Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Supporting Information). That finding suggests that ALD can be screened for by directly comparing the ion peak intensity ratios of C24:0/C22:0 when stable isotope-labeled internal standards are not available in the laboratory. Although GC-MS is a full quantitative method, quantification of VLCFAs in serum or plasma is typically time-consuming. 25,27 For example, esterification of fatty acids with methylating reagent can take up to 16 h. Ultrasound-assisted transmethylation of fatty acids has been reported 28,29 to simplify the sample preparation procedure and improve the efficiency of transmethylation of fatty acids. However, the VLCFAs are not all detected in these reports (especially the specific markers C22:0 and C24:0 for the screening of peroxisomal disorder). CONCLUSION The described method has a number of advantages that satisfy the increased demand for selective and sensitive high-throughput techniques. Derivative VLCFAs make desorption/ionization in the SALDI process more efficient, and the neutral loss in PSD allows 6820 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 for the specific identification of VLCFAs in plasma matrix. Enrichment of VLCFAs by MWCNT without eluting and evaporation procedures is an easy method for prepurification and concentration of samples. Use of VLCFAs-adsorbed MWCNTs as the SALDI substrate works well for detection in the low mass range without background peak interference and suppression. ACKNOWLEDGMENT The study was funded by a grant from the National Research Council of the Republic of China and the China Medical University Hospital (DMR 93-018). 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 25, 2010. Accepted July 2, 2010. AC100772J
Anal. Chem. 2010, 82, 6821–6829 MacroSEQUEST: Efficient Candidate-Centric Searching and High-Resolution Correlation Analysis for Large-Scale Proteomics Data Sets Brendan K. Faherty † and Scott A. Gerber* ,†,‡ Department of Genetics, Dartmouth Medical School, Lebanon, New Hampshire 03756, and Norris Cotton Cancer Center, Lebanon, New Hampshire 03756 Modern mass spectrometers are now capable of producing tens of thousands of tandem mass (MS/MS) spectra per hour of operation, resulting in an ever-increasing burden on the computational tools required to translate these raw MS/MS spectra into peptide sequences. In the present work, we describe our efforts to improve the performance of one of the earliest and most commonly used algorithms, SEQUEST, through a wholesale redesign of its processing architecture. We call this new program MacroSEQUEST, which exhibits a dramatic improvement in processing speed by transiently indexing the array of MS/MS spectra prior to searching FASTA databases. We demonstrate the performance of MacroSEQUEST relative to a suite of other programs commonly encountered in proteomics research. We also extend the capability of SEQUEST by implementing a parameter in MacroSE- QUEST that allows for scalable sparse arrays of experimental and theoretical spectra to be implemented for highresolution correlation analysis and demonstrate the advantages of high-resolution MS/MS searching to the sensitivity of large-scale proteomics data sets. Mass spectrometry (MS) coupled with computer-assisted database spectral matching has evolved into a cornerstone technology that drives research for the field of proteomics. 1 Recent developments in MS instrumentation have resulted in commercial mass spectrometers capable of generating tens of thousands of tandem mass spectra (MS/MS) per penultimate online reversephase liquid chromatography (LC) separation. 2-4 When coupled with biochemical prefractionation methods, a complete data set for a proteomics experiment (including technical and biological replicates) can consist of millions of MS/MS spectra per biological * To whom correspondence should be addressed. E-mail: scott.a.gerber@ dartmouth.edu. † Dartmouth Medical School. ‡ Norris Cotton Cancer Center. (1) Aebersold, R.; Mann, M. Nature 2003, 422, 198–207. (2) Makarov, A.; Denisov, E.; Kholomeev, A.; Balschun, W.; Lange, O.; Strupat, K.; Horning, S. Anal. Chem. 2006, 78, 2113–2120. (3) Olsen, J. V.; Schwartz, J. C.; Griep-Raming, J.; Nielsen, M. L.; Damoc, E.; Denisov, E.; Lange, O.; Remes, P.; Taylor, D.; Splendore, M.; Wouters, E. R.; Senko, M.; Makarov, A.; Mann, M.; Horning, S. Mol. Cell. Proteomics 2009, 8, 2759–2769. (4) Second, T. P.; Blethrow, J. D.; Schwartz, J. C.; Merrihew, G. E.; MacCoss, M. J.; Swaney, D. L.; Russell, J. D.; Coon, J. J.; Zabrouskov, V. Anal. Chem. 2009, 81, 7757–7765. sample. Importantly, although the absolute sensitivity of new instruments to detect a single peptide species has improved, it has been suggested that much of the credit for increased depth of peptide and protein coverage, and improved detection of substoichiometric species belongs to the increased rate of MS/ MS spectral acquisition of these instruments, which allows them to penetrate transient rasters of precursor ions to greater ion peak depth per chromatographic unit time. 5 Indeed, the number of candidate precursor ions (MS1 features) is often at least an order of magnitude greater than the number of MS/MS sequencing events in a typical LC-MS analysis. 6 Given the current level of success with this strategy, it is only reasonable to predict that this trend of increasing MS/MS bandwidth to improve overall peptide identification rates per sample will continue, which places an additional burden on the computational tools required to search these larger data sets. A single raw MS/MS spectrum is translated into a peptide spectral match (PSM) through the use of algorithms that first search translated genomic databases from an organism of interest for candidate peptides by a defined enzyme specificity (based on the protease used for digestion) and precursor mass (based on the MS1 feature mass from which the MS/MS spectrum was derived and a desired mass precision). 7 Other parameters (fixed protein modifications, variable post-translational modifications, number of missed enzyme cleavage loci, maximum and/or minimum peptide size, etc.) may also be included. This search results in a list of candidate peptides that may contain the correct peptide sequence from which the MS/MS spectrum was derived. These candidate peptides are then evaluated for correctness by comparing the observed MS/MS spectrum with a dynamically generated theoretical spectrum for each candidate peptide and given a score that reflects the quality of their match. These scores are then ranked and, in some cases, further evaluated 8 before reporting the “best” candidate peptide match (PSM) for the MS/ MS spectrum. In a collection of MS/MS spectra from a single LC-MS run, a series of LC-MS runs for a given sample or from (5) Haas, W.; Faherty, B. K.; Gerber, S. A.; Elias, J. E.; Beausoleil, S. A.; Bakalarski, C. E.; Li, X.; Villen, J.; Gygi, S. P. Mol. Cell. Proteomics 2006, 5, 1326–1337. (6) Hoopmann, M. R.; Finney, G. L.; MacCoss, M. J. Anal. Chem. 2007, 79, 5620–5632. (7) Nesvizhskii, A. I. Methods Mol. Biol. 2007, 367, 87–119. (8) Kall, L.; Canterbury, J. D.; Weston, J.; Noble, W. S.; MacCoss, M. J. Nat. Methods 2007, 4, 923–925. 10.1021/ac100783x © 2010 American Chemical Society 6821 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Published on Web 07/28/2010
