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• W2035901782 abstract "This report describes an integrated and modular microsystem providing rapid analyses of trace-level tryptic digests for proteomics applications. This microsystem includes an autosampler, a microfabricated device comprising a large channel (2.4 μl total volume), an array of separation channels, together with a low dead volume enabling the interface to nanoelectrospray mass spectrometry. The large channel of this microfluidic device provides a convenient platform to integrate C18 reverse phase packing or other type of affinity media such as immobilized antibodies or immobilized metal affinity chromatography beads thus enabling affinity selection of target peptides prior to electrophoretic separation and mass spectrometry analyses on a quadrupole/time-of-flight instrument. Sequential injection, preconcentration, and separation of peptide standards and tryptic digests are achieved with a throughput of up to 12 samples/per h and a concentration detection limit of ∼5 nm (25 fmol on chip). Replicate injections of peptide mixtures indicated that reproducibility of migration time was 1.2–1.8%, whereas relative standard deviation ranging from 9.2 to 11.8% are observed on peak heights. The application of this device for trace-level protein identification is demonstrated for two-dimensional gel spots obtained from extracts of human prostatic cancer cells (LNCap) using both peptide mass-fingerprint data base searching and on-line tandem mass spectrometry. Enrichment of target peptides prior to mass spectral analyses is achieved using c-myc-specific antibodies immobilized on protein G-Sepharose beads and facilitates the identification of antigenic peptides spiked at a level of 20 ng/ml in human plasma. Affinity selection is also demonstrated for gel-isolated protein bands where tryptic phosphopeptides are captured on immobilized metal affinity chromatography beads and subsequently separated and characterized on this microfluidic system. This report describes an integrated and modular microsystem providing rapid analyses of trace-level tryptic digests for proteomics applications. This microsystem includes an autosampler, a microfabricated device comprising a large channel (2.4 μl total volume), an array of separation channels, together with a low dead volume enabling the interface to nanoelectrospray mass spectrometry. The large channel of this microfluidic device provides a convenient platform to integrate C18 reverse phase packing or other type of affinity media such as immobilized antibodies or immobilized metal affinity chromatography beads thus enabling affinity selection of target peptides prior to electrophoretic separation and mass spectrometry analyses on a quadrupole/time-of-flight instrument. Sequential injection, preconcentration, and separation of peptide standards and tryptic digests are achieved with a throughput of up to 12 samples/per h and a concentration detection limit of ∼5 nm (25 fmol on chip). Replicate injections of peptide mixtures indicated that reproducibility of migration time was 1.2–1.8%, whereas relative standard deviation ranging from 9.2 to 11.8% are observed on peak heights. The application of this device for trace-level protein identification is demonstrated for two-dimensional gel spots obtained from extracts of human prostatic cancer cells (LNCap) using both peptide mass-fingerprint data base searching and on-line tandem mass spectrometry. Enrichment of target peptides prior to mass spectral analyses is achieved using c-myc-specific antibodies immobilized on protein G-Sepharose beads and facilitates the identification of antigenic peptides spiked at a level of 20 ng/ml in human plasma. Affinity selection is also demonstrated for gel-isolated protein bands where tryptic phosphopeptides are captured on immobilized metal affinity chromatography beads and subsequently separated and characterized on this microfluidic system. Proteomics research entails the global characterization of proteins expressed in cells under defined conditions. Such studies are particularly important in view of the conflicting evidence regarding the correlation between the abundance of expressed proteins and gene-expression levels obtained from mRNA microarrays (1Anderson L. Seilhamer J. A comparison of selected mRNA and protein abundances in human liver.Electrophoresis. 