The synthesis and characterization of the probes were previously reported by Ahmed et al.25 (link) In brief, Para-isothiocyanatobenzyl-deferoxamine (p-NCS-Bz-DFO; CheMatech, Dijon, France) or fluorescein isothiocyanate (FITC; Sigma-Aldrich, St. Louis, MO, USA) were conjugated to human serum albumin (HSA; Sigma-Aldrich) by adding p-NCS-Bz-DFO or FITC to HSA and stirring at room temperature (RT) and stable pH. The products were purified on a desalting column, and the product was freeze-dried. To conjugate the maleic anhydride (MalA), solid MalA was added while stirring at RT to a solution of HSA-DFO or HSA-FITC at pH 9. The resulting Mal-HSA-DFO was purified by dialysis and the Mal-HSA-FITC was purified on a desalting column. Solutions of the products were sterile filtered and freeze-dried. The approximate average numbers of conjugated FITC, DFO, and Mal groups could be determined by comparisons of the average molecular masses of the different HSA modifications with the molecular mass of unconjugated HSA. The respective masses were analyzed by mass spectrometry (MS) (Bruker ultrafleXtreme MALDI-TOF/TOF with a Smartbeam-II laser (Bruker Daltonik, Bremen, Germany)).
Ultraflextreme maldi tof tof
Manufactured by Bruker
Sourced in Germany, United States
The UltrafleXtreme MALDI-TOF/TOF is a high-performance mass spectrometry instrument designed for advanced protein and peptide analysis. It combines MALDI-TOF and tandem TOF/TOF techniques to provide accurate mass determination and structural characterization of biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Peptide calibration standard 2, by Bruker (5 mentions)
Flexanalysis 3, by Bruker (5 mentions)
Flexcontrol 3, by Bruker (4 mentions)
Nicolet 6700 ft ir, by Thermo Fisher Scientific (3 mentions)
Micro bio spin p 6 gel column, by Bio-Rad (2 mentions)
Imageprep, by Bruker (2 mentions)
Biotools 3, by Bruker (2 mentions)
Ethyl glyoxylate, by Thermo Fisher Scientific (2 mentions)
Toluene, by Sinopharm (2 mentions)
400 mhz spectrometer, by Bruker (2 mentions)
Sep pak c18 cartridge, by Waters Corporation (2 mentions)
C18 ziptip, by Merck Group (2 mentions)
Mbt compass library, by Bruker (2 mentions)
Maldi biotyper compass mbt compass, by Bruker (2 mentions)
Double sided tape, by 3M (2 mentions)
Maldi 7090, by Shimadzu (2 mentions)
Human serum albumin (hsa), by Merck Group (1 mentions)
Smartbeam 2 laser, by Bruker (1 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
5800 maldi tof tof, by AB Sciex (1 mentions)
65 protocols using ultraflextreme maldi tof tof
1
Synthesis and Characterization of Protein Conjugates
2
MALDI-TOF-TOF Mass Spectrometry for Small Molecule Binding
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An AB SCIEX 5800 MALDI TOF-TOF (AB Sciex, Framingham, MA) or a Bruker UltraFleXtreme MALDI TOF-TOF (Bruker Daltonics, Bremen, Germany) was used to acquire data in the 100 to 600 m/z range in reflector positive mode. Specifically, the AB SCIEX 5800 MALDI TOF-TOF mass spectrometer was operated with a 400 Hz YAG laser (power 4000, 600 laser shots). Similarly, for the Bruker UltraFleXtreme MALDI-TOF-TOF mass spectrometer, a 1 kHz laser (66% power, 500 laser shots) was used.
Binding of small molecules was quantitated using the area under the curve (AUC) of the small molecule compound peak and the AUC of the (ethylene glycol) 3 -SH (EG3 thiol) monolayer component with MW 335.2 Da. The compound peak AUC data were normalized to the EG3 thiol monolayer peak AUC, and MS data were analyzed with Genedata Expressionist software (Genedata, Basel, Switzerland) in batch load mode. Compounds were identified and annotated in the spectra based on experimental versus theoretical input mass through database searching by referencing a database file containing compound masses and chemical formulas loaded into Genedata Expressionist. Samples within a given pool of compounds are compared with and without target protein. Compounds with equivalent signal in the presence and absence of protein were identified as nonspecific compound binders and eliminated as hits.
Binding of small molecules was quantitated using the area under the curve (AUC) of the small molecule compound peak and the AUC of the (ethylene glycol) 3 -SH (EG3 thiol) monolayer component with MW 335.2 Da. The compound peak AUC data were normalized to the EG3 thiol monolayer peak AUC, and MS data were analyzed with Genedata Expressionist software (Genedata, Basel, Switzerland) in batch load mode. Compounds were identified and annotated in the spectra based on experimental versus theoretical input mass through database searching by referencing a database file containing compound masses and chemical formulas loaded into Genedata Expressionist. Samples within a given pool of compounds are compared with and without target protein. Compounds with equivalent signal in the presence and absence of protein were identified as nonspecific compound binders and eliminated as hits.
3
MALDI-TOF/TOF Analysis of Zebrafish Larvae
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Saturated solution of sinapinic acid (SA) was prepared in ethanol and 1 µL of the sample was dried on a ground steel MALDI plate. Another saturated solution of SA was prepared in acetonitrile and trifluoroacetic acid (30:70, v/v) and mixed with the zebrafish larvae head homogenate at a 1:1 ratio. In all, 0.5 µL of this mixture was applied to previously dried SA layer. The dried layer was analyzed by a Bruker ultraflextreme MALDI-TOF/TOF in the linear positive mode. The instrument was calibrated using protein calibration standards I and II. A total of 8000 shots were gathered across the sample spots using Flexcontrol software (3.4) in the range of 1–20 kDa. The acquired spectra were processed by baseline subtraction and peak picking using Flexanalysis software (3.4).
