that are otherwise inaccessible due to the extension of the FP PSM distribution into that score space. Equally dramatic was the change in TP PSM dCn scores, whose median values increased from 0.25 to 0.42 across the entire data set (Figure 5d); for those “rescued” TP PSMs, the median dCn value jumped from 0.06 to 0.33. As dCn is a direct measure of the relative Xcorr score separation between the highest-ranked PSM and its nearest neighbor (de facto a FP PSM), our observations of overall reduced FP Xcorr scores and increased TP dCn scores are consistent with this relationship. To further describe the nature of some of these “rescued” TP PSMs, we examined their scores and individual PSM rankings. Figure 6a depicts the top two ranked PSMs for a particular MS/ MS spectrum that yielded an excellent Xcorr score (4.3), but a very low dCn score of 0.005 when scored with a 1.0 m/z bin width. Note that these PSMs differ only by a K/Q (mass difference ) 0.036 Da) in the middle of the peptide sequences. Although distinguishable by Orbitrap precursor ion mass precision, for correlation analysis at unit resolution fragment ion correlation, this difference is transparent and results in almost identical Xcorr (24) Beausoleil, S. A.; Jedrychowski, M.; Schwartz, D.; Elias, J. E.; Villen, J.; Li, J.; Cohn, M. A.; Cantley, L. C.; Gygi, S. P. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 12130–12135. (25) Hsieh, E. J.; Hoopmann, M. R.; Maclean, B.; Maccoss, M. J. J. Proteome Res. 2010, 9, 1138–1143.
Figure 4. High-resolution MS/MS spectra and Macro. (a) A typical, raw high-mass accuracy MS/MS spectrum generated using an LTQ- Orbitrap (R ) 15 000). (b) The raw spectrum in part a preprocessed for fast Xcorr using 1 m/z-wide bins to define the spectrum array. (c) The same spectrum preprocessed for fast Xcorr using 0.01 m/z-wide bins. Note in the inset that multiple ion features persist in a 2 m/z-wide space in the higher resolution array, while they are compressed into two features when searches are performed at unit resolution. scores for these two sequences. However, when searched by Macro using 0.025 m/z bin widths, this K/Q difference is readily distinguished by dCn, likely for all singly and doubly charged fragment ions from this quadruply charged precursor that contain the Lys residue. Figure 6b describes the general behavior of all candidate PSMs for this MS/MS spectrum at the two fragment ion tolerances. It is worth noting again in this plot that the discriminating power of these high-resolution correlations lies predominantly in positive overlap between the theoretical and experimental arrays and not in the “signal-to-noise” of this overlap relative to neighboring offsets (±75 m/z equivalents), an important aspect of the unit resolution Xcorr. This can be observed in the large decrease in the number of negative Xcorr values for lowranked PSMs between the high- and low-resolution searches. Indeed, of the 819 “rescued” PSMs, 26% of them were for peptide sequences that were correctly assigned (top-ranked) in the lower resolution correlation analysis but with extremely low dCn/Xcorr combinations such that they precluded a definitive assignment when score cutoffs were applied to achieve a 1% FDR. The remaining rescued peptide matches were reranked from lower ranks to the top-ranked candidate PSM. Figure 6c depicts the distribution across the entire run of original candidate PSM ranks when searches were performed with 1.0 m/z-wide fragment ion bins that are “rescued” when the search is done at 0.025 m/z bin widths. Of those PSMs that are reranked between the two searches, the median rank in the low-resolution search is 9; the lowest reranked candidate PSM moved from the 4 517th position to the top-ranked spot when searched at high resolution. Although this MS/MS spectrum is assigned a PSM (AASVHTVGEDTEET- PHR, M + 4H + , MMA ) 0.1 ppm) with an Xcorr of only 0.67, the dCn is 0.08 (dCn = 0.001 at unit fragment ion resolution). This peptide is from the protein hPOP1 with a molecular weight of 115 kDa, which is in the range of molecular weights (80-125 kDa) of SDS-PAGE-fractionated human cell lysate from which the sample was derived. Figure 6d displays a reciprocal plot of the endogenous, rescued MS/MS spectrum from the lysate with the matched ions noted and a mirrored spectrum from a synthetic peptide that we analyzed under identical LC-MS/MS conditions, confirming this rescued sequence as being a correct match. Additionally, there were two other peptides from the same protein that were identified in both the high- and low-resolution correlation analyses with scores and mass measurement accuracy assignments that pass cutoffs (KTHQPSDEVGTSIEHPR, M + 4H + , Xcorr ) 3.78, dCn ) 0.43, MMA ) -0.2 ppm and IPILLIQQPGK, M + 2H + , Xcorr ) 3.31, dCn ) 0.51, MMA ) 0.1 ppm), supporting the likelihood that this PSM is a valid match. Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6827
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: Figure 2. Productivity of SEQUST an
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 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?