Ultimate 3000 rslcnano liquid chromatography system

Manufactured by Thermo Fisher Scientific

Sourced in United States

The UltiMate 3000 RSLCnano is a liquid chromatography system designed for the separation and analysis of complex samples, with a focus on nano-scale separations. It provides high-performance liquid chromatography capabilities for a wide range of applications, including proteomics, metabolomics, and other analytical techniques that require precise separation and detection of small-volume samples.

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15 protocols using ultimate 3000 rslcnano liquid chromatography system

IgG Glycopeptide Analysis by LC-MS

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The IgG glycopeptide samples were analysed using liquid chromatography coupled to mass spectrometry (LC-MS), in a setup described previously^{41 (link)}. An amount of 2.5 µL of the samples was injected in an Ultimate 3000 RSLCnano liquid chromatography system (Dionex, Sunnyvale, CA). The samples were first washed on an Acclaim PepMap100 C18 trap column (5 mm × 300 µm i.d., Dionex, Sunnyvale, CA), and subsequently separated on an Ascentis Express C18 nanoLC column (50 mm × 75 µm i.d., 2.7 µm HALO fused core particles; Supelco, Bellefonte, PA) with a flow rate of 0.9 µL/min. The following linear gradient was used, with solvent A consisting of 0.1% trifluoroacetic acid and B of 95% acetonitrile (ACN): t = 0, 3% solvent B; t = 2, 6%; t = 4.5, 18%; t = 5, 30%; t = 7, 30%; t = 8, 0%; t = 11, 0%.
Via a sheath-flow electrospray (ESI) interface (Agilent Technologies, Santa Clara, CA), the LC was coupled to a Maxis Impact quadrupole time-of-flight (QTOF)-MS system (micrOTOF-Q; Bruker Daltonics, Bremen, Germany). A sheath-flow consisting of 50% isopropanol, 20% propionic acid (Merck) and 30% MilliQ-purified water was applied at 2 µL/min, and nitrogen gas was applied at 4 L/min. MS1 spectra were acquired with a frequency of 0.5 Hz and within an m/z range of 600-2000. An IgG standard and two blank injections were run in between every 12 runs.

Plomp R., Ruhaak L.R., Uh H.W., Reiding K.R., Selman M., Houwing-Duistermaat J.J., Slagboom P.E., Beekman M, & Wuhrer M. (2017). Subclass-specific IgG glycosylation is associated with markers of inflammation and metabolic health. Scientific Reports, 7, 12325.

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Orbitrap Fusion Lumos Mass Spectrometry

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Gel purified samples were analyzed using an Ultimate 3000 RSLC-Nano liquid chromatography system (Dionex) connected an Orbitrap Fusion Lumos mass spectrometer (Thermo Electron). Additional details regarding the sample processing are provided in the Supplementary Materials and Methods.

Kim D.S., Camacho C.V., Setlem R., Kim K., Malladi S., Hou T.Y., Nandu T., Gadad S.S, & Kraus W.L. (2022). Functional Characterization of lncRNA152 as an Angiogenesis-Inhibiting Tumor Suppressor in Triple-Negative Breast Cancers. Molecular cancer research : MCR, 20(11), 1623-1635.

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Proteomic Characterization via MS/MS

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Gel bands were trypsinized overnight following reduction and alkylation with DTT and iodoacetamide (Sigma-Aldrich). Following solid-phase extraction cleanup with Oasis HLB plates (Waters), the resulting samples were analyzed by LC/MS/MS using an Orbitrap Elite or Q Exactive Plus mass spectrometer (Thermo Electron) coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system (Dionex). Peptides were eluted with a gradient 0%–28% buffer B (80% [v/v] ACN, 10% [v/v] trifluoroethanol, and 0.08% formic acid in water) over 60 min.
Raw MS data files were converted to a peak list format and analyzed using the central proteomics facilities pipeline (CPFP), version 2.0.3 (Trudgian et al. 2010 (link); Trudgian and Mirzaei 2012 (link)). Peptide identification was performed using the X!Tandem (Craig and Beavis 2004 (link)) and open MS search algorithm (OMSSA) (Geer et al. 2004 (link)) search engines against the human protein database from Uniprot, with common contaminants and reversed decoy sequences appended (Elias and Gygi 2007 (link)). The detailed proteomics method is provided in the Supplemental Material.

Akinci B., Sankella S., Gilpin C., Ozono K., Garg A, & Agarwal A.K. (2017). Progeroid syndrome patients with ZMPSTE24 deficiency could benefit when treated with rapamycin and dimethylsulfoxide. Cold Spring Harbor Molecular Case Studies, 3(1), a001339.