4
MALDI-TOF/TOF Mass Spectrometry Protocol
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Sample preparation and matrix coating were performed as previously described (Aiyar et al., 2017 (link)). Samples were analyzed (Aiyar et al., 2017 (link)) in an UltrafleXtreme MALDI TOF/TOF (Bruker Daltonics, Bremen, Germany), in reflector positive mode with the following modifications: 100 – 3000 Da range, 30% laser intensity (laser type 4) and raster width 200 µm. The experiments were repeated three times (2nd and 3rd replicates with 250 µm raster width). Calibration of the acquisition method, spectra procession, visualization, analysis and illustration were performed as described before (Aiyar et al., 2017 (link)). Chemical images were obtained using Median normalization and weak denoising.
5
MALDI-ToF Analysis of Tattooed Skin and Lymph Nodes
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In total 8 skin and 8 lymph node samples of tattooed donors as well as 2 skin and 2 lymph node samples of non-tattooed donors were analyzed. Samples between 50–200 µg were lysed using 1 mg/ml collagenase from Clostridium histolyticum Type IA (Sigma Aldrich, Munich, Germany) with an incubation time of at least 24 hours at 37 °C. Lysates were heat-inactivated at 90–95 °C for at least 12 hours. Precipitated pigment particles were washed twice with PBS. Centrifugation was carried out with 500× g for 10 min. Precipitates were applied as thin films to a ground steel target plate with a plastic pipette tip and measured using an UltrafleXtreme MALDI-ToF/ToF (Bruker Daltonik, Bremen, Germany). Spectra were obtained by averaging 3000 individual spectra, with a laser rate of 1000 Hz in positive reflector mode. The instrument was calibrated prior to each measurement with an external ProteoMass™ MALDI Calibration Kit (Sigma Aldrich, Munich, Germany). Data were processed using the flexControl 3.4 and flexAnalysis 3.4 software (Bruker Daltonik, Bremen, Germany).
6
iTRAQ-based Proteomic Analysis
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For each iTRAQ experiment, 5 μl of the respective KCl fraction was analyzed in duplicate. The samples from each iTRAQ experiment were subjected to nano-LC separation using an EASY-nLC Proxeon (Bruker Daltonics, Germany) coupled to a Proteineer fc II (Bruker Daltonics, Germany) fraction collector, as previously described [28 (link)]. MS/MS analyses were performed using a UltrafleXtreme MALDI-TOF/TOF (Bruker Daltonics, Germany) instrument, as previously described [28 (link)].
7
MALDI-TOF Imaging Protocol for Peptides
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Once re-crystallised, samples were mounted into a slide adaptor bracket and were imaged in an UltraFlextreme MALDI TOF/TOF (Bruker Daltonics Breman Germany) with the following settings: reflector positive mode, laser power—65%, laser attenuation—30%, detector gain—27×, mass range—750–3500 m/z, sample rate/Digitizer—1.25 GS/s, realtime smoothing—Off, smartbeam parameter set—2_small, frequency—1000 Hz, laser Shots—500 and raster width 50 µm. Prior to imaging, a co-mounted serial section was used for on tissue calibration using the same settings: 1 µL of Standard Peptide Mix 2 (Sciex, Framingham, MA, USA) was pipetted onto the tissue surface and allowed to dry. Calibration was performed until a <50 ppm accuracy was achieved.
8
Proteomic Profiling of Crude Venom
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The crude venom extract was solubilised in Acetonitrile: Water mix and filtered using a 0.2 μM filter. ESI-MS (Esquire 3000-plus mass spectrometer- Bruker Daltonics, Germany) was performed on the extract to discover the peptide components in the venom complex (Rajesh, 2015 (link), Rajesh et al., 2019 (link)). The crude venom components were separated in HPLC (Agilent 1100 series) using an analytical HPLC column [Agilent Zorbax analytical C18 column, 150 × 4.6 mm, 5 µm, 90 Å pore size] with a binary gradient solvent system (H2O with 0.1% TFA): (acetonitrile with 0.1% TFA) at a flow rate of 0.2 mL min−1. Data acquisition was performed over m/z 100–2000 in positive ion mode. All MALDI-TOF analysis was performed in Ultraflextreme MALDI-TOF-TOF (Bruker Daltonics, Germany) with CCA being the matrix (Rajesh, 2015 (link), Rajesh et al., 2019 (link)).
9
Synthesis and Purification of 7-EAA Oligonucleotides
10
MALDI-TOF MS Analysis of Extracellular Vesicles
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The sample preparation and MALDI-TOF MS analytical procedures were the same as reported by Zhu et al.20 Briefly, the obtained EVs were deposited on the MALDI target plate (1 μL for each spot). The matrix was dropped onto the dried sample spots to overlay the EVs (1 μL for each spot). After that, the target plate was loaded into a MALDI-TOF MS for measurement. With a laser energy absorption matrix's assistance, a mass spectrometry fingerprint was generated within a few minutes under pulsed laser irradiation. Bruker UltrafleXtreme MALDI TOF/TOF (MA, USA) MS was utilized for protein marker analysis. All measurements were conducted under positive linear mode with 20 kV accelerating voltage. Instrumental parameters were set as mass range m/z 2000–50 000, laser intensity 70%, laser attenuator with 30% offset and 40% range, 300 laser shots accumulation for each spot, 20.0 Hz laser frequency, 20× detector gain, suppress up to 1000 Da, 350 ns pulsed ion extraction.
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