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10 protocols using ultraflex 3 maldi tof tof instrument

1

MALDI-TOF/TOF Protein Identification Protocol

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The peptide mixture obtained was mixed in 1:1 ratio with 5 mg/ml HCAA in 1:2 ratio of 0.1% TFA and 100% ACN. Two microliters of sample was spotted onto the MALDI plate [(MTP 384 ground steel (Bruker Daltonics, Germany)]. The samples were analyzed on the MALDI-TOF/TOF ULTRAFLEX III instrument (Bruker Daltonics, Germany). FLEX ANALYSIS SOFTWARE (Version 3.3) was used to analyze the molecular weight of 500 laser shots with reflectron ion mode ranging between 500 and 5000 m/z for obtaining the MS-MS data. The masses obtained in the MS-MS were submitted for Mascot search in the “Viridiplantae” database to identify the protein (Koenig et al., 2008 (link)).
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2

MALDI-TOF/TOF Protein Identification

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The sample was mixed with an equal volume of HCCA (α-Cyano-4-hydroxycinnamic acid) matrix (5 mg/mL HCCA 1:2 ratio of 0.1% TFA and 100% ACN) and 1.5 μl of air-dried sample was analyzed by the MALDI TOF/TOF ULTRAFLEX III instrument (Bruker Daltonics, Germany). PEPMIX mixture was used for external calibration from mass range 1046 Da to 3147 Da. Further analysis was performed with Flex Analysis Software (Version 3.3) in reflectron ion mode. The masses obtained in the MS-MS were subjected to Mascot (Version 3.3) search against Swiss-Prot “Homo Sapiens” database for identification of proteins. Identified proteins having a minimum of 2 matching unique peptides were further analyzed. A confidence interval of ≥ 95% was maintained for protein identification.
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3

Chemoselective Conjugation of GBS67

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GBS67 was modified as previously described [34] . Briefly, 1.52 mg of EMCS were dissolved in 50 μL of DMSO, and 11 μL of the prepared mixture (6 eq-v) was added to a solution of GBS67 (327 µL of 18.4 mg/mL stock solution) in 670 μL of 100 mM NaPi 1 mM EDTA pH 8.1. Reaction was incubated for 3 hours at RT. After 3 hours, GBS67-EMCS was purified using 30 kDa Vivaspin filter unit 0.5 mL (x5 cycles) dialysing against 50 mM NaPi, 1 mM EDTA pH 7.5. Protein content was determined by BCA colorimetric assay. The linker/protein molar ratio was determined by MALDI-TOF mass spectrometry analysis run in an UltraFlex III MALDI-TOF/TOF instrument (Bruker Daltonics) in linear mode and with positive ion detection. The samples for analysis were prepared by mixing 2.5 μL of product and 2.5 μL of sinapinic acid matrix. 2.5 μL of mixture was deposited on a samples plate, dried at RTfor 10 min, and subjected to the spectrometer.
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4

Enzymatic Assays for TtAA10 Activity

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TtAA10 activity assays were set up using PASC [prepared according to Wood (1988 ▸ )] or Avicel as the main substrate. First, 1 ml samples were prepared in 50 mM sodium acetate pH 6.0 buffer containing 1 mg ml−1 Avicel or PASC and 1 µM TtAA10. Then, 1 mM ascorbate was used as the electron donor in positive controls. Where TtX183A, TtX183B or TtX183C were used as the electron source, the haem was chemically reduced first by the addition of 1 mM ascorbate, which was subsequently removed by passing the protein down a PD-10 desalting column before the protein was added to assays at 100 µM. Reactions were incubated rotating end-over-end on a tube rotator (Stuart Scientific) overnight at room temperature. Prior to mass-spectrometric analysis, the samples were centrifuged at 10 000g for 1 min to pellet any solid material.
For MALDI-MS measurements, 1 µl of sample was mixed with an equivalent volume of 10 mg ml−1 2,5-di­hydroxy­benzoic acid in 50% aceto­nitrile, 0.1% tri­fluoro­acetic acid on a Bruker SCOUT-MTP 384 target plate. The spotted samples were then dried in air under a lamp before being analysed by mass spectrometry on an Ultraflex III MALDI-TOF/TOF instrument (Bruker), as described by Hemsworth et al. (2014 ▸ ).
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5

MALDI-TOF Mass Spectrometry Analysis of Glycans

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One microliter of reaction supernatant was mixed with an equal volume of 20 mg mL−1 2,5-dihydroxybenzoic acid (DHB) in 50% acetonitrile, 0.1% TFA on a SCOUT-MTP 384 target plate (Bruker). The spotted samples were then dried in a vacuum desiccator before being analyzed by mass spectrometry on an Ultraflex III matrix-assisted laser desorption ionization-time of flight/time of flight (MALDI/TOF-TOF) instrument (Bruker)54 (link).
Sample permethylation was carried out according to Ciucanu and Kerek55 (link). Spotted samples were analyzed by MS using 2,5-DHB matrix with 0.1% TFA on an AB-Sciex 4700 (for MALDI-TOF) and Ultraflex III MALDI/TOF-TOF instrument (Bruker). Data were collected using a 2-kHz smartbeam-II laser and acquired on reflector mode (mass range 300–3000 Da) for MS analysis and on LIFT-CID for MS/MS analysis using argon as collision gas. FlexControl and FlexAnalysis softwares were used for data acquisition and analysis. On average, about 10,000 shots were used to obtain high-enough resolution. MS/MS fragmentation patterns were named according to Domon and Costello56 (link).
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6