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Quantitative LC-MS/MS Analysis of Phosphorylated Peptides

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Peptide and purified PKR protein mixture was diluted to 1 ug/uL in 2% acetonitrile (ACN), 0.1% formic acid, and analyzed by LC/MS/MS, using an Orbitrap Fusion Lumos mass spectrometer (Thermo Electron) coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system (Dionex). Samples were injected onto a 75 μm i.d., 50-cm long EasySpray column (Thermo), and eluted with a gradient from 1%–28% buffer B over 60 min. Buffer A contained 2% (v/v) ACN and 0.1% formic acid in water, and buffer B contained 80% (v/v) ACN, 10% (v/v) trifluoroethanol, and 0.1% formic acid in water. The mass spectrometer operated in positive ion mode with a source voltage of 2.2 kV and an ion transfer tube temperature of 275°C. MS scans were acquired at 120,000 resolution in the Orbitrap and up to 10 MS/MS spectra were obtained in the ion trap for each full spectrum acquired using higher-energy collisional dissociation (HCD) for ions with charges 2–7. Phosphorylated peptides of interest were observed by generating an extracted ion chromatogram corresponding to a 0.01 Da window around the theoretical m/z of the +3 charged monoisotopic mass of the singly phosphorylated peptide of interest.

Zaman A., Wu X., Lemoff A., Yadavalli S., Lee J., Wang C., Cooper J., McMillan E.A., Yeaman C., Mirzaei H., White M.A, & Bivona T.G. (2021). Exocyst protein subnetworks integrate Hippo and mTOR signaling to promote virus detection and cancer. Cell reports, 36(5), 109491.

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Histone H2B Immunoprecipitation and Mass Spectrometry

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MDA-MB-231 cells expressing FLAG-tagged wild-type or mutant histone H2B were seeded at ~5×10⁶ cells per 15 cm diameter plate and cultured as described above. Nucleosome immunoprecipitation was performed as described above. The immunoprecipitated material was electrophoresed on a precast SDS-PAGE gel and the regions containing histone proteins were excised from the gel. The proteins were digested with trypsin and processed for mass spectrometry as described previously (22 (link)). The resulting peptides were injected onto an Orbitrap Fusion Lumos mass spectrometer (Thermo Electron) coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system (Dionex). The samples were eluted with a gradient from 1–28% Buffer B over 90 minutes. Buffer A contained 2% (v/v) ACN and 0.1% formic acid in water, and Buffer B contained 80% (v/v) ACN, 10% (v/v) trifluoroethanol, and 0.1% formic acid in water. MS scans were acquired at a 120,000 resolution in the Orbitrap and up to 10 MS/MS spectra were obtained in the ion trap for each full spectrum acquired using higher-energy collisional dissociation (HCD) for ions with charges 2–7. Raw MS data files were analyzed using Proteome Discoverer v2.2 (Thermo), with peptide identification performed using Sequest HT searching against the Homo sapiens database from UniProt.

Huang D., Camacho C.V., Martire S., Nagari A., Setlem R., Gong X., Edwards A.D., Chiu S.P., Banaszynski L.A, & Kraus W.L. (2022). Oncohistone Mutations Occur at Functional Sites of Regulatory ADP-ribosylation. Cancer research, 82(13), 2361-2377.

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Identification of ERG-Interacting Proteins

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ERG-V5 was ectopically expressed in HEK293T cells, immunoprecipitated with a V5 antibody, and used as bait for pulldown of ERG-binding proteins from VCaP cell lysates. Eluted proteins were separated by SDS-PAGE. For Mass spectrometry, PAGE gel slices were digested with trypsin, and high performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS) analysis of tryptic peptides was performed with a Thermo Fusion Lumos mass spectrometer (Thermo) coupled to an Ultimate 3000 RSLC-Nano liquid chromatography systems (Dionex).

Wang S., Kollipara R.K., Humphries C.G., Ma S.H., Hutchinson R., Li R., Siddiqui J., Tomlins S.A., Raj G.V, & Kittler R. (2016). The ubiquitin ligase TRIM25 targets ERG for degradation in prostate cancer. Oncotarget, 7(40), 64921-64931.

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Quantitative Proteomic Analysis of AR Interactome

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Following treatment of R1-D567 cells with 2Gy RT, cell lysates were incubated with Dynabeads Protein G (Invitrogen) pre-coupled with AR antibody (Sigma) or an irrelevant IgG control, eluted at 95°C, separated by SDS-PAGE, proteolytically digested overnight with trypsin (Promega) following reduction and alkylation with DTT and iodoacetamide (Sigma–Aldrich). Samples then underwent solid-phase extraction cleanup with Oasis HLB plates (Waters) and were analyzed by LC/MS/MS, using an Orbitrap Fusion Lumos (Thermo Electron) coupled to an Ultimate 3000 RSLC-Nano liquid chromatography systems (Dionex). MS operated in positive ion mode with a source voltage of 2.2 kV and capillary temperature of 275°C. MS scans were acquired at 120,000 resolution and up to 10 MS/MS spectra were obtained in the ion trap for each full spectrum acquired using higher- energy collisional dissociation (HCD) for ions with charge 2–7. Dynamic exclusion was set for 25s. Raw MS data files were converted to a peak list format and analyzed using the central proteomics facilities pipeline (CPFP), version 2.0.3(18 (link)). Label-free quantitation of proteins across samples was performed using SINQ normalized spectral index Software (18 (link)).

