Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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For the analysis of complex sample mixtures, as for example encountered in proteomics research, the peak capacities that can be realized with one-dimensional ( 1 D) LC often do not suffice to achieve complete separation of all compounds. Multidimensional separation approaches have the potential to separate thousands of components within a reasonable time. In first approximation, the maximum peak capacity in two-dimensional LC is the product of the peak capacities of the individual dimensions, provided that the two retention mechanisms are orthogonal. 15 Comprehensive two-dimensional liquid chromatography, where all essential fractions of the sample are being analyzed in both dimensions, can be subdivided into two main categories, i.e., online comprehensive two-dimensional LC (or LC×LC) and off-line comprehensive two-dimensional LC (or LC/×/LC). 16 In LC×LC, column dimensions must be selected such that the eluent composition and transfer volume match the LC conditions used in the second dimension. Schoenmakers showed that the maximum flow rate in the first-dimension and, consequently, the diameter of the 1 D column depends on the maximum injection volume in the second dimension and on the second-dimension analysis time. 17 The optimization of sampling time in online two-dimensional LC has recently been reviewed by Guiochon et al. 18 When the sampling rate is too low, resolution achieved in the first dimension is partially lost. However, more time is available to achieve a high peak capacity in the second dimension. The fraction of potential peak capacity of the twodimensional combination of columns that is lost due to the selection of a too small modulation frequency is described by the “Nobuo factor” (modulation efficiency). 19 Typically, for LC×LC, two cuts per first-dimension peak provides the best trade-off between the loss of resolution in the first dimension and the analysis time in the second dimension. 19,20 However, from a practical point of view, this is difficult to achieve since modulation-phase effects and the random sampling process must be considered. The off-line approach (LC/×/LC) offers more flexibility for the selection of column dimensions and elution conditions, since the second-dimension analysis time can be optimized independently from the first-dimension sampling time. In addition, the organic modifier can be evaporated, so that fractions can be concentrated or dissolved in a different solvent prior to reinjection. 18,21 To enhance the preconcentration of peptides or proteins on a trap column, a strong ion-pairing agent can be added prior to the second-dimension separation. 21 As a result, the flow rates, transfer volumes, and the compatibility of (first and second dimension) eluent composition are less critical issues in LC/×/LC. The LC/×/LC setup can be optimized for proteomics applications. A large I.D. first-dimension column can be used, which provides (15) Giddings, J. C. Anal. Chem. 1984, 65, 1258A–1270A. (16) Schoenmakers, P. J.; Marriott, P.; Beens, J. LC-GC Eur. 2003, 16, 335– 339. (17) van der Horst, A.; Schoenmakers, P. J. J. Chromatogr., A 2003, 1000, 693– 709. (18) Guiochon, G.; Marchetti, M.; Mriziq, K.; Shalliker, R. A. J. Chromatogr., A 2008, 1189, 109–168. (19) Horie, K.; Kimura, K.; Ikegami, T.; Iwatsuka, A.; Saad, N.; Fiehn, O.; Tanaka, N. Anal. Chem. 2007, 79, 3764–3770. (20) Davis, J. M.; Stoll, D. R.; Carr, P. W. Anal. Chem. 2008, 80, 461–473. (21) Eeltink, S.; Dolman, S.; Swart, R.; Ursem, M.; Schoenmakers, P. J. J. Chromatogr., A 2009, 1216, 7368–7374. 7016 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 sufficient loadability for the analysis of samples with a broad dynamic range. A small I.D. column can be applied in the second dimension, minimizing chromatographic dilution and maximizing ionization efficiency when coupling LC with mass spectrometry via an electrospray interface. Huber and co-workers compared the sequence coverage obtained after an LC/×/LC-MS/MS separation of a tryptic digest of C. glutamicum using either a strong cationexchange (SCX) column or a reversed-phase (RP) column operated at high pH in the first dimension and a reversed-phase monolith operated at low pH in the second dimension. 22,23 They observed that the SCX/×/RP and RP/×/RP approaches complemented each other. Recently, our group compared the 1 D-LC performance of a 50 mm long monolithic column with that of an LC/×/LC approach employing a weak-anion exchange and an RP monolithic column in the first and second dimensions, respectively. 