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Ultimate 3000 rslcnano liquid chromatograph

Manufactured by Thermo Fisher Scientific
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The UltiMate 3000 RSLCnano liquid chromatograph is a high-performance instrument designed for nano-flow liquid chromatography applications. It is capable of delivering precise and reliable liquid flow rates for the separation and analysis of small sample volumes.

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Lab products found in correlation

C18 acclaim pepmap100 precolumn, by Thermo Fisher Scientific (6 mentions) Q exactive plus hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (5 mentions) Easy spray column, by Thermo Fisher Scientific (2 mentions) Reprosil pur c18 aq materials, by Dr. Maisch (2 mentions) Orbitrap fusion lumos tribrid mass spectrometer, by Thermo Fisher Scientific (2 mentions) Proteome discoverer software, by Thermo Fisher Scientific (1 mentions) Ltq orbitrap xl mass spectrometer, by Thermo Fisher Scientific (1 mentions) Xcalibur software, by Thermo Fisher Scientific (1 mentions) Q exactive hf hybrid quadrupole orbitrap, by Thermo Fisher Scientific (1 mentions) Ltq orbitrap velos pro mass spectrometer, by Thermo Fisher Scientific (1 mentions) Rapigest sf, by Waters Corporation (1 mentions)

11 protocols using ultimate 3000 rslcnano liquid chromatograph

1

Ultra-sensitive Peptide Analysis by LC-MS/MS

Cited 2 times
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All peptide samples were analyzed by 1D-LC-MS/MS as previously described (96 (link)), with the modification that a 75-cm analytical column was used. Briefly, an UltiMate 3000 RSLCnano liquid chromatograph (Thermo Fisher Scientific) was used to load peptides with loading solvent A (2% acetonitrile, 0.05% trifluoroacetic acid) onto a 5-mm, 300-μm-internal-diameter (i.d.) C18 Acclaim PepMap100 precolumn (Thermo Fisher Scientific). Since peptide concentrations were very low, complete peptide samples (80 μl) were loaded onto the precolumn. Peptides were eluted from the precolumn onto a 75-cm by 75-μm analytical EASY-Spray column packed with PepMap RSLC C18, 2-μm material (Thermo Fisher Scientific) heated to 60°C. Separation of peptides on the analytical column was achieved at a flow rate of 225 nl min−1 using a 460-min gradient going from 98% buffer A (0.1% formic acid) to 31% buffer B (0.08% formic acid, 80% acetonitrile) in 363 min and then to 50% B in 70 min and to 99% B in 1 min and ending with 26 min 99% B. Eluting peptides were analyzed in a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). Carryover was reduced by running two wash runs (injection of 20 μl acetonitrile) between samples. Data acquisition in the Q Exactive Plus was done as previously described (5 (link)).
Seah B.K., Antony C.P., Huettel B., Zarzycki J., Schada von Borzyskowski L., Erb T.J., Kouris A., Kleiner M., Liebeke M., Dubilier N, & Gruber-Vodicka H.R. (2019). Sulfur-Oxidizing Symbionts without Canonical Genes for Autotrophic CO2 Fixation. mBio, 10(3), e01112-19.
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2

Nanoflow LC-MS/MS Proteomics Analysis

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A nanoLC/MS/MS system consisting of an UltiMate™ 3000 RSLCnano liquid chromatograph and an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) was employed. The purified peptides (250 ng) were injected onto a self-pulled analytical column (150 mm length × 100 μm i.d.) packed with ReproSil-Pur C18-AQ materials (3 μm, Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). A gradient condition with flow rate of 500 nL/min was employed, that is, 5–10% B in 1 min, 10–40% B in 64 min, 40–100% B in 5 min, 100% B for 10 min, and 5% B for 30 min (Solvent A was 0.5% acetic acid, and solvent B was 0.5% acetic acid in 80% acetonitrile). Peptides were ionized at 2400 V. The MS scan range was m/z 300–1500 at a resolution of 120,000 (at m/z 200) at orbitrap using an automatic gain control (AGC) set to 4 × 105 ions and the maximum injection time (IT) set to 50 ms, followed by product ion scans of the 20 most intense precursors for 3 s with 1.6 m/z isolation at quadrupole, normalized HCD collision energy of 30%, and a resolution of 15,000 (at m/z 200), 5 × 104 AGC and 100 ms maximum IT. Dynamic exclusion was applied for 20 s.
Tajima K., Ikeda K., Chang H.Y., Chang C.H., Yoneshiro T., Oguri Y., Jun H., Wu J., Ishihama Y, & Kajimura S. (2019). Mitochondrial lipoylation integrates age-associated decline in brown fat thermogenesis. Nature metabolism, 1(9), 886-898.
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3

Protein Identification by LC-MS/MS

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Trypsin-digested protein samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a Thermo-Dionex UltiMate3000 RSLCnano liquid chromatograph that was connected in line with an LTQ-Orbitrap-XL mass spectrometer equipped with a nanoelectrospray ionization source (Thermo Fisher Scientific, Waltham, MA). Data acquisition was controlled using Xcalibur software (version 2.0.7, Thermo) and data analysis was performed using Proteome Discoverer software (version 1.3, Thermo). LC-MS/MS method details have been published elsewhere [30 (link)].
Liu T.Y., Iavarone A.T, & Doudna J.A. (2017). RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System. PLoS ONE, 12(1), e0170552.
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4