Yin Y., Li R., Xu K., Ding S., Li J., Baek G., Ramanand S.G., Ding S., Liu Z., Gao Y., Kanchwala M., Li X., Hutchinson R., Liu X., Woldu S., Xing C., Desai N.B., Feng F.Y., Burma S., de Bono J., Dehm S.M., Mani R.S., Chen B.P, & Raj G.V. (2017). Androgen Receptor Variants Mediate DNA Repair Following Radiation in Prostate Cancer. Cancer research, 77(18), 4745-4754.

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Membrane Protein Extraction and Mass Spectrometry

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Cells were subjected to Dounce homogenation on ice in 250 mM sucrose/10 mM Tris pH 7.4 with cOmplete protease inhibitor (Roche). Cell debris was pelleted and the supernatant was spun at 2000 g for 60 min to pellet membrane. Membranes were washed and reconstituted with 4 X Laemmli buffer for SDS-PAGE. Protein bands were excised and digested overnight with trypsin (Pierce) following reduction and alkylation with DTT and iodoacetamide (Sigma-Aldrich). The samples underwent solid-phase extraction cleanup with Oasis HLB plates (Waters) and the resulting samples were analyzed by LC/MS/MS, using an Orbitrap Fusion Lumos mass spectrometer (Thermo Electron) coupled to an Ultimate 3000 RSLC-Nano liquid chromatography systems (Dionex). Raw MS data files were converted to a peak list format and analyzed using the central proteomics facilities pipeline (CPFP), version 2.0.3^{43 (link),44 (link)}. Peptide identification was performed using the X!Tandem^{45 (link)} and open MS search algorithm (OMSSA) search engines against the human protein database from Uniprot, with common contaminants and reversed decoy sequences appended^{46 (link)}. Label-free quantitation of proteins across samples was performed using SINQ normalized spectral index Software^{47 (link)}.

Bricogne C., Fine M., Pereira P.M., Sung J., Tijani M., Wang Y., Henriques R., Collins M.K, & Hilgemann D.W. (2019). TMEM16F activation by Ca2+ triggers plasma membrane expansion and directs PD-1 trafficking. Scientific Reports, 9, 619.

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Mass Spectrometry-based Proteomic Workflow

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Neuronal CM samples were subjected to SDS-PAGE, followed by in-gel trypsin digestion (Shevchenko et al., 2006 (link)). The extracted peptides were analyzed on an Orbitrap Fusion Lumos Tribrid Mass Spectrometer coupled with the UltiMate 3000 RSLCnano liquid chromatography system (Thermo Fisher Scientific). The peptides were loaded on Acclaim PepMap100 Nano-Trap Column (100 μm × 2 cm, Thermo Fisher Scientific). Peptides were resolved at 300-nl/min flow rate using a linear gradient of 10% to 35% solvent B (0.1% formic acid in 95% acetonitrile) over 95 minutes on an EASY-Spray column (50 cm × 75 μm ID, Thermo Fisher Scientific). MaxQuant (v1.5.5.1) software was used for quantitation and identification of proteins from the mass spectrometry data using mouse UniProt database (released on May 2018) with common contaminant proteins (Cox and Mann, 2008 (link)). Search parameters included, a) trypsin as a proteolytic enzyme with up to 2 missed cleavages; b) first search peptide mass error tolerance of 20 ppm and the main search peptide mass error tolerance of 4 ppm; c) fragment mass error tolerance of 20 ppm; d) carbamidomethylation of cysteine (+57.02146 Da) as a fixed modification: e) oxidation of methionine (+15.99492 Da) and protein acetyl (+42.01056 Da) on N terminus as dynamic modifications. Peptides and proteins were filtered at 1% false-discovery rate.

Choi B.R., Cave C., Na C.H, & Sockanathan S. (2020). GDE2-Dependent Activation of Canonical Wnt Signaling in Neurons Regulates Oligodendrocyte Maturation. Cell reports, 31(5), 107540.

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Circadian Proteomic Profiling of Fetal SCN

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Samples (n = 16) of pooled fetal SCN were processed at Laboratory of Mass Spectrometry, Biocev (Prague, Czech Republic). Briefly, total proteins were extracted and digested, and peptides were subsequently analyzed on Orbitrap Fusion Tribrid Mass Spectrometer coupled with UltiMate 3000 RSLCnano liquid chromatography system (Thermo Fisher Scientific, USA). MaxQuant software package v.1.6.10.43 was used to gain final LFQ intensity values of 3,707 unique proteins in all samples; additional 183 peptides were assigned to 2 or more proteins and ca. 1,940 peptides showed undetectable LFQ in one or more samples; these were excluded from the analysis. Normalized data were then evaluated by Biocycle with P < 0.01 required to report potentially significant circadian change in protein levels across time points.

Greiner P., Houdek P., Sládek M, & Sumová A. (2022). Early rhythmicity in the fetal suprachiasmatic nuclei in response to maternal signals detected by omics approach. PLoS Biology, 20(5), e3001637.

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