1997; 18: 533-537Google Scholar, 2Gygi S.P. Rochon Y. Franza B.R. Aebersold R. Correlation between protein and mRNA abundance in yeast.Mol. Cell. Biol. 1999; 19: 1720-1730Google Scholar). The monitoring of protein expression profiles remains a very challenging task because of the wide dynamic range of expressed proteins and the variability of gene products (splicing variants, N- and C-terminal truncations, co- and post-translational modifications, etc.), which may change between and within the tissues of an organism (3Harry J.L. Wilkins M.R. Herbert B.R. Packer N.H. Gooley A.A. Williams K.L. Proteomics: capacity versus utility.Electrophoresis. 2000; 21: 1071-1081Google Scholar). The traditional approach to isolating and characterizing proteins from biological samples has been separation by two-dimensional gel electrophoresis (2-D 1The abbreviations used are: 2-D, two-dimensional; 2-D gel, 2-D gel electrophoresis; MS-MS, tandem mass spectrometry; LC, liquid chromatography; BCQ, [(acryloylamino)propyl]trimethylammonium chloride; TEMED, N,N,N′,N′-tetramethylethylenediamine; DMP, dimethyl pimelimidate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; RIE, reconstructed ion electrophoregram(s); IMAC, immobilized metal affinity chromatography; CE, capillary electrophoresis; nESMS, nanoelectrospray mass spectrometry. 1The abbreviations used are: 2-D, two-dimensional; 2-D gel, 2-D gel electrophoresis; MS-MS, tandem mass spectrometry; LC, liquid chromatography; BCQ, [(acryloylamino)propyl]trimethylammonium chloride; TEMED, N,N,N′,N′-tetramethylethylenediamine; DMP, dimethyl pimelimidate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; RIE, reconstructed ion electrophoregram(s); IMAC, immobilized metal affinity chromatography; CE, capillary electrophoresis; nESMS, nanoelectrospray mass spectrometry. gel), followed by identification of protein spots using sensitive mass spectrometry techniques and data base searching (3Harry J.L. Wilkins M.R. Herbert B.R. Packer N.H. Gooley A.A. Williams K.L. Proteomics: capacity versus utility.Electrophoresis. 2000; 21: 1071-1081Google Scholar, 4Abbott A. A post-genomic challenge: learning to read patterns of protein synthesis.Nature. 1999; 401: 715-720Google Scholar, 5Quadroni M. James P. Proteomics and automation.Electrophoresis. 1999; 20: 664-677Google Scholar). An alternate approach to 2-D gel includes a comprehensive chromatographic separation of the proteolytic fragments derived from intact proteins, followed by mass spectral identification and data base searching. This can be achieved using a two-dimensional liquid chromatography approach whereby peptides are fractionated on a strong cation exchange column, followed by an extended gradient elution on a C18 reverse phase column (6Opitek G.J. Lewis K.C. Jorgensen J.W. Moseley M.A. Comprehensive two-dimensional high-performance liquid chromatography for the isolation of overexpressed proteins and proteome mapping.Anal. Biochem. 1998; 258: 349-361Google Scholar, 7Link A.J. Eng J. Schieltz D.M. Carmack E. Mize G.J. Morris R. Garvik B.M. Yates III, J.R. Direct analysis of protein complexes using mass spectrometry.Nat. Biotechnol. 1999; 17: 676-682Google Scholar). This two-dimensional chromatography approach has been described recently for the comprehensive identification of the yeast proteome (6,113 proteins) and provided the identification of 1484 proteins among which were 131 proteins with three or more predicted transmembrane domains (8Washburn M.P. Wolters D. Yates III, J.R. Large scale analysis of the yeast proteome by multidimensional protein identification technology.Nat. Biotechnol. 