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• W2113550007 abstract "Hybrid quadrupole time-of-flight (QTOF) mass spectrometry is one of the two major principles used in proteomics. Although based on simple fundamentals, it has over the last decades greatly evolved in terms of achievable resolution, mass accuracy, and dynamic range. The Bruker impact platform of QTOF instruments takes advantage of these developments and here we develop and evaluate the impact II for shotgun proteomics applications. Adaption of our heated liquid chromatography system achieved very narrow peptide elution peaks. The impact II is equipped with a new collision cell with both axial and radial ion ejection, more than doubling ion extraction at high tandem MS frequencies. The new reflectron and detector improve resolving power compared with the previous model up to 80%, i.e. to 40,000 at m/z 1222. We analyzed the ion current from the inlet capillary and found very high transmission (>80%) up to the collision cell. Simulation and measurement indicated 60% transfer into the flight tube. We adapted MaxQuant for QTOF data, improving absolute average mass deviations to better than 1.45 ppm. More than 4800 proteins can be identified in a single run of HeLa digest in a 90 min gradient. The workflow achieved high technical reproducibility (R2 > 0.99) and accurate fold change determination in spike-in experiments in complex mixtures. Using label-free quantification we rapidly quantified haploid against diploid yeast and characterized overall proteome differences in mouse cell lines originating from different tissues. Finally, after high pH reversed-phase fractionation we identified 9515 proteins in a triplicate measurement of HeLa peptide mixture and 11,257 proteins in single measurements of cerebellum−the highest proteome coverage reported with a QTOF instrument so far. Hybrid quadrupole time-of-flight (QTOF) mass spectrometry is one of the two major principles used in proteomics. Although based on simple fundamentals, it has over the last decades greatly evolved in terms of achievable resolution, mass accuracy, and dynamic range. The Bruker impact platform of QTOF instruments takes advantage of these developments and here we develop and evaluate the impact II for shotgun proteomics applications. Adaption of our heated liquid chromatography system achieved very narrow peptide elution peaks. The impact II is equipped with a new collision cell with both axial and radial ion ejection, more than doubling ion extraction at high tandem MS frequencies. The new reflectron and detector improve resolving power compared with the previous model up to 80%, i.e. to 40,000 at m/z 1222. We analyzed the ion current from the inlet capillary and found very high transmission (>80%) up to the collision cell. Simulation and measurement indicated 60% transfer into the flight tube. We adapted MaxQuant for QTOF data, improving absolute average mass deviations to better than 1.45 ppm. More than 4800 proteins can be identified in a single run of HeLa digest in a 90 min gradient. The workflow achieved high technical reproducibility (R2 > 0.99) and accurate fold change determination in spike-in experiments in complex mixtures. Using label-free quantification we rapidly quantified haploid against diploid yeast and characterized overall proteome differences in mouse cell lines originating from different tissues. Finally, after high pH reversed-phase fractionation we identified 9515 proteins in a triplicate measurement of HeLa peptide mixture and 11,257 proteins in single measurements of cerebellum−the highest proteome coverage reported with a QTOF instrument so far. Building on the fundamental advance of the soft ionization techniques electrospray ionization and matrix-assisted laser desorption/ionization (1.Karas M. Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons.Anal. Chem. 1988; 60: 2299-2301Crossref PubMed Scopus (4836) Google Scholar, 2.Fenn J.B. Mann M. Meng C.K. Wong S.F. Whitehouse C.M. Electrospray ionization for mass spectrometry of large biomolecules.Science. 1989; 246: 64-71Crossref PubMed Scopus (6334) Google Scholar), MS-based proteomics has advanced tremendously over the last two decades (3.Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref PubMed Scopus (5596) Google Scholar, 4.Cravatt B.F. Simon G.M. Yates 3rd, J.R. The biological impact of mass-spectrometry-based proteomics.Nature. 2007; 450: 991-1000Crossref PubMed Scopus (572) Google Scholar, 5.Altelaar A.F. Munoz J. Heck A.J. Next-generation proteomics: towards an integrative view of proteome dynamics.Nat. Rev. Genet. 2013; 14: 35-48Crossref PubMed Scopus (521) Google Scholar, 6.Richards A.L. Merrill A.E. Coon J.J. Proteome sequencing goes deep.Curr. Opin. Chem. Biol. 2014; 24C: 11-17Google Scholar). Bottom-up, shotgun proteomics is usually performed in a liquid chromatography-tandem MS (LC-MS/MS)1 format, where nanoscale liquid chromatography is coupled through electrospray ionization to an instrument capable of measuring a mass spectrum and fragmenting the recognized precursor peaks on the chromatographic time scale. Fundamental challenges of shotgun proteomics include the very large numbers of peptides that elute over relatively short periods and peptide abundances that vary by many orders of magnitude. Developments in mass spectrometers toward higher sensitivity, sequencing speed, and resolution were needed and helped to address these critical challenges (7.Domon B. Aebersold R. Challenges and opportunities in proteomics data analysis.Mol. Cell. Proteomics. 2006; 5: 1921-1926Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 8.Mann M. Kelleher N.L. Precision proteomics: the case for high-resolution and high mass-accuracy.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 18132-18138Crossref PubMed Scopus (353) Google Scholar). Especially the introduction of the Orbitrap mass analyzers has advanced the state of the art of the field because of their very high resolution and mass accuracy (9.Makarov A. Electrostatic axially harmonic orbital trapping: a high-performance technique of mass analysis.Anal. Chem. 2000; 72: 1156-1162Crossref PubMed Scopus (646) Google Scholar, 10.Zubarev R.A. Makarov A. Orbitrap mass spectrometry.Anal. Chem. 2013; 85: 5288-5296Crossref PubMed Scopus (359) Google Scholar). A popular configuration couples a quadrupole mass filter for precursor selection to the Orbitrap analyzer in a compact benchtop format (11.Michalski A. Damoc E. Hauschild J.P. Lange O. Wieghaus A. Makarov A. Nagaraj N. Cox J. Mann M. Horning S. Mass spectrometry-based proteomics using Q Exactive, a high-performance benchtop quadrupole Orbitrap mass spectrometer.Mol. Cell. Proteomics. 2011; 10M111.011015Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar, 12.Scheltema R.A. Hauschild J.P. Lange O. Hornburg D. Denisov E. Damoc E. Kuehn A. Makarov A. Mann M. The Q exactive HF, a benchtop mass spectrometer with a pre-filter, high-performance quadrupole and an ultra-high-field Orbitrap analyzer.Mol. Cell. Proteomics. 2014; 13: 3698-3708Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 13.Kelstrup C.D. Jersie-Christensen R.R. Batth T.S. Arrey T.N. Kuehn A. Kellmann M. Olsen J.V. Rapid and deep proteomes by faster sequencing on a benchtop quadrupole ultra-high-field Orbitrap mass spectrometer.J. Proteome Res. 2014; 13, 12: 6187-6195Crossref Scopus (137) Google Scholar). In addition to the improvements in MS instrumentation, there have been key advances in the entire proteomics workflow, from sample preparation through improved LC systems and in computational proteomics (14.Zhou H. Ning Z. Wang F. Seebun D. Figeys D. Proteomic reactors and their applications in biology.FEBS J. 2011; 278: 3796-3806Crossref PubMed Scopus (34) Google Scholar, 15.Kocher T. Swart R. Mechtler K. Ultra-high-pressure RPLC hyphenated to an LTQ-Orbitrap Velos reveals a linear relation between peak capacity and number of identified peptides.Anal. Chem. 2011; 83: 2699-2704Crossref PubMed Scopus (116) Google Scholar, 16.Cox J. Mann M. Quantitative, high-resolution proteomics for data-driven systems biology.Annu. Rev. Biochem. 