Mass spectrometra were acquired on a Bruker Autoflex®Speed TOF/TOF Mass Spectrometer (Bruker Daltonics Inc.) in negative reflective mode with delayed extraction. The ion-accelerating voltage was set at 20 kV. Each spectrum was an average of 300 shots. A peptide calibration standard (Bruker Daltonics Inc.) was used to calibrate the Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF), and lipid A extracted from E. coli strain MG1655 grown in LB medium at 37°C.
Peptide calibration standard
Manufactured by Bruker
Sourced in Germany, United States, Japan
The Peptide Calibration Standard is a reference material used to calibrate and verify the performance of mass spectrometry instrumentation. It contains a mixture of synthetic peptides with known molecular weights, enabling accurate mass measurement and calibration of the instrument.
Automatically generated - may contain errors
Lab products found in correlation
Ultraflextreme, by Bruker (7 mentions)
Flexanalysis 3, by Bruker (6 mentions)
Flexcontrol 3, by Bruker (6 mentions)
Flexcontrol software, by Bruker (4 mentions)
Flexanalysis software, by Bruker (4 mentions)
Autoflex 3 smartbeam, by Bruker (3 mentions)
Maldi tof mass spectrometer, by Bruker (2 mentions)
Acrylate peg n hydroxysuccinimide, by JenKem Technology (2 mentions)
Flexcontrol 4, by Bruker (2 mentions)
Smartbeam 3d laser, by Bruker (2 mentions)
Anchorchip target plate, by Bruker (2 mentions)
Norharmane 9h pyrido 3 4 b indole, by Merck Group (2 mentions)
Dihydroxybenzoic acid matrix, by Merck Group (2 mentions)
Autoflex speed tof tof mass spectrometer, by Merck Group (2 mentions)
Build 108, by Bruker (2 mentions)
Mtp anchorchip 800 384 tf, by Bruker (2 mentions)
800 μm anchorchip, by Bruker (2 mentions)
Mascot 2.4.1 search engine, by Matrix Science (2 mentions)
Ultraflextreme maldi tof tof mass spectrometer, by Bruker (2 mentions)
Protein calibration standard 1, by Bruker (2 mentions)
56 protocols using peptide calibration standard
1
Lipid A Extraction from E. coli
2
MALDI-TOF/TOF Mass Spectrometry Protocol
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1 μl of fragments collected from RP-HPLC was sported onto an 800 μm Anchor Chip target plate (Bruker Daltonics, Bremen, Germany) separately and air dried. 1 μl of matrix [alpha-Cyano-4-hydroxycinnamic acid, 0.5 mg/mL in water/acetonitrile/TFA 10/90/0.1] was spotted subsequently and air dried. Mass spectra were acquired on an ultraflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics) operating in reflective positive mode. Instrument settings were set in flexControl software (Version 3.4, Bruker Daltonik GmbH). Sample m/z range was set to 300–4000 Da. 1000 shots were collected for the external calibration and sample measurement. External calibration was performed using a 1:20 dilution of peptide calibration standard (Bruker Daltonics). Laser intensity and detector gain were manually adjusted for optimal resolution. The MS spectra obtained were analysed using the FlexAnalysis software (Version 3.3, Bruker Daltonics) employing smoothing, background subtraction and peak detection algorithms.
3
Protein Identification by MALDI-TOF MS
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CBB-stained proteins were excised from the gels, destained, dried, and rehydrated in 100 μL 50 mmol/L NH4HCO3. A total of 15 μL of 0.1 mg/mL trypsin in 1 mmol/L HCl was added and digestion was performed overnight at 37°C. Peptides were extracted with 20 μL 0.5% Trifluoroacetic acid (TFA) and 0.5 μL of each sample was applied onto the MALDI plate. Matrix-assisted laser desorption/ionization time of fligth mass spectrometry (MALDI-TOF MS) spectra were acquired on an Autoflex apparatus (Bruker Daltonics, Billerica, MA USA). External calibration was performed using Peptide Calibration Standard (Bruker Daltonics) and the trypsin autodigestion products of m/z values 842.5094 and 2211.1046 were used for internal calibration. Proteins were identified as the highest ranked result by searching the databases NCBInr or MSDB, including all species, using the MASCOT search engine (Matrix Science, London, UK). The mass tolerance was of 100 ppm and one missed cleavage was allowed. Carbamidomethylation of cysteines, oxidation of methionine, and acrylamide-modified cysteines were considered for PMF searches. For accepting the identification, the cutoff value for the Probability-Based Mowse score calculated by MASCOT (at P < 0.05) was used. For MS/MS data, the peptide mass tolerance was of 0.5 Da, MS/MS ion mass tolerance of 0.5 Da, allowance of one missed cleavage, and charge state +1.
4
MALDI-TOF Mass Spectrometry Protocol
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A MALDI-TOF
Autoflex Speed Bruker spectrometer was used for MALDI-TOF analysis
using an already published methodology36 (link) with modifications. Samples (2 mg) were dissolved with 0.1% aqueous
TFA and diluted to 1:10 with DHB matrix solution (0.1% TFA). Aqueous
sodium chloride (1 mg/mL, 1 μL) was added to the solution. The
samples were applied to the MALDI plate (duplicate) and analyzed after
drying. The analyses were made in the positive mode, using a peptide
calibration standard (Bruker). Data were processed using mMass 5.5.0
software (Strohalm M.).
Autoflex Speed Bruker spectrometer was used for MALDI-TOF analysis
using an already published methodology36 (link) with modifications. Samples (2 mg) were dissolved with 0.1% aqueous
TFA and diluted to 1:10 with DHB matrix solution (0.1% TFA). Aqueous
sodium chloride (1 mg/mL, 1 μL) was added to the solution. The
samples were applied to the MALDI plate (duplicate) and analyzed after
drying. The analyses were made in the positive mode, using a peptide
calibration standard (Bruker). Data were processed using mMass 5.5.0
software (Strohalm M.).