2001; 19: 242-247Google Scholar). Although questions remain concerning the ability of the 2-D gel approach to analyze hydrophobic proteins and to quantitate and characterize the full dynamic range of protein expression from a given genome (9Gygi S.P. Corthals G.L. Zhang Y. Rochon Y. Aebersold R. Evaluation of two-dimensional gel electrophoresis based proteome analysis technology.Proc. Nat. Acad. Sci. U. S. A. 1999; 97: 9390-9395Google Scholar), protein identification via 2-D gel and mass spectrometry is still widely used in numerous proteomics core facilities. This approach enables the visualization of a very large number of proteins simultaneously, and in contrast to 2-D chromatographic separation it facilitates the identification of post-translational modifications and proteolytic processing in a convenient reference format. Furthermore, differential protein expression profiles can be compared easily for large data sets and for protein extracts obtained under different growth conditions, biological perturbations, or following pre-fractionation through organelle-enrichment methods. Unambiguous identification of gel-isolated proteins typically relies on sensitive tandem mass spectrometry (MS-MS) techniques to obtain partial amino acid sequences, which in combination with the mass of the precursor ion and that of the unidentified N- and C-terminal segments, can be used to search protein data bases (10Eng J.K. McCormack A.L. Yates III, J.R. An approach to correlate tandem mass spectra data of peptides with amino acid sequences in a protein database.J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Google Scholar, 11Mann M. Wilm M. Error-tolerant identification of peptides in sequence databases by peptide sequence tags.Anal. Chem. 1994; 66: 4390-4399Google Scholar, 12Mørtz E. O'Connor P.B. Roepstorff P. Kelleher N. Wood T.D. McLafferty F.W. Mann M. Sequence tag identification of intact proteins by matching tandem mass spectral data against sequence data bases.Proc. Nat. Acad. Sci. U. S. A. 1996; 93: 8264-8267Google Scholar). The combination of microscale LC column (50–180 μm inner diameter column) to nanoelectrospray MS-MS has also become a widely used workhorse system for proteomics applications involving the identification of 50–100 fmol of in-gel digests with a duty cycle of <15 min/sample (13Davis M.T. Lee T.D. Rapid protein identification using a microscale electrospray LC/MS system on an ion trap mass spectrometer.J. Am. Soc. Mass Spectrom. 1998; 9: 194-201Google Scholar, 14Moseley A., Blackburn K., Burkhart W., Vissers, H., and Bordoli, R. (2000) in Proceedings of the 48th ASMS Conference, Long Beach, CA, 2000, Abstract WPF-232, American Society for Mass Spectrometry, Santa Fe, NMGoogle Scholar). The coupling of microfluidic devices to mass spectrometry also offers an efficient means of handling small liquid volumes while simultaneously performing separation and sensitive detection on devices of small footprint. These microfabricated devices do not involve moving parts or high pressure liquid chromatography pumps as the analysis format involved in the precise control of voltages and electrical fields across small separation channels. The microchip interface to mass spectrometry has been achieved by creating a guided channel to the nanoelectrospray emitter using butted capillary (15Figeys D. Aebersold R. Nanoflow solvent gradient delivery from a microfabricated device for protein identifications by electrospray ionization mass spectrometry.Anal. Chem. 1998; 70: 3721-3727Google Scholar), the double-etching procedure (16Zhang B. Liu H. Karger B.L. Foret F. Microfabricated devices for capillary electrophoresis-electrospray mass spectrometry.Anal. Chem. 1999; 71: 3258-3264Google Scholar), polymer casting (17Liu H. Felten C. Xue Q. Zhang B. Jedrzejewski P. Karger B.L. Foret F. Development of multichannel devices with an array of electrospray tips for high-throughput mass spectrometry.Anal. Chem. 2000; 72: 3303-3310Google Scholar), and microdrilling (18Bings N.H. Wang C. Skinner C.D. Colyer C.L. Thibault P. Harrison D.J. Microfluidic devices connected to glass capillaries with minimal dead volume.Anal. Chem. 1999; 71: 3292-3296Google Scholar, 19Li J. Thibault P. Bings N.H. Skinner C.D. Wang C. Colyer C.L. Harrison D.J. Integration of microfabricated devices to capillary electrophoresis-electrospray mass spectrometry using a low dead volume connection; application to rapid analyses of proteolytic digests.Anal. Chem. 1999; 71: 3036-3045Google Scholar). Rapid separations on these microfluidic devices have been demonstrated using time-of-flight (20Lazar I.M. Ramsey R.S. Sundberg S. Ramsey J.M. Subattomole-sensitivity microchip nanoelectrospray source with time-of-flight mass spectrometry detection.Anal. Chem. 1999; 71: 3627-3631Google Scholar, 21Li J. Kelly J.F. Chernushevich I. Harrison J.D. Thibault P. Rapid separation of peptides from gel-isolated membrane proteins using a microfabricated device coupled to a high performance quadrupole/time-of-flight mass spectrometer.Anal. Chem. 2000; 72: 599-609Google Scholar, 22Pinto D.M. Ning Y. Figeys D. Microdevices for bioapplications - an enhanced microfluidic chip coupled to an electrospray Q-Star mass spectrometer for protein identification.Electrophoresis. 