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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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Page 27 and 28: on a substrate are preferred. 20-24
Page 29 and 30: Figure 4. SERS analysis of NAADP co
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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
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Page 45 and 46: esult in a big SPR signal change wi
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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
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Page 57 and 58: from the specimen and enclosed in a
Page 59 and 60: Figure 4. (A) IRMS mass-44 chromato
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Page 65 and 66: Polymerase (1 U per sample). Reacti
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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
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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
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Page 117 and 118: data as well. The 41 genes in Table
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Page 123 and 124: Table 1. Absolute Quantification Re
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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.
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solution under appropriate incubati
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Figure 4. Standardization curve for
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Anal. Chem. 2010, 82, 6919-6925 Ele
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the ×10 objective, to have a large
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Figure 2. With a suitable removal o
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the NB signal in a much better foot
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Scheme 1. Reactions of Selenium Rea
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Figure 2. (a) ESI-MS spectrum showi
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Figure 4. (a) ESI-MS spectrum showi
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Anal. Chem. 2010, 82, 6933-6939 Dif
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trode 28 by a finite element using
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Figure 4. Comparison between simula
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Figure 6. Comparison between simula
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educed in the vicinity of double bo
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Figure 2. Normalized product ion ab
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Figure 4. EID (a) and IRMPD (b) of
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Anal. Chem. 2010, 82, 6947-6957 Ide
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Figure 1. Schematic flowchart showi
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difference, ppm compound Table 1. I
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isoforms, its successful use, in th
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Figure 5. Extracted ion current ESI
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m/z 1172.935 was observed for Ser14
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linked products via affinity tags.
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Scheme 2. Fragmentation Mechanism o
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Figure 1. (A) ESI-LTQ-CID-MS 2 prod
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Figure 3. (A) ESI-LTQ-CID-MS 2 prod
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Figure 5. (A) MALDI-TOF/TOF product
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Anal. Chem. 2010, 82, 6969-6975 Ana
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Figure 3. Equilibrium response as a
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Figure 6. The average measured resp
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for the fill time, and we find that
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nucleotide tails. 3-5 Thus, the amo
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allow the use of higher aptamer con
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quent ligation of the aptamers afte
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Anal. Chem. 2010, 82, 6983-6990 Imp
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Figure 2. Configuration editor wind
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Figure 4. Dependencies between volu
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Since the time for liquid expulsion
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Anal. Chem. 2010, 82, 6991-6999 Int
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Figure 1. Representation of the Car
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Table 2. Capillary Electrophoresis
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Figure 4. (A) Typical raw electroph
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field. DNA profiles were delivered
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Another approach to handle this lim
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(principal components, PCs) in whic
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Figure 5. Relevant score plots and
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CONCLUSIONS In this paper we have i
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electroactive-species loaded liposo
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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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