A custom-built nano electrospray unit (nESI) was coupled to a Maxis Impact HD Q-TOF mass spectrometer (Bruker, Billerica, MA) for all the native mass spectrometry analysis. Quartz glass capillaries (O.D.: 1.0 mm and I.D.: 0.70 mm) were pulled utilizing a P-2000 micropipette laser puller (Sutter Instruments, Novato, CA) and loaded with 10 µL aliquot of the sample solution. Sample solutions consisted of 1–10 µM HMGA2 in 10 mM ammonium acetate solution at physiological pH (pH = 6.7). For the observation of the HMGA2-Ligand complexes, a 1:1, 1:3 and 1:10 ratio of 5 µM concentration of the HMGA2 and Ligand (suramin) were prepared in 10 mM ammonium acetate and let it rest for 10 min prior infusion. A typical nESI source voltage of +/− 600–1200 V was applied between the pulled capillary tips and the MS instrument inlet. Ions were introduced via a stainless-steel tube (1/16 × 0.020″, IDEX Health Science, Oak Harbor, WA) held at room temperature into the TIMS cell. Solvents, methanol, and ammonium acetate salts utilized in this study were analytical grade or better and purchased from Fisher Scientific (Pittsburgh, PA). A Tuning Mix calibration standard (G24221A) was obtained from Agilent Technologies (Santa Clara, CA) and used as received. Mass spectra were processed using Bruker Compass Data Analysis version 5.1 (Bruker Daltonik GmbH).
Tuning mix calibration standard
Manufactured by Agilent Technologies
Sourced in Canada
The Tuning Mix calibration standard is a product offered by Agilent Technologies for laboratory applications. It is designed to provide a reliable and consistent reference for instrument calibration and performance verification. The Tuning Mix contains a pre-determined composition of chemical compounds that can be used to assess the accuracy and precision of analytical equipment.
Automatically generated - may contain errors
Lab products found in correlation
P 2000 micropipette laser puller, by Sutter Instruments (1 mentions)
D glucose 13c6, by Merck Group (1 mentions)
Equisplash lipidomix, by Avanti Polar Lipids (1 mentions)
Ammonium acetate salts, by Thermo Fisher Scientific (1 mentions)
Fl876, by Eurofins (1 mentions)
Labview, by National Instruments (1 mentions)
Infinity icr cell, by Bruker (1 mentions)
5 protocols using tuning mix calibration standard
1
Native MS Analysis of HMGA2-Ligand Complexes
2
Calibration Standards for Lipidomics
3
Synthesis and Characterization of AT-Rich DNA Hairpins
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AT-rich DNA oligomers, FL876 (sequence: 5′-GGATATTGCCCCCGCAATATCC-3′ (C212H270N79O130P21, MW 6655.1561)), FL876T1 (sequence: 5′-GGATATTGCCTCCGCAATATCC-3′ (C213H272N78O131P21, MW 6670.3326), and FL876T2 (sequence: 5′-GGATATTGCCTTCGCAATATCC-3′ (C214H273N77O132P21, MW6685.3440)) were purchased from Eurofins Genomics (Luxembourg, Luxembourg) and used as received (Scheme 1 and Supplementary Scheme S1 ). These three 22 nucleotide DNA hairpins contain a 9-base pair stem including a 5 base pair AT DNA in the middle of the stem. AT-hook peptides 1 (Lys-Arg-Gly-Arg-Gly-Arg-Pro-Arg-Lys), 2 (Pro-Lys-Arg-Pro-Arg-Gly-Arg-Pro-Lys) and 3 (Lys-Arg-Pro-Arg-Gly-Arg-Pro-Arg-Lys-Trp), which correspond to the first, second, and third ‘AT-hook’ motifs of HMGA2, were purchased from Advanced ChemTech Inc. (Louisville, KY) and used as received. Solvents, 1xBPE buffer (6 mM Na2HPO4, 2 mM NaH2PO4, and 1 mM Na2EDTA, pH 7.0), and ammonium acetate salts utilized in this study were analytical grade or better and purchased from Fisher Scientific (Pittsburgh, PA). A Tuning Mix calibration standard (G24221A) was obtained from Agilent Technologies (Santa Clara, CA) and used as received.
4
High-Resolution TIMS-MS Proteomics Workflow
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Experiments were performed using a custom TIMS analyzer coupled to a maXis Impact Q-UHRToF instrument (Bruker Daltonics Inc., Billerica, MA). [47] Briefly, TIMS separation is based on using an electric field to hold the ions stationary against a moving gas flow. [48, 49] The separation in a TIMS cell works by the center of the mass frame using the same principles as in a conventional IMS drift tube. The. Data acquisition was controlled using an in-house software, written in National Instruments Lab VIEW (2012, version 12.0f3) and synchronized with the maXis Impact acquisition program. TIMS separation was performed using nitrogen as a bath gas at 300 K, and typical P1 and P2 values were 2.5 and 0.9 mbar, respectively. [50] An 880 kHz radiofrequency and 200-350 Vpp was applied to the TIMS entrance funnel, analyzer section, and exit funnel. A custom-built, nano electrospray ionization source was coupled to the TIMS-TOF MS and further used for all experiments. A typical source voltage of 600-1200 V (positive ion mode) was applied. Both mass and mobility calibrations were performed using Tuning Mix calibration standard (G24221A, Agilent Technologies, Santa Clara, CA). The TIMS operation was controlled using an in-house software, written in National Instruments Lab VIEW, and synchronized with the maXis Impact Q-ToF acquisition program.
5
Ultrahigh-Resolution Mass Spectrometry of NCS1
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Ultrahigh resolution MS experiments were performed in a Solarix 9.4T FT-ICR MS spectrometer equipped with an infinity ICR cell (Bruker Daltonics Inc., MA) and a custom-built nanoESI source operated in positive ion mode. Small aliquots (5-10 mL) of NCS1 native protein samples were loaded into a glass capillary tip located at the instrument inlet. Typical operating conditions were 1600 V capillary voltage, skimmer 30V, funnel RF amplitude 220 Vpp, transfer line RF 400 Vpp and collision cell RF 1100 Vpp. Broadband MS spectra (600-800 scans) in the mass range 600-3500 Da were collected at 1 MW data acquisition size. External mass calibration was performed using Agilent Tuning Mix calibration standard.
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