5
MALDI-MS Analysis of Tryptic Peptides
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Conk‐S3 was digested with sequencing‐grade modified trypsin (Merck, NJ, USA) using an enzyme‐to‐substrate ratio of 1:50 (w/w), in 50 mM ammonium bicarbonate, at 37°C overnight. For reduction of disulphides, peptides were incubated in 50 mM ammonium bicarbonate containing 15 mM DTE (1,4‐dithioerythritol) at 56°C for 45 min, and subsequently, free cysteines were alkylated by addition of iodoacetamide (to 50 mM) and incubated for 30 min in the dark at room temperature. Prior to MALDI‐MS analyses, peptides were desalted and concentrated on a Zip‐tip column containing C18 reversed‐phase material (Merck). MALDI‐MS was performed using a Bruker Autoflex III instrument (Bruker Daltonics, Bremen, Germany). The analysis was performed using a saturated solution of α‐cyano‐4‐hydroxycinnamic acid. The instrument was operated in linear and reflected positive ionization mode and calibrated in the mass range 1000 to 3200 Da using a peptide calibration standard (Bruker Daltonics). The theoretical peptide masses were calculated using the General Protein/Mass Analysis for Windows software (Lighthouse Data, Odense, Denmark).
6
Methylation Profiling of Histone H3 and H1.5 Peptides
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The methylation reactions were performed using the unmodified H3K36 (aa 26-44), H3K36me1 (aa 26-44), or H1.5 (aa 160–176) peptide (4.5 μM) in methylation buffer (50 mM Tris/HCl, pH 9, 5 mM MgCl2 and 1 mM DTT) supplemented with 1 mM unlabeled AdoMet (Sigma-Aldrich) and 6.7 μM NSD1 for 4 h at 37 °C. The reactions were halted by the addition of 0.1% trifluoroacetic acid (TFA). All the samples were cleaned using C18 tips (Agilent Technologies). The eluted samples were spotted onto an anchor chip plate (Bruker-Daltonics) followed by drying. Next, 1 μl of HCCA matrix (0.7 mg/ml α-cyano-4 hydroxycinnamic acid dissolved in 85% acetonitrile, 0.1% TFA, 1 mM ammonium dihydrogen phosphate) was added to the dried sample spots and allowed to dry again. Afterward, the dried spots on the anchor plate were analyzed using an Autoflex Speed MALDI-TOF mass spectrometer (Bruker-Daltonics). The mass spectra were collected using the Flex control software (Bruker-Daltonics). For calibration, the peptide calibration standard (Bruker-Daltonics) with peptides ranging from 700 to 3200 Da was used. The collected spectra were analyzed with Flex analysis software (Bruker-Daltonics).
7
Lipid A Analysis by MALDI-TOF
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Lipid A was precipitated from GMMA using mild-acid hydrolysis and was analyzed with an Ultraflex matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometer (Bruker Daltonics) in negative-ion reflectron mode as reported previously (20 (link)). A peptide calibration standard (Bruker Daltonics) was included in each analysis. The samples and the standard were mixed with Super DHB solution (Sigma-Aldrich), a matrix substance for MALDI-TOF analyses. The m/z ratios were determined by flexAnalysis software in comparison to the peptide standard.
8
MALDI-TOF/MS Peptide Analysis Protocol
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Cell culture media were first desalted and purified by ZipTip C18 pipette tips, following the procedure described in the ZipTip user’s guide. Five microliters of each purified peptide solution were added to 5 μl of α-cyano-4-hydroxycinnamic acid (HCCA) and 1 μl of the resulting mixture was deposited in duplicate on the steel sample holder and allowed to dry. MALDI-TOF/MS measurements were performed using an Ultraflex II MALDI-TOF instrument (BrukerDaltonics, Bremen, Germany), operating in reflectron positive ion mode. Ions were formed using a pulsed UV laser (λ = 337 nm) beam. The instrumental conditions were: IS1 = 25 kV; IS2 = 21.65 kV; reflectron potential = 26.3 kV; delay time = 0 nsec. External mass calibration (Peptide Calibration Standard, BrukerDaltonics) was based on monoisotopic values of [M + H] + of Angiotensin II, AngiotensinI, Substance P, Bombesin, ACTH clip (1–17), ACTH clip (18–39), Somatostatin 28 at m/z 1046.5420, 1296.6853, 1347.7361, 1619.8230, 2093.0868, 2465.1990 and 3147.4714, respectively.
9
Covalent Attachment of RGDS Peptide to PEG Hydrogels
10
Isolation and Characterization of Lipid A from GMMA
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Lipid A was precipitated from GMMA as previously described [25 (link)] using mild acid hydrolysis with 1% acetic acid for 2 h at 100°C. Samples were centrifuged at 14,000 g for 15 min, the pellets resuspended in water, and washed twice with water. The pellets were dried overnight using a Speedvac, resuspended in chloroform-methanol 4:1, and mixed with an equal volume of Super DHB (Sigma-Aldrich) solution in water/acetonitrile 1:1 (vol/vol). Two μL of the mixture were loaded to the target plate (MTP 384 target plate ground steel BC, Bruker Daltonics) and analyzed by Ultraflex MALDI-TOF (Bruker Daltonics) in reflectron ion-negative mode. A Peptide Calibration Standard (Bruker Daltonics), mixed with the Super DHB solution, was included in each analysis. The m/z rations were determined by Flex Analysis software in comparison to the Peptide Standard.
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