2000; 21: 181-190Google Scholar) and ion trap (15Figeys D. Aebersold R. Nanoflow solvent gradient delivery from a microfabricated device for protein identifications by electrospray ionization mass spectrometry.Anal. Chem. 1998; 70: 3721-3727Google Scholar, 16Zhang B. Liu H. Karger B.L. Foret F. Microfabricated devices for capillary electrophoresis-electrospray mass spectrometry.Anal. Chem. 1999; 71: 3258-3264Google Scholar) mass analyzers for submicromolar sample concentrations (15Figeys D. Aebersold R. Nanoflow solvent gradient delivery from a microfabricated device for protein identifications by electrospray ionization mass spectrometry.Anal. Chem. 1998; 70: 3721-3727Google Scholar, 17Liu H. Felten C. Xue Q. Zhang B. Jedrzejewski P. Karger B.L. Foret F. Development of multichannel devices with an array of electrospray tips for high-throughput mass spectrometry.Anal. Chem. 2000; 72: 3303-3310Google Scholar, 21Li J. Kelly J.F. Chernushevich I. Harrison J.D. Thibault P. Rapid separation of peptides from gel-isolated membrane proteins using a microfabricated device coupled to a high performance quadrupole/time-of-flight mass spectrometer.Anal. Chem. 2000; 72: 599-609Google Scholar). On-line stacking or adsorption preconcentration can also be applied prior to electrophoretic separation to enhance sensitivity (23Li J. Wang C. Kelly J.F. Harrison D.J. Thibault P. Rapid and sensitive separation of trace level protein digests using microfabricated device coupled to a high performance quadrupole/time-of-flight mass spectrometer.Electrophoresis. 2000; 21: 198-210Google Scholar). Obviously, significant advances in automation are still needed to provide the required throughput and ruggedness sought for the rapid and positive identification of proteins. To this end, efforts are also devoted to microfabricated chip designs that enable sample introduction from autosampler or microwell plates (24Zhang B. Foret F. Karger B.L. High-throughput microfabricated CE/ESI-MS: automated sampling from a microwell plate.Anal. Chem. 2001; 73: 2675-2681Google Scholar, 25Li J. Tremblay T.-L. Thibault P. Wang C. Attiya S. Harrison D.J. Integrated system for high throughput protein identification using a microfabricated device coupled to capillary electrophoresis/nanoelectrospray mass spectrometry.Joint publication in Eur. J. Mass Spectrom. 2001; 7 (Proteomics 1, 975–986): 143-155Google Scholar). In an effort to accelerate the analysis of protein digests while simultaneously maintaining the flexibility of the current microfluidic device, the present investigation describes the integration of an autosampler coupled to the chip via a convenient sample port. The previous chip design (21Li J. Kelly J.F. Chernushevich I. Harrison J.D. Thibault P. Rapid separation of peptides from gel-isolated membrane proteins using a microfabricated device coupled to a high performance quadrupole/time-of-flight mass spectrometer.Anal. Chem. 2000; 72: 599-609Google Scholar, 25Li J. Tremblay T.-L. Thibault P. Wang C. Attiya S. Harrison D.J. Integrated system for high throughput protein identification using a microfabricated device coupled to capillary electrophoresis/nanoelectrospray mass spectrometry.Joint publication in Eur. J. Mass Spectrom. 2001; 7 (Proteomics 1, 975–986): 143-155Google Scholar) was modified to include a port of low flow resistance enabling the introduction of sample on and off the chip without perturbing the fluids in the analysis manifold. Such design was used previously to conduct on-chip tryptic digestion and provided a convenient format to conduct rapid in-solution digest (<10 min) (26Wang C. Oleschuk R. Ouchen F. Li J. Thibault P. Harrison D.J. Integration of immobilized trypsin bead beds for protein digestion within a microfluidic chip incorporating capillary electrophoresis separations and an electrospray mass spectrometry interface.Rapid Commun. Mass Spectrom. 