Proteomic Analysis of Microbial Samples by 1D-LC-MS/MS

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Samples were analyzed by 1D-LC–MS/MS as described in Hinzke et al. (2019) [43 (link)]. Replicate samples (e.g., replicate 1 at 1%, 5%, and 10%) were run consecutively followed by two wash runs and a blank run to reduce carryover. For 1D-LC–MS/MS, 0.4 μg (pure culture samples) or 2 μg of peptide (mock community-spike in samples) were loaded onto a 5 mm, 300 μm i.d. C18 Acclaim PepMap 100 precolumn (Thermo Fisher Scientific) using an UltiMate 3000 RSLCnano Liquid Chromatograph (Thermo Fisher Scientific). After loading, the precolumn was switched in line with either a 50 cm × 75 μm (pure culture samples) or a 75 cm × 75 μm (mock community–spike in samples) analytical EASY-Spray column packed with PepMap RSLC C18, 2 μm material. The analytical column was connected via an Easy-Spray source to a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). Peptides were separated on the analytical column using 140 (pure culture samples) or 260 (mock community-spike in) min gradients and mass spectra were acquired in the Orbitrap. The resolution used on the Q Exactive Plus for MS1 scans, which provide the isotope pattern information used by Calis-p, was 70,000.
Kleiner M., Kouris A., Violette M., D’Angelo G., Liu Y., Korenek A., Tolić N., Sachsenberg T., McCalder J., Lipton M.S, & Strous M. (2023). Ultra-sensitive isotope probing to quantify activity and substrate assimilation in microbiomes. Microbiome, 11, 24.
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5

Nanoflow LC-MS/MS Proteomics Analysis

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A nanoLC/MS/MS system consisting of an UltiMate™ 3000 RSLCnano liquid chromatograph and an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) was employed. The purified peptides (250 ng) were injected onto a self-pulled analytical column (150 mm length × 100 μm i.d.) packed with ReproSil-Pur C18-AQ materials (3 μm, Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). A gradient condition with flow rate of 500 nL/min was employed, that is, 5–10% B in 1 min, 10–40% B in 64 min, 40–100% B in 5 min, 100% B for 10 min, and 5% B for 30 min (Solvent A was 0.5% acetic acid, and solvent B was 0.5% acetic acid in 80% acetonitrile). Peptides were ionized at 2400 V. The MS scan range was m/z 300–1500 at a resolution of 120,000 (at m/z 200) at orbitrap using an automatic gain control (AGC) set to 4 × 105 ions and the maximum injection time (IT) set to 50 ms, followed by product ion scans of the 20 most intense precursors for 3 s with 1.6 m/z isolation at quadrupole, normalized HCD collision energy of 30%, and a resolution of 15,000 (at m/z 200), 5 × 104 AGC and 100 ms maximum IT. Dynamic exclusion was applied for 20 s.
Tajima K., Ikeda K., Chang H.Y., Chang C.H., Yoneshiro T., Oguri Y., Jun H., Wu J., Ishihama Y, & Kajimura S. (2019). Mitochondrial lipoylation integrates age-associated decline in brown fat thermogenesis. Nature metabolism, 1(9), 886-898.
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6

Peptide Analysis by 1D LC-MS/MS

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Four hundred nanograms of peptides for each sample was analyzed by one-dimensional liquid chromatography-tandem mass spectrometry (1D LC-MS/MS) as described by Speare et al. (44 (link)). Briefly, peptides were loaded with an UltiMate 3000 RSLCnano liquid chromatograph (Thermo Fisher Scientific) in loading solvent A (2% acetonitrile, 0.05% trifluoroacetic acid) onto a 5-mm, 300-μm ID C18 Acclaim PepMap100 precolumn (Thermo Fisher Scientific). Peptides were separated on the analytical column (75-cm by 75-μm analytical EASY-Spray column packed with PepMap RSLC C18, 2-μm material; Thermo Fisher Scientific) by using a 140-min gradient, and mass spectrometry analyses was performed on a Q Exactive HF hybrid quadrupole Orbitrap (Thermo Fisher Scientific) are described by Speare et al. (44 (link)).
Smith S., Salvato F., Garikipati A., Kleiner M, & Septer A.N. (2021). Activation of the Type VI Secretion System in the Squid Symbiont Vibrio fischeri Requires the Transcriptional Regulator TasR and the Structural Proteins TssM and TssA. Journal of Bacteriology, 203(21), e00399-21.
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7