2011; 80: 273-299Crossref PubMed Scopus (532) Google Scholar). Together, such advances are making shotgun proteomics increasingly comprehensive and deep analyses can now be performed in a reasonable time (13.Kelstrup C.D. Jersie-Christensen R.R. Batth T.S. Arrey T.N. Kuehn A. Kellmann M. Olsen J.V. Rapid and deep proteomes by faster sequencing on a benchtop quadrupole ultra-high-field Orbitrap mass spectrometer.J. Proteome Res. 2014; 13, 12: 6187-6195Crossref Scopus (137) Google Scholar, 17.Nagaraj N. Kulak N.A. Cox J. Neuhauser N. Mayr K. Hoerning O. Vorm O. Mann M. System-wide perturbation analysis with nearly complete coverage of the yeast proteome by single-shot ultra HPLC runs on a bench top Orbitrap.Mol. Cell. Proteomics. 2012; 11M111.013722Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 18.Hebert A.S. Richards A.L. Bailey D.J. Ulbrich A. Coughlin E.E. Westphall M.S. Coon J.J. The one hour yeast proteome.Mol. Cell. Proteomics. 2014; 13: 339-347Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 19.Kulak N.A. Pichler G. Paron I. Nagaraj N. Mann M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells.Nat. Methods. 2014; 11: 319-324Crossref PubMed Scopus (1002) Google Scholar). Nevertheless, complete analysis of all expressed proteins in a complex system remains extremely challenging and complete measurement of all the peptides produced in shotgun proteomics may not even be possible in principle (20.Michalski A. Cox J. Mann M. More than 100,000 detectable peptide species elute in single shotgun proteomics runs but the majority is inaccessible to data-dependent LC-MS/MS.J. Proteome Res. 2011; 10: 1785-1793Crossref PubMed Scopus (480) Google Scholar, 21.Savitski M.M. Nielsen M.L. Zubarev R.A. ModifiComb, a new proteomic tool for mapping substoichiometric post-translational modifications, finding novel types of modifications, and fingerprinting complex protein mixtures.Mol. Cell. Proteomics. 2006; 5: 935-948Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Therefore, an urgent need for continued improvements in proteomics technology remains. Besides the Orbitrap analyzer and other ion trap technologies, the main alternative MS technology is time-of-flight, a technology that has been used for many decades in diverse fields. The configuration employed in proteomics laboratories combines a quadrupole mass filter via a collision cell and orthogonal acceleration unit to a reflectron and a multichannel plate (MCP) detector (22.Morris H.R. Paxton T. Dell A. Langhorne J. Berg M. Bordoli R.S. Hoyes J. Bateman R.H. High sensitivity collisionally-activated decomposition tandem mass spectrometry on a novel quadrupole/orthogonal-acceleration time-of-flight mass spectrometer.Rapid Commun. Mass Spectr. 1996; 10: 889-896Crossref PubMed Scopus (390) Google Scholar). TOF scans are generated in much less than a millisecond (ms), and a number of these “pulses” are added to obtain an MS or MS/MS spectrum with the desired signal to noise ratio. Our own laboratory has used such a quadrupole time-of-flight (QTOF) instrument as the main workhorse in proteomics for many years, but then switched to high-resolution trapping instruments because of their superior resolution and mass accuracy. However, TOF technology has fundamental attractions, such as the extremely high scan speed and the absence of space charge, which limits the number of usable ions in all trapping instruments. In principle, the high spectra rate makes TOF instruments capable of making use of the majority of ions, thus promising optimal sensitivity, dynamic range and hence quantification. It also means that TOF can naturally be interfaced with ion mobility devices, which typically separate ions on the ms time scale. Data independent analysis strategies such as MSE, in which all precursors are fragmented simultaneously (23.Silva J.C. Denny R. Dorschel C. Gorenstein M.V. Li G.Z. Richardson K. Wall D. Geromanos S.J. Simultaneous qualitative and quantitative analysis of the Escherichia coli proteome: a sweet tale.Mol. Cell. Proteomics. 2006; 5: 589-607Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 24.Silva J.C. Gorenstein M.V. Li G.Z. Vissers J.P. Geromanos S.J. Absolute quantification of proteins by LCMSE: a virtue of parallel MS acquisition.Mol. Cell. Proteomics. 2006; 5: 144-156Abstract Full Text Full Text PDF PubMed Scopus (1144) Google Scholar) or SWATH, in which the precursor ion window is rapidly cycled through the entire mass range (25.Gillet L.C. Navarro P. Tate S. Rost H. Selevsek N. Reiter L. Bonner R. Aebersold R. Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis.Mol. Cell. Proteomics. 