2000; 14: 1377-1383Google Scholar). In the present investigation we have used the large channel of this microfluidic to integrate C18 reverse phase packing or other types of affinity selection media to select target peptides prior to CE separation and mass spectrometry analyses. Application of this integrated system is demonstrated for the analysis of trace-level tryptic peptides obtained from gel-isolated proteins of human prostatic cell extracts. Fused-silica capillary was purchased from Polymicro Technologies (Phoenix, AZ), and Teflon tubing was from LC Packing (San Francisco, CA). Peptides and protein standards were purchased from Sigma and used without further purification. Gold-electroplating solution was prepared with 24K Bright English gold Plating Salts (Grobet File Co. of America, Inc., Caristadt, NJ). [(Acryloylamino)propyl]trimethylammonium chloride (BCQ) was obtained from Chemische Fabrik Stockhausen (Krefeld, Germany). 7-Oct-1-enyltrimethoxysilane was purchased from United Chemical Technologies Inc. (Bristol, PA). N,N,N′,N′-Tetramethylethylenediamine (TEMED) was obtained from Aldrich, and formic acid was from BDH Inc. (Toronto, ON, Canada). Dimethyl pimelimidate (DMP), ethanolamine, and sodium azide used for antibody coupling were obtained from Aldrich. The C18 reverse phase packing material of 40 μm was excised from Sep-Pak cartridges obtained from Waters (Milford, MA), and 5 μm Poros was from Applied Biosystems (Framingham, MA). IMAC beads were purchased from Amersham Biosciences, Inc. Mouse ascites fluid containing the monoclonal anti-c-myc (mouse IgG1 isotype) was obtained from Dr. R. MacKenzie (Institute for Biological Sciences, Ottawa, Ontario, Canada). The ascites fluid was diluted 3-fold in 20 m m phosphate buffer (pH 7) and centrifuged at 10,000 rpm for 20 min to remove lipids. The supernatant was purified using a HiTrap protein A column (Amersham Biosciences, Inc.) under isocratic elution using 0.1 m citric acid (pH 3). The UV absorbance was monitored at 280 nm. The antibody fraction was adjusted to pH 6 using 0.2 m Tris buffer (pH 8) and dialyzed against phosphate-buffered saline. The antibody was cross-linked to protein G-Sepharose 4 fast flow gel (Amersham Biosciences, Inc.) using DMP. Briefly, 2 mg of IgG1 antibody was added to 1 ml of the protein G gel and incubated for 1 h at room temperature with occasional mild shaking on a vortex. A DMP solution (in 0.2 m sodium borate) was added to the protein G-bound antibody gel slurry to a final concentration of 20 mm borate and incubated at room temperature for 30 min. The DMP solution was removed, the gel was washed once with borate solution and resuspended in 0.2 m ethanolamine (pH 8) for a 2-h incubation period. The ethanolamine solution was replaced by 0.02% NaN3 in phosphate-buffered saline and stored at 4 °C prior to use. Human prostatic cancer LNCap cells (CRL-1740) were obtained from the American Type Culture Collection (Manassas, VA). LNCap cells were grown in RPMI 1640 medium supplemented with 8% fetal bovine serum (Sigma) at 37 °C and 8% carbon dioxide. Cells were harvested by gentle scraping and washed three times in phosphate-buffered saline at 4 °C. Cell pellets were then frozen at −80 °C until further processing. LNCap cells were resuspended in lysis buffer (7 m urea, 2 m thiourea, 4% CHAPS, 1% dithiothreitol). After shaking at room temperature for 1 h, the lysate was centrifuged for 10 min at 12,000 × g to pellet unbroken cells and nuclei. Protein content in supernatant was then evaluated by Bradford assay. Samples containing 300 μg of total cell lysate was used to rehydrate immobilized pH gradient strips (pH 5–8) (Bio-Rad, Hercules, CA). First dimension electrophoresis was performed using the following program: 200-V rapid ramp for 1 h, 500-V rapid ramp for 1 h, 5000-V linear ramp for 5 h, 5000-V focusing for 80000 Vh. Prior to second dimension electrophoresis, proteins on immobilized pH gradient (IPG) strips were reduced (1% dithiothreitol) and alkylated (4% iodoacetamide) in SDS equilibration buffer (6 m urea, 30% glycerol, 2% SDS, 50 mm Tris-HCl (pH 8.8)). The second dimension is performed on a 10% gel (1-mm thick) at a constant 24 mA per gel. Separated proteins were then fixed in the gel using 50% ethanol, 5% acetic acid, stained with silver nitrate, and scanned using the