Ultimate 3000 nlc

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Ultimate 3000 nLC is a nano-liquid chromatography system designed for high-performance separations of complex samples. It features precise flow control, robust construction, and intuitive software for reliable and reproducible results.

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7 protocols using ultimate 3000 nlc

Comprehensive Proteome Analysis by LC-MS/MS

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We used two main LC‐MS/MS setups: System 1 and System 2. System 1 comprised an Easy nLC‐1000 (Thermo Fisher Scientific) coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Here the peptides (∼1 μg) were initially loaded onto a trap column (Acclaim PepMap 100 precolumn, 75 μm i.d. × 2 cm, C18, 3 μm, 100 Å; ThermoFisher Scientific, San Jose, CA) and then separated on an analytical column (EASY‐Spray column, 75 μm i.d. × 25 cm, PepMap RSLC C18, 2 μm, 100 Å; ThermoFisher Scientific, San Jose, CA). System 2 comprised an Ultimate 3000 nLC (Thermo Scientific, San José, CA, USA, Bremen Germany) coupled to a Q Exactive HF‐X mass spectrometer (Thermo Scientific). For this case, the peptides (∼1 μg) were loaded in a trap column (Acclaim1 PepMap 100 precolumn, 75 μm, 2 cm, C18, 3 μm, 100 Å, Thermo Scientific) and then separated on an analytical column (EASY‐Spray column 25 or 50 cm, 75 μm i.d., PepMap RSLC C18, 2 μm, 100Å, Thermo Scientific). Both systems used a flow rate of 300 nL/min and a water/ACN gradient in 0.1% formic acid and samples were measured in DDA and DIA modes. The DIA‐MS Spectral library was built out of DDA‐LC‐MS/MS analyses of samples from tissue and cultured cell origin, with spiked in iRT peptides (Biognosis AG). This also included the analysis of the mixture of samples previously fractionated by HpH RP‐HPLC.

Betancourt L.H., Gil J., Sanchez A., Doma V., Kuras M., Murillo J.R., Velasquez E., Çakır U., Kim Y., Sugihara Y., Parada I.P., Szeitz B., Appelqvist R., Wieslander E., Welinder C., de Almeida N.P., Woldmar N., Marko‐Varga M., Eriksson J., Pawłowski K., Baldetorp B., Ingvar C., Olsson H., Lundgren L., Lindberg H., Oskolas H., Lee B., Berge E., Sjögren M., Eriksson C., Kim D., Kwon H.J., Knudsen B., Rezeli M., Malm J., Hong R., Horvath P., Szász A.M., Tímár J., Kárpáti S., Horvatovich P., Miliotis T., Nishimura T., Kato H., Steinfelder E., Oppermann M., Miller K., Florindi F., Zhou Q., Domont G.B., Pizzatti L., Nogueira F.C., Szadai L., Németh I.B., Ekedahl H., Fenyö D, & Marko‐Varga G. (2021). The Human Melanoma Proteome Atlas—Complementing the melanoma transcriptome. Clinical and Translational Medicine, 11(7), e451.

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High-Throughput Proteome and Phosphoproteome Analysis

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The proteome and phosphoproteome fractions were analysed by LC/MS/MS using a Thermo Ultimate 3000 nLC coupled to an Exploris480 mass spectrometer (Thermo Scientific). Samples were injected onto an Ion Opticks Aurora C18 column (75 μm internal diameter × 15 cm, 1.6 μm particle size) and separated over a 70- or 100-min method. The gradient for separation consisted of 5–42% mobile phase B at a 250 nl/min flow rate, where mobile phase A was 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in 80% acetonitrile. The Exploris480 was operated in turboTMTpro mode with a cycle time of 3 s. Resolution for the precursor scan (m/z 375–1,400) was set to 60,000 with a automatic gain control (AGC) target set to standard and a maximum injection time set to auto. MS2 scans (30,000 resolution) consisted of higher collision dissociate set to 38; AGC target set to 300%; maximum injection time set to auto; isolation window of 0.7 Da; fixed first mass of 110 m/z.