2012; 11O111.016717Abstract Full Text Full Text PDF PubMed Scopus (1795) Google Scholar), also make use of the high scanning speed offered by QTOF instruments. It appears that QTOFs are set to make a comeback in proteomics with recent examples showing impressive depth of coverage of complex proteomes. For instance, using a variant of the MSE method, identification of 5468 proteins was reported in HeLa cells in single shots and small sample amounts (26.Distler U. Kuharev J. Navarro P. Levin Y. Schild H. Tenzer S. Drift time-specific collision energies enable deep-coverage data-independent acquisition proteomics.Nat. Methods. 2014; 11: 167-170Crossref PubMed Scopus (306) Google Scholar). In another report, employing ion mobility for better transmission of fragment ions to the detector led to the identification of up to 7548 proteins in human ovary tissue (27.Helm D. Vissers J.P. Hughes C.J. Hahne H. Ruprecht B. Pachl F. Grzyb A. Richardson K. Wildgoose J. Maier S.K. Marx H. Wilhelm M. Becher I. Lemeer S. Bantscheff M. Langridge J.I. Kuster B. Ion mobility tandem mass spectrometry enhances performance of bottom-up proteomics.Mol. Cell. Proteomics. 2014; 13: 3709-3715Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In this paper, we describe the impact II™, a benchtop QTOF instrument from Bruker Daltonics, and its use in shotgun proteomics. This QTOF instrument is a member of an instrument family first introduced in 2008, which consists of the compact, the impact, and the maXis. The original impact was introduced in 2011 and was followed by the impact HD, which was equipped with a better digitizer, expanding the dynamic range of the detector. With the impact II, which became commercially available in 2014, we aimed to achieve a resolution and sequencing speed adequate for demanding shotgun proteomics experiments. To achieve this we developed an improved collision cell, orthogonal accelerator scheme, reflectron, and detector. Here we measure ion transmission characteristics of this instrument and the actually realized resolution and mass accuracy in typical proteomics experiments. Furthermore, we investigated the attainable proteome coverage in single shot analysis and we ask if QTOF performance is now sufficient for very deep characterization of complex cell line and tissue proteomes. HeLa cells (ATCC, S3 subclone) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 20 mm glutamine and 1% penicillin-streptomycin (all from PAA Laboratories, Freiburg, Germany). Cells were collected by centrifugation at 200 × g for 10 min, washed once with cold phosphate buffered saline (PBS) and centrifuged again. Supernatant was carefully discarded and the cell pellet shock frozen in liquid nitrogen and stored at −80 °C until further use. A pellet containing 5 × 107 cells was resuspended in 1.5 ml of ice cold Milli-Q water, then an equal volume of trifluoroethanol (Sigma-Aldrich, Taufkirchen, Germany) was added. The cell suspension was kept on ice for 10 min, vortexed for 1 min and sonicated for 2 min at 20% duty cycle and output control 3 (Branson Ultrasonics sonifier, Danbury, CT; model 250). After the addition of 200 μl Tris (pH 8.5, final concentration: 100 mm), 400 μl TCEP (final concentration: 10 mm) and 400 μl 2-chloroacetamide (CAA) (final concentration: 40 mm) the lysate was incubated for 10 min at 95 °C. Then the sample was diluted to 15 ml with 50 mm ammonium bicarbonate. The mixture was digested by adding LysC (Wako Chemicals GmbH, Neuss, Germany; ratio 1 μg LysC:100 μg sample protein) for 2 h at 37 °C, followed by adding trypsin (ratio 1 μg trypsin:75 μg sample protein, Promega GmbH, Mannheim, Germany) at 37 °C overnight. After a further digestion with trypsin (ratio 1:125) for 5 h at 37 °C, the digested peptides with an estimated concentration of 1 μg/μl were diluted 1:4 with water and acidified by adding formic acid (FA) (final concentration: 0.2%) and purified on Sep-Pak tC18 cartridges (Waters, Milford, MA) according to manufacturer's instructions. Peptide concentration was determined using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE). Saccharomyces cerevisiae strains BY4742 and BY4743 (EUROSCARF) were grown at 30 °C in yeast extract peptone dextrose (YPD) media (10 g/l BactoYeast extract, 20 g/l BactoTM peptone (BD), 2% w/v glucose). Cells were grown to log phase (OD600 of 0.6), harvested by centrifugation at 1600 × g for 10 min at 4 °C, washed with cold Milli-Q water and then collected again by centrifugation at 10,000 × g for 5 min at 4 °C. Cells were lysed in 1% sodium deoxycholate, 10 mm TCEP, 40 mm CAA in 100 mm Tris pH 8.5, boiled for 10 min at 95 °C and sonicated for 3 min at 30% duty cycle and output control 3 (Branson Ultrasonics sonifier; model 250). Protein concentrations were determined by tryptophan fluorescence emission assay. Cell lysates were diluted 1:2 with Milli-Q water and digested by adding LysC (Wako Chemicals GmbH, ratio 1 μg LysC:50 μg sample protein) for 4 h at 37 °C, followed by adding again LysC (ratio 1:50) overnight at 37 °C. An equal volume of ethyl acetate acidified with 1% TFA was added to the solution, samples were vortexed for 2 min and digested peptides were purified with SDB-RPS StageTips as described in Kulak et al. (19.Kulak N.A. Pichler G. Paron I. Nagaraj N. Mann M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells.Nat. Methods. 2014; 11: 319-324Crossref PubMed Scopus (1002) Google Scholar). Peptide concentrations were determined using a NanoDrop spectrophotometer. Spinal cord neuron-neuroblastoma (NSC-34) (CED-CLU140, Biozol, Eching, Germany), mouse embryonic fibroblasts (MEFs) (American Type Culture Collection, Manassas, VA), and mouse hepatoma (liver cancer, Hepa 1–6) (CRL-1830, American Type Culture Collection) cell lines were cultured and proteins prepared as previously described (28.Hornburg D. Drepper C. Butter F. Meissner F. Sendtner M. Mann M. Deep proteomic evaluation of primary and cell line motoneuron disease models delineates major differences in neuronal characteristics.Mol. Cell. Proteomics. 2014; 13: 3410-3420Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Briefly, the cells were lysed in lysis buffer (4% SDS, 10 mm Hepes, pH 8.0) during sonication for 15 min (level 5, Bioruptor; Diagenode, Seraing (Ougrée) - Belgium). Cell lysis was followed by reduction of disulfide bonds with 10 mm DTT for 30 min and subsequent alkylation with 55 mm IAA for 45 min. To remove the detergent, cold acetone (−20 °C) was added to 100 μg of proteins to a final concentration of 80% v/v, and proteins were precipitated for at least 2 h at −20 °C. The suspension was centrifuged for 15 min (4 °C, 16,000 × g) and the precipitate was washed with 80% acetone (−20 °C) prior to re-suspension in 50 μl of 6 m urea/2 m thiourea, 10 mm Hepes, pH 8.0. An initial digestion step (3 h) was carried out after the addition of 1 μg of LysC, followed by dilution with four volumes of 50 mm ammonium bicarbonate and the final digestion with 1 μg of trypsin overnight at room temperature. The resulting peptide mixtures were desalted on SDB-RPS StageTips (29.Rappsilber J. Mann M. Ishihama Y. Protocol for micro-purification, enrichment, prefractionation, and storage of peptides for proteomics using StageTips.Nature Protoc. 2007; 2: 1896-1906Crossref PubMed Scopus (2589) Google Scholar) and subjected to single shot LC-MS/MS analysis. Cerebellum from a single mouse (strain: C57Bl6) was homogenized in 4% SDS in 100 mm Tris pH 7.6 using a FastPrep 24 homogenizer (MP Biomedicals, Eschwege, Germany), incubated for 10 min at 95 °C and sonicated for 3 min at 30% duty cycle and output control 3 (Branson Ultrasonics sonifier; model 250). To remove the detergent, acetone (−20 °C) was added to a final concentration of 80% v/v and proteins were precipitated overnight at −20 °C. Supernatants were carefully discarded after centrifugation at 1600 × g for 20 min at 4 °C, and the pellets were washed with 80% acetone (−20 °C). The protein pellets were dissolved in 8 m Urea in 10 mm Hepes and protein concentrations were determined by the tryptophan fluorescence emission at 350 nm using an excitation wavelength of 295 nm. Proteins were reduced with 10 mm DTT for 30 min and alkylated with 55 mm iodoacetamide for 20 min. After addition of thiourea to a final concentration of 0.1 m, samples were digested by adding LysC (Wako Chemicals, ratio 1 μg LysC:100 μg sample protein) for 3 h at RT, diluted with four volumes of 50 mm ammonium bicarbonate, and further