A lipid standard solution containing 12.5 µM of Ceramide/Sphingoid internal standard mixture (Avanti Polar Lipids, Alabaster, AL, USA, LM6002) was mixed with freshly prepared DHB matrix solution (10 mg/mL in 100% MeOH and 0.1% TFA) in a 1:1 ratio (v/v). One µL of the mix was spotted onto a MALDI AnchorChip target (Bruker Daltonics) with six technical repeats per day, and the spotted target was allowed to air dry before MALDI-MS analysis. Each MALDI spot was analyzed with the same instrumental method as described for MALDI-MSI, and the spectrum was obtained from the sum of 5 scans in ‘smart walk’ mode with a 500 µm grid width and 10% grid increment.
Maldi anchorchip target
Manufactured by Bruker
Sourced in United States
The MALDI AnchorChip target is a specialized sample preparation device used in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry analysis. Its core function is to provide a uniformly coated and patterned sample surface, which enhances the sensitivity and reproducibility of MALDI-based analyses.
Automatically generated - may contain errors
Lab products found in correlation
Ceramide sphingoid internal standard mixture, by Avanti Polar Lipids (2 mentions)
Flexanalysis 3, by Bruker (2 mentions)
Rapiflex maldi tof tof mass spectrometer, by Bruker (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
Ultraflex 3 maldi tof tof, by Bruker (1 mentions)
Mascot 2, by Matrix Science (1 mentions)
Flexanalysis program, by Bruker (1 mentions)
Microflex lrf mass spectrometer, by Bruker (1 mentions)
6 protocols using maldi anchorchip target
1
MALDI-MS Lipid Standard Analysis
2
MALDI-MS Lipid Standard Analysis
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A lipid standard solution containing 12.5 µM of Ceramide/Sphingoid internal standard mixture (Avanti Polar Lipids, Alabaster, AL, USA, LM6002) was mixed with freshly prepared DHB matrix solution (10 mg/mL in 100% MeOH and 0.1% TFA) in a 1:1 ratio (v/v). One µL of the mix was spotted onto a MALDI AnchorChip target (Bruker Daltonics) with six technical repeats per day, and the spotted target was allowed to air dry before MALDI-MS analysis. Each MALDI spot was analyzed with the same instrumental method as described for MALDI-MSI, and the spectrum was obtained from the sum of 5 scans in ‘smart walk’ mode with a 500 µm grid width and 10% grid increment.
3
Aβ1-28 Peptide Quantification by MALDI-TOF
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Beads were resuspended in 15 μL of SA matrix solution (38 mg/mL in water/ACN/TFA 20/80/2.5 (v/v/v)), and 30 nM Aβ1‐28 was added (internal standard). The sample was vortexed for 1 min and centrifuged for 5 min at ~1,000 g. The supernatant (matrix‐analyte mix) was collected, and 1 μl (9 technical replicates) was applied on a MALDI AnchorChip Target (Bruker Daltonics, Billerica, MA, USA) using dried droplet preparation and air‐dried. All mass spectra were acquired on a rapifleX MALDI‐TOF/TOF mass spectrometer (Bruker Daltonics) equipped with a 10 kHz Smartbeam™ laser using the AutoXecute function of the FlexControl 4.2.
4
In-Gel Trypsin Digestion for MALDI-MS
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All selected spots were manually removed from the gel and transferred to tubes containing 200 µL destaining solution (50% acetonitrile, 25 mM ammonium bicarbonate) for 1 h. The supernatant was removed, and the gel pieces were subjected to two 5-min dehydration steps with acetonitrile. For proteolytic digestion, the spots were rehydrated in 10 µL buffer containing 40 mM ammonium bicarbonate, 10% acetonitrile and 15 ng/µL trypsin (Sequencing Grade Modified Trypsin; Promega, Fitchburg, WI, USA) in an ice-cold bath for 30 min. The digestion was performed at 37°C overnight. The solution containing peptides was mixed (1:1) with 50% acetonitrile and 0.1% trifluoroacetic acid saturated with HCCA matrix (a-cyan-4-hydroxinnamic acid) and spotted onto the MALDI AnchorChip target (Bruker Daltonics, Billerica, MA, USA).
The mass of the digested peptides was determined using a MALDI-ToF/ToF/MS/MS Autoflex II (Bruker Daltonics) mass spectrometer. MALDI mass data were externally calibrated with a peptide calibration standard kit. Internal calibration was performed using trypsin autolysis fragments at an m/z ratio of 842.50 and 2211.10 Da. The analysis and treatment of the spectra were performed using FlexAnalysis 3.0 (Bruker Daltonics).
The mass of the digested peptides was determined using a MALDI-ToF/ToF/MS/MS Autoflex II (Bruker Daltonics) mass spectrometer. MALDI mass data were externally calibrated with a peptide calibration standard kit. Internal calibration was performed using trypsin autolysis fragments at an m/z ratio of 842.50 and 2211.10 Da. The analysis and treatment of the spectra were performed using FlexAnalysis 3.0 (Bruker Daltonics).
5
MALDI-TOF/TOF Proteomic Analysis
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Each protein solution digested with trypsin was mixed 1∶1 with 10 mg/mlof α-cyano-4-hydroxycinnamic acid dissolved in 50% acetonitrile/0.1% trifluoracetic acid, and spotted on AnchorChip MALDI target (Bruker Daltonics GmbH, Bremen, Germany) until dry. Peptides were analyzed by MALDI-TOF/TOF UltraflexIII (Bruker Daltonics) under 20 KV with positive model, and the peak data were transferred to FlexAnalysis™ 3.0 software (Bruker Daltonics) for advanced calculation and calibration. MASCOT 2.2 (Matrix Science) was used to match the peptides with NCBI or Swiss-Prot database for protein identification. The setting was restricted to human taxonomy parameters.Meanwhile, carbamidomethyl cysteine was used as a fixed modification, and oxidized methionine as a variable modification. The probability (P) was based on mowse score calculated from −10×Log (P). Protein score greater than 56 was significant (p<0.05). Moreover, one of the major peptide peaks appearing on the spectrum was used to confirm the identical result by identifying the amino acid sequence, which is called peptide fragment fingerprinting method.
6
MALDI-TOF MS Analysis of Biomolecules
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A sample and NSSA matrix (α-cyano-4-hydroxycoric acid in an aqueous solution of acetonitrile and trifluoroacetic acid) in 1:1 v/v were mixed and applied to the target surface (AnchorChip MALDI Target, Bruker Daltonics GmbH, Fällanden, Switzerland). The sample was dried and analyzed by MALDI-TOF MS in a Microflex LRF mass spectrometer (Bruker Daltonics GmbH) in a linear regimen. The produced mass spectra were analyzed with the FlexControl and FlexAnalysis software. Detectable masses ranged between 2 and 6 kDa. An individual mass spectrum was obtained by 40 laser pulses (60 Hz). The total mass spectrum obtained at 10 target points (400 laser pulses) was used for analysis. The laser power depended on the laser status to achieve optimal resolution and signal intensity. 19 Mass spectrometry strains analysis. Sample preparation was made as described by Hummel 20 . Protein extraction was carried out according to the protocol proposed by Bruker (Bruker Daltonics GmbH). The method of direct application of the culture to the target plate was also used 2 . The results were summarized using the FlexAnalysis program (Bruker Daltonics GmbH).
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