24 At the conditions applied, the WAX/×/RP approach provided a better peak-capacity-per-analysis-time ratio for separations requiring a peak capacity of 400 or higher. The present contribution discusses how to maximize the peakproduction rate in off-line comprehensive two-dimensional liquid chromatography for the reversed-phase separations (RP/×/RP) of peptides performed at high and low pH using monolithic column technology. The effects of the first-dimension ( 1 D) column dimensions (length and diameter) and LC conditions (flow rate and gradient time) on peak width and sampling volume are discussed. In addition, the effects of 2 D column length and gradient time on peak-production rate in 2 D-LC are demonstrated at “undersampling” conditions (i.e., containing fewer fractions than required to essentially maintain the first-dimension separation). Finally, the potential of RP/×/RP is demonstrated with separations of proteomic samples of varying complexity. EXPERIMENTAL SECTION Chemicals and Materials. Acetonitrile (ACN, HPLC supragradient quality), heptafluorobutyric acid (HFBA, ULC/MS quality), and trifluoroacetic acid (TFA, ULC/MS quality) were purchasedfromBiosolve(Valkenswaard,TheNetherlands).Ammonium bicarbonate (min. 99%), dithiothreitol (min. 99%), iodoacetic acid (approximately 99%), guanidine-HCl, sodium chloride (analytical reagent grade), cytochrome c (bovine heart), and apo-transferrin (bovine, g98%) were purchased from Sigma-Aldrich (Steinheim, Germany). Lysozyme (hen egg white), alcohol dehydrogenase (yeast), serum albumin (bovine, assay >96%), �-galactosidase, and sodium phosphate monobasic dihydrate (analytical reagent grade) were obtained from Fluka (Buchs, Switzerland). Escherichia coli (E. coli, strain K12) protein sample (lyophilized) was obtained from Bio-Rad Laboratories (Veenendaal, The Netherlands). Preconcentration and desalting of peptides prior to the analytical separation was performed ona5mm× 0.2 mm I.D. monolithic trap column (Pepswift RP, Dionex Benelux, Amsterdam, The Netherlands). HPLC separations were performed with 50 mm × 0.2 mm and 250 mm × 0.2 mm monolithic PepSwift RP columns (22) Delmotte, N.; Lasaosa, M.; Tholey, A.; Heinzle, E; Huber, C. G. J. Proteome Res. 2007, 6, 4363–4373. (23) Toll, H.; Oberacher, H.; Swart, R.; Huber, C. G. J. Chromatogr., A 2005, 1079, 274–286. (24) Eeltink, S.; Dolman, S.; Detobel, F.; Desmet, G.; Swart, R.; Ursem, M. J. Sep. Sci. 2009, 32, 2504–2509.
Table 1. Effect of 1 D-LC Conditions on Sampling Time and Number of Fractions Collected When Applying a Modulation Ration (MR) ∼2 per 1 D Peak a I.D. (mm) flow rate (µL/min) gradient time (min) 4σ peak width (s) sampling time (s) sampling volume (µL) no. fractions 0.2 2 10 5.5 2.8 0.09 214 1 60 10 5.8 2.6 2.4 231 a Column length is 50 mm; 1 µL injection of the six-protein digest; aqueous acetonitrile gradient from 1-26% (ACN) at pH ) 8; detection at 214 nm. and with a 50 mm × 1 mm monolithic ProSwift RP-10R column (Dionex). Preparation of Tryptic Digests. The six-protein mixture prepared from transferrin, bovine serum albumin, �-galactosidase, alcohol dehydrogenase, lysozyme, cytochrome, and E. coli proteins were digested according the following procedure. Proteins were reduced for1hat60°C in the presence of 7 mol/L guanidine and 1 mol/L dithiothreitol, followed by alkylation for 30 min at room temperature by adding 1 mol/L iodoacetic acid. To consume any unreacted iodoacetic acid, 1 mol/L dithiothreitol was added. The reduced and alkylated proteins were then dialyzed against 50 mM ammonium bicarbonate (pH 8) for 24 h in dialysis sacks (Sigma) with a cutoff 0.997). Therefore, the relationship 2 wb ) a + b( 2 tG) can be established, which holds at a constant length. After fitting, the a and b parameters were used to predict the peak capacity as a function Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 7017
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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.
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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
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
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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
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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: Anal. Chem. 2010, 82, 7015-7020 Sel
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
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