digested with trypsin (ratio 1 μg trypsin:100 μg sample protein, Promega) at RT overnight. After a further digestion with LysC and trypsin (ratio 1:100) for 8 h at RT, digested peptides were acidified by adding TFA (final concentration: 0.5%) and purified on Sep-Pak tC18 cartridges (Waters) according to manufacturer's instructions. Peptide concentrations were determined using a NanoDrop spectrophotometer. Universal Proteomics Standard (UPS-1, Sigma-Aldrich) and Proteomics Dynamic Range Standard (UPS-2, Sigma-Aldrich), both containing 48 human proteins, either at equimolar concentrations (UPS-1) or formulated into a dynamic range of concentrations, covering five orders of magnitude (UPS-2), were prepared according to ref (30.Wang H. Qian W.J. Mottaz H.M. Clauss T.R. Anderson D.J. Moore R.J. Camp 2nd, D.G. Khan A.H. Sforza D.M. Pallavicini M. Smith D.J. Smith R.D. Development and evaluation of a micro- and nanoscale proteomic sample preparation method.J. Proteome Res. 2005; 4: 2397-2403Crossref PubMed Scopus (144) Google Scholar). Predigested yeast sample (Promega) was re-suspended in 0.1% trifluoroacetic acid to a final concentration of 500 ng/μl. Digested UPS-2 sample was spiked in two different amounts of 250 fmol to 2.5 amol peptide amount for sample 1 and 500 fmol to 5 amol for sample 2 into 500 ng yeast background, thereby creating two samples with a theoretical ratio 1:1 for the yeast proteome and 1:2 for the UPS peptides. In another sample, digested UPS-1 sample (25 fmol for all components) was spiked into 500 ng yeast. We performed high-pH reversed-phase peptide prefractionation with fraction concatenation on 175 μg HeLa or cerebellum peptides on a 2.1 × 300 mm Acquity UPLC Peptide BEH column packed with 130 Å pore, 1.7 μm particle size C18 beads (Part No. 186005792, Waters). A gradient of basic reversed-phase buffers (Buffer A: 0.1% formic acid, ammonium hydroxide pH 10; Buffer B: 0.1% formic acid, 80% acetonitrile, ammonium hydroxide pH 10) was run on a Prominence HPLC system (Shimadzu, Duisburg, Germany) at a flow rate of 150 μl/min at 60 °C. The LC run lasted for 240 min with a starting concentration of 5% buffer B increasing to 30% over the initial 120 min and a further increase in concentration to 60% over 70 min. This elution gradient was followed by a 95% wash and re-equilibration. Fraction collection started after 0.2 ml elution and fractions were collected every 140 s resulting in 72 fractions used for concatenation into 24 fractions as described previously (31.Dwivedi R.C. Spicer V. Harder M. Antonovici M. Ens W. Standing K.G. Wilkins J.A. Krokhin O.V. Practical implementation of 2D HPLC scheme with accurate peptide retention prediction in both dimensions for high-throughput bottom-up proteomics.Anal. Chem. 2008; 80: 7036-7042Crossref PubMed Scopus (138) Google Scholar). In our instrument, in contrast to many other commercial ion source designs, the high voltage for the electrospray (ES) process is applied to the vacuum capillary inlet, whereas the sprayer is kept at ground, which allows for a simpler source design (supplemental Fig. S1A). To electrically decouple the ES voltage and the electrical potential of the vacuum section, we use an inlet capillary made from high resistive glass (∼1GOhm). Positioning the ES voltage at the capillary entrance means that the ions are transported opposite to the electrical gradient by the gas flow (32.Whitehouse C.M. Dreyer R.N. Yamashita M. Fenn J.B. Electrospray interface for liquid chromatographs and mass spectrometers.Anal. Chem. 1985; 57: 675-679Crossref PubMed Scopus (1304) Google Scholar). In this configuration, charged molecules travel somewhat slower than the surrounding gas. According to Bernoulli's law, ions are then focused toward the area of highest gas velocity along the center axis of the capillary. The set-up tends to reduce the contamination of the inner capillary walls (33.Franzen J. Method and device for transport of ions in gas through a capillary.Google Patents. 