Wasko U.N., Jiang J., Dalton T.C., Curiel-Garcia A., Edwards A.C., Wang Y., Lee B., Orlen M., Tian S., Stalnecker C.A., Drizyte-Miller K., Menard M., Dilly J., Sastra S.A., Palermo C.F., Hasselluhn M.C., Decker-Farrell A.R., Chang S., Jiang L., Wei X., Yang Y.C., Helland C., Courtney H., Gindin Y., Muonio K., Zhao R., Kemp S.B., Clendenin C., Sor R., Vostrejs W.P., Hibshman P.S., Amparo A.M., Hennessey C., Rees M.G., Ronan M.M., Roth J.A., Brodbeck J., Tomassoni L., Bakir B., Socci N.D., Herring L.E., Barker N.K., Wang J., Cleary J.M., Wolpin B.M., Chabot J.A., Kluger M.D., Manji G.A., Tsai K.Y., Sekulic M., Lagana S.M., Califano A., Quintana E., Wang Z., Smith J.A., Holderfield M., Wildes D., Lowe S.W., Badgley M.A., Aguirre A.J., Vonderheide R.H., Stanger B.Z., Baslan T., Der C.J., Singh M, & Olive K.P. (2024). Tumour-selective activity of RAS-GTP inhibition in pancreatic cancer. Nature, 629(8013), 927-936.

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Proteomic Analysis of Retina Samples

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For proteome analysis, immunoprecipitated proteins were reduced, alkylated and trypsin digested as previously described [57 (link)]. Desalted tryptic peptides were analyzed on an Orbitrap Lumos Tribrid mass spectrometer coupled with a UltiMate 3000-nLC (Thermo Fisher Scientific). Raw data files were processed with Proteome Discoverer (v2.2, Thermo Fisher Scientific) software, using Mascot v2.5.1 (Matrix Sciences) search node for peptide/protein identification. Target Decoy was used to calculate the false discovery rate (FDR) of peptide spectrum matches, set to a p-value <0.05 [58 (link)]. Geneset enrichment analysis with fold change ratio of peptide spectral match counts between KO and WT retina was performed as described in the RNA seq analyses section below.

Hargrove-Grimes P., Mondal A.K., Gumerson J., Nellissery J., Aponte A.M., Gieser L., Qian H., Fariss R.N., Bonifacino J.S., Li T, & Swaroop A. (2020). Loss of endocytosis-associated RabGEF1 causes aberrant morphogenesis and altered autophagy in photoreceptors leading to retinal degeneration. PLoS Genetics, 16(12), e1009259.

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FAIMS-Enabled High-Resolution Mass Spectrometry

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Samples were analyzed on a Q Exactive Exploris 480 (Thermo Scientific, Waltham, MA, USA) mass spectrometer equipped with a FAIMSpro differential ion mobility device that was coupled to an UltiMate 3000 nLC (all Thermo Scientific Waltham, MA, USA). Samples were loaded onto a 5 µm PepMap Trap cartridge precolumn (Thermo Scientific, Waltham, MA, USA) and reverse-flushed onto an in-house packed analytical pulled-tip column (30–75 µm I.D., filled with 2.7 µm Poroshell EC120 C18, Agilent, Santa Clara, CA, USA). Peptides were chromatographically separated at a constant flow rate of 300 nL/min and the following gradient: initial 2% B (0.1% formic acid in 80% acetonitrile), up to 6 and in 1 min, up to 32% B in 72 min, up to 55% B within 7.0 min and up to 95% solvent B within 2.0 min, followed by a 6 min column wash with 95% solvent B. The FAIMS pro was operated at −50 compensation voltage and electrode temperatures of 99.5 °C for the inner and 85 °C for the outer electrode.

Meshko B., Volatier T.L., Hadrian K., Deng S., Hou Y., Kluth M.A., Ganss C., Frank M.H., Frank N.Y., Ksander B., Cursiefen C, & Notara M. (2023). ABCB5+ Limbal Epithelial Stem Cells Inhibit Developmental but Promote Inflammatory (Lymph) Angiogenesis While Preventing Corneal Inflammation. Cells, 12(13), 1731.

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LC-MS/MS Proteomic Workflow for DDA and DIA Analysis

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We used two main LC‐MS/MS setups. System 1 comprised an Easy nLC‐1000 (Thermo Fisher Scientific) coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). Here the peptides ( ´∼1 μg) were initially loaded onto a trap column (Acclaim PepMap 100 precolumn, 75 μm i.d. × 2 cm, C18, 3 μm, 100 Å; ThermoFisher Scientific) and then separated on an analytical column (EASY‐Spray column, 75 μm i.d. × 25 cm, PepMap RSLC C18, 2 μm, 100 Å; ThermoFisher Scientific). System 2 comprised an Ultimate 3000 nLC (Thermo Scientific Bremen Germany) coupled to a Q Exactive HF‐X mass spectrometer (Thermo Scientific). For this case the peptides (´∼1 μg) were loaded in a trap column (Acclaim1 PepMap 100 pre‐column, 75 μm, 2 cm, C18, 3 m, 100 Å, Thermo Scientific, San José, CA) and then separated on an analytical column (EASY‐Spray column 25 or 50 cm, 75 μm i.d., PepMap RSLC C18, 2 μm, 100Å, Thermo Scientific). Both systems used a flow rate of 300 nL/min and a water/ACN gradient in 0.1% formic acid and samples were measured in DDA and DIA modes. The DIA‐MS Spectral library was built out of DDA‐LC‐MS/MS analyses of samples from tissue and cultured cell origin, with spiked in iRT peptides (Biognosis AG). This also included the analysis of a mixture of samples previously fractionated by HpH RP‐HPLC.