Fluo-S imager (Bio-Rad). Selected spots were excised manually under a laminar flow hood. Excised spots were placed in a 96-well plate with a pierced-well bottom. Protein spots were processed used a Progest automated digestion unit (Genomics Solutions, Ann Arbour, MI). Briefly the procedure involved spots destaining with potassium ferricyanide solution (15 m m potassium ferricyanide, 50 mm sodium thiosulfate). Gel pieces were then rinsed three times with water and shrunk with acetonitrile. The gel pieces were reswelled with 10 to 20 μl of trypsin solution (0.01 μg/μl in 50 mm ammonium carbonate), and 30–50 μl of 50 mm ammonium carbonate is added to the gel pieces prior to overnight incubation (37 °C) with trypsin (Promega). Digestion solution containing peptide fragments was combined with organic extracts (30–50 μl of 50% methanol, 5% acetic acid) of the gel pieces. Peptide extracts were then evaporated to dryness on a Savant preconcentrator. The microfluidic device shown in Fig. 1a was fabricated at the University of Alberta Microfab laboratory (Edmonton, Alberta, Canada), as described previously (25Li J. Tremblay T.-L. Thibault P. Wang C. Attiya S. Harrison D.J. Integrated system for high throughput protein identification using a microfabricated device coupled to capillary electrophoresis/nanoelectrospray mass spectrometry.Joint publication in Eur. J. Mass Spectrom. 2001; 7 (Proteomics 1, 975–986): 143-155Google Scholar, 26Wang C. Oleschuk R. Ouchen F. Li J. Thibault P. Harrison D.J. Integration of immobilized trypsin bead beds for protein digestion within a microfluidic chip incorporating capillary electrophoresis separations and an electrospray mass spectrometry interface.Rapid Commun. Mass Spectrom. 2000; 14: 1377-1383Google Scholar). Channels were etched on Corning 0211 glass (Corning Glass) using standard photolithography and wet chemical etching techniques. Channels were etched on one glass plate to a 10-μm depth and 30-μm-width for the separation channels, with 230-μm-wide segments near the reservoirs. Channel lengths were essentially the same as PCRD2, described previously (21Li J. Kelly J.F. Chernushevich I. Harrison J.D. Thibault P. Rapid separation of peptides from gel-isolated membrane proteins using a microfabricated device coupled to a high performance quadrupole/time-of-flight mass spectrometer.Anal. Chem. 2000; 72: 599-609Google Scholar). The injector to capillary distance was 45 mm, the double-T injector had a 100-μm offset, and the additional channel attached to reservoir D was 24 mm from the junction. A large volume channel, 800-μm-wide, 150-μm-deep, and 22-mm-long, was etched into the cover plate, after which access holes were drilled into the cover hole. The channels on the chip were covalently modified with BCQ coating prior to inserting the nanoelectrospray emitter into a flat-bottom hole at the exit of the separation channel (21Li J. Kelly J.F. Chernushevich I. Harrison J.D. Thibault P. Rapid separation of peptides from gel-isolated membrane proteins using a microfabricated device coupled to a high performance quadrupole/time-of-flight mass spectrometer.Anal. Chem. 2000; 72: 599-609Google Scholar). Small plastic pipette tubes were inserted through small holes made in the center of the septa (Thermogreen LB-1 from Supelco or Septa 77 from Chromatographic Specialties) for buffer (B) and waste (A) reservoirs. Teflon tubes (180-μm inner diameter) were inserted in the center of septa for well D and used to seal a capillary transfer line. In this configuration the chip lay on the Teflon support, and a Plexiglas top was used to compress the septa to provide an air-tight seal between the sample/buffer reservoirs and the chip device. A 0.1 m formic acid solution was used for separation of background electrolytes. All aqueous solutions were filtered through a 0.45-μm filter (Millipore, Bedford, MA) before use. The separation and waste reservoirs were filled with 30 μl of running buffer. A custom-made 96-well plate autosampler comprising a sealed pressurized inlet (27Bonneil, E., Li, J., Tremblay, T.