MALDI-TOF Mass Spectra Acquisition

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MALDI-TOF mass spectra were recorded by an UltraFlex III MALDI TOF/TOF instrument (Bruker Daltonics, Dresden, Germany) in linear mode and with positive ion detection. Before analysis, the samples (50 μg) were dialyzed against water via Vivaspin, 30 kDa cut-off. Then 2.5 µL of a product sample was mixed with 2.5 µL of sinapic acid matrix. 2.5 μL of each mixture was applied on the sampling plate, dried at room temperature for 10 min and analyzed.
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7

Mass Spectrometry and HPLC Analysis

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Reagents and solvents were purchased from Sigma Aldrich (Taufkirchen, Germany), Glen Research (Sterling, VA, USA), or Alfa Aesar (Thermo Fisher Scientific, Waltham, MA, USA) and were used without further purification. Phospholipids were purchased from Bachem AG (Bubendorf, Switzerland) or Avanti Polar Lipids (Alabaster, AL, USA). Fluorescent dyes were purchased from Sigma Aldrich (Germany). Cell culture media and additives were from Gibco® (Thermo Fisher Scientific, Waltham, MA, USA).
For the mass spectrometry analysis, an Ultraflex III MALDI-TOF-TOF instrument (Bruker Daltonics GmbH, Bremen, Germany) equipped with a Smartbeam 2 laser with a repetition rate up to 200 Hz (500 laser shots) was used. The obtained mass spectrum was analyzed with Flex-analysis version 4.0 software (Bruker Daltonics GmbH).
The HPLC separation and analysis were performed with Waters® Alliance 2695 (Waters® Corporation, Milford, MA, USA) apparatus equipped with the ultraviolet (UV) detector Waters® 486 (Waters Corporation) and analyzed with Millennium 32 software (Waters Corporation). The reverse phase analytical C8 column (Macherey-Nagel, GmbH & Co, KG, Hoerdt, France) was used for all separations.
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8

MALDI-TOF Quantification of Bcl-2 Protein

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MS analysis was performed using a Bruker Ultraflex III MALDI-TOF/TOF instrument or a Bruker Microflex LRF MALDI-TOF instrument. A mixture of angiotensin II, angiotensin I, substance P, bombesin, ACTH clip 1-17, ACTH clip 18-39, and somatostatin 28 was used as peptide calibration standard. Peptide spectra were acquired in reflectron mode. The whole detection region (five pairs of parallel wells shown with brown color in Figure 1a) was manually scanned in order to capture signal from all crystals in the well area. For each detection region, 1000 laser shots at ~35 spots were probed. The detection region was identified via the crystal rim formed by the co-crystallization of MALDI matrix and sample through the camera in the MALDI TOF instrument. Since the peptide peak m/z [M+H]+ 1210.6 (FATVVEELFR) resulted in the largest S/N in the experiments with Bcl-2 protein solution it was chosen as the sample peptide for quantification. An isotope labeled peptide m/z [M+H]+ 1220.6 (FATVVEEL(d10)FR) or m/z 1217.6 (FATVVEEL(13C,15N)FR) with the same sequence was dissolved to a known concentration as the internal standard for quantification. Peak areas of the Bcl-2 digest peptide and the internal standard peptide in the MS spectra were analyzed using OriginPro software to calculate the ratio of the two and quantify Bcl-2.48 (link)-49 (link)
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9

Purification and identification of Pdx1 protein

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The cellular extract was loaded on a 10 ml column of Ni2+-activated Chelating Sepharose FF (GE Heathcare, Italy). Fractions containing Pdx1 protein was analyzed by SDS-PAGE. A Sephadex G-25 column (GE Heathcare, Italy) was employed to remove imidazole and to exchange buffer with PBS. Mass spectrometry analyses were performed after tryptic digestion of the band of 43 kDa isolated by Coomassie blue stained gel. Mass spectra were acquired by Ultraflex III MALDI-TOF-TOF instrument (Bruker-Daltonics, Bremen, Germany), and peptide sites were searched in the NCBI database by MASCOT search engine.
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10

Purification and Characterization of Proteins

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All chemicals were from Merck KGaA (Saint Luis, MI, USA). VersaMax Microplate and SoftMax Pro were from Molecular Devices (San Jose, CA, USA). Bradford assay, Mini-PROTEAN Tetra Cell and Mini-Protean Tricine precast gels were from Bio-Rad (Hercules, CA, USA). Plus DNA Ladder and PageRuler Low range unstained protein ladder were from Life Technologies (Carlsbad, CA, USA). Stericup filters were from Millipore (Burlington, MA, USA). HiTrap SP FF cation exchange column and HiTrapOctyl FF hydrophobic interaction column were from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Vivacel 250 membrane filter were from Sartorius (Gottinga, Germany). Ultraflex III MALDI-TOF/TOF instrument and Flex Analysis software were from Bruker Daltonik GmbH (Bremen, Germany).
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