Betancourt L.H., Gil J., Kim Y., Doma V., Çakır U., Sanchez A., Murillo J.R., Kuras M., Parada I.P., Sugihara Y., Appelqvist R., Wieslander E., Welinder C., Velasquez E., de Almeida N.P., Woldmar N., Marko‐Varga M., Pawłowski K., Eriksson J., Szeitz B., Baldetorp B., Ingvar C., Olsson H., Lundgren L., Lindberg H., Oskolas H., Lee B., Berge E., Sjögren M., Eriksson C., Kim D., Kwon H.J., Knudsen B., Rezeli M., Hong R., Horvatovich P., Miliotis T., Nishimura T., Kato H., Steinfelder E., Oppermann M., Miller K., Florindi F., Zhou Q., Domont G.B., Pizzatti L., Nogueira F.C., Horvath P., Szadai L., Tímár J., Kárpáti S., Szász A.M., Malm J., Fenyö D., Ekedahl H., Németh I.B, & Marko‐Varga G. (2021). The human melanoma proteome atlas—Defining the molecular pathology. Clinical and Translational Medicine, 11(7), e473.

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Quantitative LC-MS/MS Proteomics Protocol

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The 24 fractions were analyzed by LC/MS/MS using an Ultimate 3000 nLC coupled to an Orbitrap Exploris480 mass spectrometer (Thermo Scientific) using a turboTMT method.^{33 (link)} Samples were injected onto an Ion Opticks Aurora C18 column (75 μm id × 15 cm, 1.6 μm particle size) and separated over a 70 min method. The gradient for separation consisted of 5−42% mobile phase B at a 250 nl/min flow rate, where mobile phase A was 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in 80% ACN. The Exploris480 was operated in turboTMT mode with a cycle time of 3 s. Resolution for the precursor scan (m/z 375−1400) was set to 60 000 with a AGC target set to standard and a maximum injection time set to auto. MS2 scans (30 000 resolution) consisted of higher collision dissociate (HCD) set to 38, AGC target set to 300%, maximum injection time set to auto, isolation window of 0.7 Da, and fixed first mass of 110 m/z.

Hanley R.P., Nie D.Y., Tabor J.R., Li F., Sobh A., Xu C., Barker N.K., Dilworth D., Hajian T., Gibson E., Szewczyk M.M., Brown P.J., Barsyte-Lovejoy D., Herring L.E., Wang G.G., Licht J.D., Vedadi M., Arrowsmith C.H, & James L.I. (2023). Discovery of a Potent and Selective Targeted NSD2 Degrader for the Reduction of H3K36me2. Journal of the American Chemical Society, 145(14), 8176-8188.

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Orbitrap Fusion Lumos Tribrid Mass Spectrometry Protocol

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All fractions were analyzed on an Ultimate 3000-nLC coupled to an Orbitrap Fusion Lumos Tribrid instrument (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a nanoelectrospray source. Peptides were separated on an EASY-Spray C18 column (75 μm x 50 cm inner diameter, 2 μm particle size and 100 Å pore size, Thermo Fisher Scientific, Waltham, MA, USA). Peptide fractions were placed in an autosampler and separation was achieved by 125 min gradient from 3-24% buffer B (100% ACN and 0.1% formic acid) at a flow rate of 300 nL/min. An electrospray voltage of 1.9 kV was applied to the eluent via the EASY-Spray column electrode. The Lumos was operated in positive ion data-dependent mode, using Synchronous Precursor Selection (SPS-MS3) (56) . Full scan MS1 was performed in the Orbitrap with a precursor selection range of 380-1,500 m/z at nominal resolution of 1. performed by the quadrupole with 0.7 m/z transmission window, followed by CID fragmentation in the linear ion trap with 35% normalized collision energy in rapid scan mode and parallelizable time option was selected. SPS was applied to co-select 10 fragment ions for HCD-MS 3 analysis.
SPS ions were all selected within the 400-1,200 m/z range and were set to preclude selection of the precursor ion and TMTC ion series (57) . The AGC target and maximum accumulation time were set to 1 x 10

Chung D.J., Madison G.P., Aponte A., Singh K., Li Y., Pirooznia M., Bleck C.K.E., Darmani N.A., & Balaban R.S. (2021). Metabolic design in a model of extreme mammalian metabolism, the North American least shrew (Cryptotis parva).

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