-L., Bergeron, J. J., and Thibault, P. (2001) in Proceedings of the 49th ASMS Conference, Chicago, IL, 2001 Abstract TPG-166, American Society for Mass Spectrometry, Santa Fe, NMGoogle Scholar) was used to introduce an 8–10-μl sample plug to the chip device via a 40-cm length of transfer capillary (360-μm outer diameter, 50-μm inner diameter). The first two rows of the plate were filled with wash and organic buffers whereas the remaining 72 wells were filled with reconstituted protein digests. Manual packing of the stationary phase or affinity selection media was achieved by loading the slurry (typically 10 μl of bead suspension) to well C using a syringe while a constant vacuum was applied to well D. For C18 reverse phase packing, a mixed bed composed of 2 mm of 40-μm beads (Waters) and ∼18 mm of 5-μm Poros particles (Applied Biosystems) was filled in sequence into the large channel. The reverse phase slurry was prepared by suspending 10 mg of beads in 50 μl of methanol prior to packing on the chip. For affinity selection using IMAC the beads were washed using 1% acetic acid in 10% aqueous acetonitrile and conditioned with 3 × 10 μl of an aqueous solution of 100 mm ferric chloride (Aldrich). The beads were washed again with 1% acetic acid in 10% aqueous acetonitrile, suspended in 50 μl of the same solution, and applied to the large packing channel. The IMAC bed was conditioned with 20 μl of 0.1 m formic acid before loading the sample on the chip. Prior to sample loading (20 μl) on the chip, the sample was acidified by reconstituting the in-gel digest in 5% acetic acid for proper binding of phosphopeptides on IMAC medium. Following sample application, the beads were washed with deionized water, and the selected peptides were eluted from the beads using 2% ammonium hydroxide. To minimize bubble formation a short plug of deionized water (200 nl) was intercalated between the acidic and basic buffers and provided sample stacking. In all cases, a constant voltage of −4.5 kV was applied to well D during the electrokinetic injection whereas other wells were floated. Mass spectrometric experiments were conducted using a Q-Star quadrupole/time-of-flight instrument (MDS/Sciex). The mass spectral resolution (half-height definition) was 10,000. The interface was optimized by infusing a solution of 1 μg/ml of angiotensin I at a flow rate of 0.2 μl/min through well E using a Harvard syringe pump. During the separation, this peptide solution was introduced at a flow rate of 50 nl/min, and the [M + 3H]3+ and [M+2H]2+ ions of angiotensin I were used as internal mass markers for accurate mass measurements. Tandem mass spectra were obtained using the Q-Star, and collisional activation of selected precursor ions was obtained using nitrogen as a target gas at collision energies of typically 50 to 90 eV (laboratory frame of reference). Fragment ions formed in the RF-only quadrupole were record by a time-of-flight mass analyzer. Accurate peptide masses were determined using internal standardization on the Q-Star instrument and were transferred to the ProteinProspector program. The list of peptide masses was searched against a nonredundant protein sequence data base from NCBI or SwissProt. Parameters for all searches assumed that masses corresponded to tryptic peptides and that cysteine residues were converted to S-carbamidomethylcysteine. All peptide masses were considered monoisotopic, and the maximum deviation between the calculated and measured masses was set to <10 ppm. Alternatively, the search was conducted using the peptide sequence tag approach where the precise molecular mass of a given tryptic peptide plus the fragment ion m/z values derived from the MS-MS spectrum were used to retrieve potential protein candidates. In situations where no match was obtained from either peptide mass fingerprinting or sequence tags, sequence segments of at least six amino acids were subjected to BLAST search at the NCBI web site (www.ncbi.nlm.nih.gov). In a previous report from this laboratory (25Li J. Tremblay T.-L. Thibault P. Wang C. Attiya S. Harrison D.J. Integrated system for high throughput protein identification using a microfabricated device coupled to capillary electrophoresis/nanoelectrospray mass spectrometry.Joint publication in Eur. J. Mass Spectrom. 2001; 7 (Proteomics 1, 975–986): 143-15" @default.
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• W2035901782 title "Application of Microfluidic Devices to Proteomics Research" @default.
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