The samples were diluted in 0.1% trifluoroacetic acid (TFA), and 1 µL of each of the samples was spotted in duplicate onto the MALDI target (MTP 384 target polished steel plate; Bruker Daltonics, Bremen, Germany) and air-dried at 25 °C. Then, each spot was overlaid with 1 µL of 2,5-dihydroxy-benzoic acid (DHB) or sinapic acid (SA) matrix solution (20 mg/mL) in 50% acetonitrile and 0.1% TFA and air-dried completely prior to the MALDI–TOF MS measurement on ultrafleXtreme (Bruker Daltonics, Bremen, Germany). Reflector positive mode was used for Van and Hec, and in case of Van/Hec analysis, linear positive mode was used. Each spectrum was taken in the m/z range 500–8000 Da; one sample mass spectrum was averaged from 1000 sub-spectra per spot.
Mtp 384 polished steel target plate
Manufactured by Bruker
Sourced in Germany, United States
The MTP 384 polished steel target plate is a lab equipment product designed for use in MALDI-TOF mass spectrometry applications. It features a 384-well format and a polished steel surface.
Automatically generated - may contain errors
Lab products found in correlation
Trifluoroacetic acid, by Thermo Fisher Scientific (3 mentions)
Sinapinic acid, by Merck Group (2 mentions)
Ultraflextreme maldi tof ms, by Bruker (2 mentions)
Peptide calibration standard 2, by Bruker (2 mentions)
Ultraflextreme, by Bruker (1 mentions)
Rapiflex maldi tof ms, by Bruker (1 mentions)
Ultraflextreme maldi tof mass spectrometer, by Bruker (1 mentions)
Ultraflex 2, by Bruker (1 mentions)
Ammonium phosphate, by Merck Group (1 mentions)
Maldi matrix, by Bruker (1 mentions)
Flexanalysis 3, by Bruker (1 mentions)
Trifluoroacetic acid tfa, by Merck Group (1 mentions)
α cyano 4 hydroxycinnamic acid, by Bruker (1 mentions)
Maldi tof mass spectrometer, by Bruker (1 mentions)
Acetonitrile, by Carl Roth (1 mentions)
Biotools 3, by Bruker (1 mentions)
Ultraflex 2 maldi tof tof instrument, by Bruker (1 mentions)
2 d quant kit, by GE Healthcare (1 mentions)
Ethanol, by Merck Group (1 mentions)
Acetone, by Merck Group (1 mentions)
12 protocols using mtp 384 polished steel target plate
1
MALDI-TOF MS for Van and Hec analysis
2
Serum N-Glycan Analysis by MALDI-TOF MS
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The lyophilized purified mixture of N-glycans was reconstituted in an ultrapure aqueous solution containing 0.1% trifluoroacetic acid (TFA). The matrix, 2,5-dihydroxybenzoic acid, was dissolved in the mixture solution of 50% ACN/50% H2O/0.1% TFA at a final concentration of 10 μg/μL [24 ]. Sodium chloride solution was added to the aforementioned solution to achieve a final concentration of 1 mmol/L.
MS analysis of serum N-glycans was performed using rapifleX MALDI-TOF MS (Bruker Daltonics, Germany) equipped with a 10 kHz smartbeam 3D laser (Nd:YAG, 355 nm) operating at a frequency of 2000 Hz. Before the samples were tested by MS, external mass calibration was performed using bovine albumin digested peptides, with an error value required to be within 10 ppm. The sample and matrix solution were mixed at a volume ratio of 1:1 (v/v), spotted on an MTP 384 target polished steel plate (Bruker Daltonics, Germany), and allowed to dry naturally. MS spectra were acquired in the positive reflection mode with a laser intensity of 60%. The MS scan range was set at 1000–3000 Da. For each sample, three technical replicates were performed separately, and the data were stored for further analysis.
MS analysis of serum N-glycans was performed using rapifleX MALDI-TOF MS (Bruker Daltonics, Germany) equipped with a 10 kHz smartbeam 3D laser (Nd:YAG, 355 nm) operating at a frequency of 2000 Hz. Before the samples were tested by MS, external mass calibration was performed using bovine albumin digested peptides, with an error value required to be within 10 ppm. The sample and matrix solution were mixed at a volume ratio of 1:1 (v/v), spotted on an MTP 384 target polished steel plate (Bruker Daltonics, Germany), and allowed to dry naturally. MS spectra were acquired in the positive reflection mode with a laser intensity of 60%. The MS scan range was set at 1000–3000 Da. For each sample, three technical replicates were performed separately, and the data were stored for further analysis.
3
Protein Molecular Weight Analysis by MALDI-TOF-MS
4
Protein Mass Characterization by MALDI-TOF
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The molecular weights of protein constructs and digested fragments were confirmed using MALDI-TOF mass spectrometry. Samples were spotted on an MTP 384 polished steel target plate (Bruker). Spots consisted of 1 μL of protein solution and 1 μL matrix solution (10 mg/mL sinapinic acid dissolved in 50:50 acetonitrile:water with 0.1% trifluoroacetic acid). Spectra were collected on an Ultraflextreme MALDI-TOF mass spectrometer (Bruker).