1998; Google Scholar). The Bruker CaptiveSpray nanoflow ES source is directly attached to the vacuum inlet capillary via a short capillary extension that can be heated using the instrument's drying gas (Supplemental Fig. S1A). The spray tip is automatically mechanically aligned on axis with the capillary inlet without the need for any adjustments. The principle of the CaptiveSpray is a vortex gas (usually air) that sweeps around the emitter spray tip at three different stages. The first one is designed to assist spray formation, the second and third one help to focus the spray plume into the MS inlet capillary. All three flows are created solely by the vacuum of the MS system, which requires that the entire source is vacuum sealed. The spray emitter consists of a 2 cm long, 20 μm ID fused silica capillary. Its tip is etch-tapered, thus the inner diameter remains constant to the very end of the tip making it very robust against clogging. Furthermore, it also allows using the same emitter at flow rates ranging from 50 nl/min to 5 μl/min, thereby supporting a wide range of column types. Fused silica columns, which are often used for proteomics, are typically connected to the emitter via a low dead volume union (supplemental Fig. S1A), which also provides the electrical contact for keeping the electrospray at ground potential. Using the described design, the CaptiveSpray source provides very stable ionization; however, when we initially coupled it to the LC set-up used in the Munich laboratory (17.Nagaraj N. Kulak N.A. Cox J. Neuhauser N. Mayr K. Hoerning O. Vorm O. Mann M. System-wide perturbation analysis with nearly complete coverage of the yeast proteome by single-shot ultra HPLC runs on a bench top Orbitrap.Mol. Cell. Proteomics. 2012; 11M111.013722Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 34.Ishihama Y. Rappsilber J. Andersen J.S. Mann M. Microcolumns with self-assembled particle frits for proteomics.J. Chromatogr. A. 2002; 979: 233-239Crossref PubMed Scopus (260) Google Scholar), we observed broader LC peak elution distributions than we normally do (supplemental Fig. S1B). Furthermore, we wished to incorporate a column oven and pulled tip columns. We therefore constructed a modified source, which keeps the back end of the CaptiveSpray but replaces the front end by the standard set-up used in our department. The modified set-up incorporating the tip column is displayed in supplemental Fig. S1C. The modified design of the column holder allows for exact aligning and fixation of the column inside the CaptiveSpray source. Electrical grounding was applied using a connecting tee at the column head. This setup produced the desired, narrow LC peak distributions (supplemental Fig. S1D) and was used for the proteomic analyses described in this article. We used an Easy nLC-1000 (Thermo Fisher Scientific) on-line coupled to an impact II (Bruker Daltonics) with a CaptiveSpray ion source (Bruker Daltonics). The peptide mixtures (1 μg) were loaded onto an in-house packed column (50 cm, 75 μm inner diameter) filled with C18 material (ReproSil-Pur C18 AQ 1.9 μm reversed phase resin, Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). Chromatographic separation was carried out using a linear gradient of 5–30% buffer B (80% ACN and 0.1% FA) at a flow rate of 250 nl/min over 90 min. Because of loading and washing steps, the total time for an LC-MS/MS run was about 40 to 50 min longer. Generally, LC-MS/MS data were acquired using a data-dependent auto-MS/MS method selecting the 17 most abundant precursor ions in cycle for fragmentation and an MS/MS summation time adjusted to the precursor intensity (Compass 1.8 acquisition and processing software, Bruker Daltonics). For the deep proteome measurements of a cell line in combination with peptide fractionation, we used a “dynamic method,” with a fixed cycle time of 3 s. The mass range of the MS scan was set to extend from m/z 150 to 1750. Dynamic exclusion duration was 0.4 min. Isolation of precursor ions was performed using an m/z dependent isolation window of 1.5–5 Th. The collision energy was adjusted between 23–65 eV as a function of the m/z value. For the quantitative analysis of the UPS standards in yeast we used a trapping column set-up (" @default.
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• W2113550007 title "The Impact II, a Very High-Resolution Quadrupole Time-of-Flight Instrument (QTOF) for Deep Shotgun Proteomics *" @default.
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