Mussel Proteome Analysis by 1D-LC-MS/MS

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Samples were analyzed by 1D-LC-MS/MS. For each mussel sample, three technical replicates were run. Two wash runs and one blank run were done between samples to reduce carry over. For each run, 2 to 7 µg of peptide were loaded onto a 5 mm, 300 µm ID C18 Acclaim® PepMap100 pre-column (Thermo Fisher Scientific) using an UltiMateTM 3000 RSLCnano Liquid Chromatograph (Thermo Fisher Scientific), and desalted on the pre-column. After desalting the peptides, the pre-column was switched in line with a 50 cm x 75 µm analytical EASY-Spray column packed with PepMap RSLC C18, 2µm material (Thermo Fisher Scientific), which was heated to 45° C. The analytical column was connected via an Easy-Spray source to a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). Peptides were separated on the analytical column and mass spectra acquired in the Orbitrap as described previously70 (link). Roughly 650,000 MS/MS spectra were acquired per sample (three technical replicates combined).
Rubin-Blum M., Antony C.P., Borowski C., Sayavedra L., Pape T., Sahling H., Bohrmann G., Kleiner M., Redmond M.C., Valentine D.L, & Dubilier N. (2017). Short-chain alkanes fuel mussel and sponge Cycloclasticus symbionts from deep-sea gas and oil seeps. Nature microbiology, 2, 17093.
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8

Mussel Proteome Analysis by 1D-LC-MS/MS

Cited 1 time
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Samples were analyzed by 1D-LC-MS/MS. For each mussel sample, three technical replicates were run. Two wash runs and one blank run were done between samples to reduce carry over. For each run, 2 to 7 µg of peptide were loaded onto a 5 mm, 300 µm ID C18 Acclaim® PepMap100 pre-column (Thermo Fisher Scientific) using an UltiMateTM 3000 RSLCnano Liquid Chromatograph (Thermo Fisher Scientific), and desalted on the pre-column. After desalting the peptides, the pre-column was switched in line with a 50 cm x 75 µm analytical EASY-Spray column packed with PepMap RSLC C18, 2µm material (Thermo Fisher Scientific), which was heated to 45° C. The analytical column was connected via an Easy-Spray source to a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). Peptides were separated on the analytical column and mass spectra acquired in the Orbitrap as described previously70 (link). Roughly 650,000 MS/MS spectra were acquired per sample (three technical replicates combined).
Rubin-Blum M., Antony C.P., Borowski C., Sayavedra L., Pape T., Sahling H., Bohrmann G., Kleiner M., Redmond M.C., Valentine D.L, & Dubilier N. (2017). Short-chain alkanes fuel mussel and sponge Cycloclasticus symbionts from deep-sea gas and oil seeps. Nature microbiology, 2, 17093.
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9

Shotgun Proteomics Analysis by LC-MS/MS

Cited 2 times
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Samples were analyzed by one-dimensional Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS) on a Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific). The LC-MS/MS analysis was done as previously described (Kleiner et al., 2017 (link)). For each run, ∼1200 ng of peptide was loaded onto a 5 mm, 300 μm ID C18 Acclaim PepMap100 pre-column (Thermo Fisher Scientific) using an UltiMateTM 3000 RSLCnano Liquid Chromatograph (Thermo Fisher Scientific). Peptides were then separated on a 50 cm × 75 μm analytical EASY-Spray column packed with PepMap RSLC C18, 2-μm material (Thermo Fisher Scientific) using a 260-min gradient as described in Kleiner et al. (2017) (link). The column was heated to 45°C via an integrated heating module. The analytical column was connected via an EASY-Spray source to the Orbitrap Mass Spectrometer. In between each sample, two washes with acetonitrile and one blank were run to reduce and assess carry over. Eluting peptides were analyzed in the Orbitrap Mass Spectrometer as described by Petersen et al. (2016) (link). Roughly 140,000 MS/MS spectra were acquired per sample run (Supplementary Table 1).
Zorz J.K., Kozlowski J.A., Stein L.Y., Strous M, & Kleiner M. (2018). Comparative Proteomics of Three Species of Ammonia-Oxidizing Bacteria. Frontiers in Microbiology, 9, 938.
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10

Shotgun Proteomics Analysis by LC-MS/MS

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Protein samples were analysed on LC–MS/MS system using an UltiMate 3000 RSLCnano liquid chromatograph (Dionex) connected to LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Scientific). Chromatographic separation was performed on EASY-Spray C18 separation column (25 cm × 75 µm, 3 µm particles, Thermo Scientific) with 2 h long (label free) or 3 h long (label based) 3–36% acetonitrile gradient.
High resolution (30 000 FWHM at 400 m/z) MS spectra were acquired for the 390–1700 m/z interval in an Orbitrap analyser with an AGC target value of 1 × 106 ions and maximal injection time of 100 ms. Low resolution MS/MS spectra were acquired in Linear Ion Trap in a data-dependent manner and the top 10 precursors exceeding a threshold of 10 000 counts and having a charge state of +2 or +3 were isolated within a 2 Da window and fragmented using CID.
Sekelova Z., Stepanova H., Polansky O., Varmuzova K., Faldynova M., Fedr R., Rychlik I, & Vlasatikova L. (2017). Differential protein expression in chicken macrophages and heterophils in vivo following infection with Salmonella Enteritidis. Veterinary Research, 48, 35.
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