5
MALDI-TOF Protein Profiling Protocol
6
MALDI-TOF MS Analysis of Permethylated Glycans
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Permethylated glycans were resuspended in 25 µL of 75% methanol and spotted in a 1:1 ratio with DHB matrix on an MTP 384 polished steel target plate (Bruker Daltonics #8280781) as previously described73 (link). MALDI-TOF MS data was acquired from a Bruker Ultraflex II instrument using FlexControl Software in the reflective positive mode. For N-glycans, a mass/charge (m/z) range of 1000–5000 kD was collected, and for O-glycans, a range of 500–3000 kD. Twenty independent captures (representing 1000 shots each) were obtained from each sample and averaged to create the final combined spectra file. Data was exported in .msd format using FlexAnalysis Software for subsequent annotation. Tandem MS (MS/MS) data were collected using the same instrument for both N- and O-glycans, using the LIFT positive mode, and a +/− 1 Da range from the predicted parent m/z, and again represent the sum of twenty independent captures.
7
MALDI-TOF Analysis of Endopeptidase Digests
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MS-based Endopep analysis was done in MS-positive ion reflector mode utilizing an autoflex speed matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a smartbeam laser. Sample (2 µL) was mixed with 18 µL of MALDI-Matrix (5 mg/mL of α-Cyano-4hydroxycinnamic acid, Bruker Daltonics) in 0.1% trifluoroacetic acid (TFA, Sigma-Aldrich, Seelze, Germany) and 50% acetonitrile (Carl Roth, Karlsruhe, Germany) in HPLC water, 1 mM ammoniumphosphate (Merck, Darmstadt, Germany). Of this mixture 1 µL was spotted onto a MTP 384 polished steel target plate (Bruker Daltonics). For matrix suppression, deflection was set to m/z 500, mass spectra were acquired over the mass range m/z 600 to 4800. External mass calibration was performed with peptide calibration standard II (Bruker Daltonics). Each spectrum represents an average of 4,000 laser shots. Spectra were processed by flexAnalysis 3.4 software (Bruker Daltonics).
8
MALDI-TOF/TOF Peptide Characterization
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1 μl of each sample was spotted on a MTP 384 target plate polished steel (Bruker Daltonics), together with 1 μl of HCCA matrix (cyano-4-hydroxycinnamic acid, 10 mg/ml Sigma Aldrich). Mass spectrometry analyses were performed in positive reflector mode with an UltraFlex II MALDI-TOF/TOF instrument (Bruker Daltonics, Bremen, Germany) equipped with a Smartbeam laser having a repetition rate up to 200 Hz and controlled by FlexControl 3.0 (Build184) software (Bruker Daltonics, Bremen, Germany). The spectra were treated with FlexAnalysis 3.0 (Build 96) software.
A total of 2000 spectra were acquired for each sample. Interesting peptides were fragmented in MS/MS (positive ion mode) and spectra were annotated with Biotools 3.1 (Bruker Daltonics) and Rapid de novo sequencing 3.1 (Bruker Daltonics, Bremen).
A total of 2000 spectra were acquired for each sample. Interesting peptides were fragmented in MS/MS (positive ion mode) and spectra were annotated with Biotools 3.1 (Bruker Daltonics) and Rapid de novo sequencing 3.1 (Bruker Daltonics, Bremen).
9
Mass Spectrometry Sample Preparation
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Research-grade acetonitrile (ACN), trifluoroacetic acid (TFA), ammonium bicarbonate, ammonium acetate, acetone, ethanol, and methanol were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). The 2D Quant Kit was purchased from GE Healthcare (Piscataway, New Jersey, USA). Sequencing Grade Modified Trypsin was purchased from Promega (Madison, Wisconsin, USA). The UltrafleXtreme MALDI-TOF/TOF mass spectrometer and MTP 384 target plate polished steel were acquired from Bruker Daltonics Inc (Billerica, Massachusetts, USA). The following software programs were used: FlexControl Version 3.3, FlexAnalysis Version 3.3, BioTools Version 3.2 (Bruker Daltonics Inc, Billerica, Massachusetts, USA).
10
MALDI-TOF MS Characterization of Creatine Kinase
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For Matrix-Assisted Laser Dissociation-Ionization—Time-Of-Flight mass spectrometry ((MALDI-TOF MS) of creatine kinase digest, 1 μL of digest was manually spotted in triplicate on a polished steel target (MTP 384 Target Plate Polished Steel, Bruker Daltonics GmbH, Bremen, Germany). The matrix α-cyano-4-hydroxy-cinnamic acid (CHCA) was used at 7 mg/mL in water/acetonitrile 50:50 (v/v) with 0.2% trifluoroacetic acid, at a ratio of 1:1. Peptide spectra were acquired on an Autoflex Speed MALDI-TOF/TOF, equiped with a Smartbeam laser, using FlexControl software (Version 3.4; Bruker Daltonics GmbH, Bremen, Germany).
A total of 2500 spectra were accumulated randomly per sample, in triplicate for each technical triplicate. The laser power was constant for all the samples, and the laser focus was set at medium. Ion detection was done in reflectron mode at a mass range of m/z 750–3500. External calibration of spectra was done through the deposition of a peptide standard (Peptide Calibration Standard II, Bruker Daltonics) before each measurement on the same target.
A total of 2500 spectra were accumulated randomly per sample, in triplicate for each technical triplicate. The laser power was constant for all the samples, and the laser focus was set at medium. Ion detection was done in reflectron mode at a mass range of m/z 750–3500. External calibration of spectra was done through the deposition of a peptide standard (Peptide Calibration Standard II, Bruker Daltonics) before each measurement on the same target.
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