The peptides were analyzed using an ULTRAFLEX II (Bruker Daltonics, Bremen, Germany) MALDI-TOF/TOF mass spectrometer in positive ion reflector mode. 0.4 µL of the sample was mixed with 0.4 µL alpha-cyano-4-hydroxycinnamic acid (Bruker Daltonics) matrix solution (10 mg mL−1 in 0.1% trifluoroacetic acid/acetonitrile) in a 1:1 ratio and spotted onto a MALDI plate. For some peptides, the samples were purified using a µ-C18 ZipTip (Millipore, Billerica, MA, USA) and directly spotted onto the MALDI plate with 0.6 µL alpha-cyano-4-hydroxycinnamic acid. The mass spectra were internally calculated from the raw mass spectra using the SNAP algorithm in FlexAnalysis 2.4 (Bruker Daltonics). Expected masses were calculated using the PeptideSynthetics Peptide mass calculator [42 ].
Alpha cyano 4 hydroxycinnamic acid
Manufactured by Bruker
Sourced in Germany
Alpha-cyano-4-hydroxycinnamic acid is a chemical compound used as a matrix compound in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. It is a common matrix for the analysis of proteins, peptides, and other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Ultraflex 2, by Bruker (2 mentions)
Flexanalysis 2, by Bruker (2 mentions)
C18 ziptip, by Merck Group (1 mentions)
Maldi biotyper microflex system, by Bruker (1 mentions)
Maldi tof ms spectrometer, by Bruker (1 mentions)
Msp 96 polished steel target plate, by Bruker (1 mentions)
Hplc plus, by Merck Group (1 mentions)
Trifluoroacetic acid, by Thermo Fisher Scientific (1 mentions)
Microflex, by Bruker (1 mentions)
Minispin f 45 12 11, by Eppendorf (1 mentions)
Clinprot purification reagent sets, by Bruker (1 mentions)
Mb wcx, by Bruker (1 mentions)
Chymotrypsin, by Roche (1 mentions)
Calcium chloride cacl2, by Merck Group (1 mentions)
Trifluoroacetic acid tfa, by Thermo Fisher Scientific (1 mentions)
Milli q reagent grade system, by Merck Group (1 mentions)
Ammonium acetate, by Merck Group (1 mentions)
Trypsin, by Roche (1 mentions)
Acetonitrile, by Carlo Erba (1 mentions)
Isopropanol, by Carlo Erba (1 mentions)
8 protocols using alpha cyano 4 hydroxycinnamic acid
1
MALDI-TOF/TOF Mass Spectrometry of Peptides
2
Microbial Identification Using MALDI-TOF MS
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The obtained isolates were identified with a MALDI Biotyper Microflex system (Bruker Daltonics, Bremen, Germany) according to the manufacturer’s instructions. The protein extraction method was applied using ethanol/formic acid sample preparation [14 (link)]. 1 µL of the respective protein extract of each isolated colony was added to a spot on the Biotyper steel target plate. After air drying, the samples were overlayed with 1 μL MALDI-matrix solution (alpha-Cyano-4-hydroxycinnamic acid, Bruker Daltonics, Bremen, Germany). After further air drying, the samples were analyzed. The obtained mass spectra were compared against the internal MALDI Biotyper reference libraries: MBT Compass Library, revision F, v. 9, containing 8468 main spectra (MSPs); MBT Filamentous Fungi Library (revision No. 2, containing 468 MSPs); MBT Security Related Library (SR Library, revision No. 1; containing 104 MSPs). Matches with the respective spectra in the databases were displayed as scores ranging from 0.0 to 3.0. Scores ≥ 1.7 indicated a secure genus identification and scores ≥ 2.0 a secure genus and probable species identification [15 (link)].
3
MALDI-TOF MS Analysis of VuEI Peptide
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The sample containing VuEI (16 pmol) was spotted onto a MSP 96 polished steel target plate (Bruker Daltonics, Germany) and air-dried at room temperature. The spots were covered with 2 μL of saturated matrix solution (alpha-cyano-4-hydroxycinnamic acid, Bruker Daltonics, Germany) prepared in 50% acetonitrile HPLC Plus (Sigma Aldrich) and 0.1% trifluoroacetic acid (Pierce Biotechnology, USA). The mass spectra were acquired using MALDI-TOF MS spectrometer in a linear positive mode (Microflex, Bruker Daltonics, Germany). Bacterial test standard (BTS, Bruker) was used for instrument calibration. The mass spectra data was analyzed in a range of 2,000 to 20,000 m/z.
4
Serum Proteome Analysis by MALDI-TOF MS
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Serum samples were separated using MB-WCX chromatography (Bruker Daltonics, Bremen, Germany) according to the manufacturer’s protocol. With the magnet lowered, 5 μl serum samples were diluted in 10 μl binding solution in a standard thin well PCR tube, added to 10 μl of MB-WCX beads and mixed by pipetting up and down. After incubated for 5 min at room temperature, the tube was placed into the magnetic separator and the beads were collected on the wall of the tube until the supernatant was clear (~1 min). The supernatant was then removed carefully and the magnetic beads were washed three times with washing buffer. Next, we eluted the peptide fraction from the magnetic beads with 5 μl of elution solution and 5 μl stabilization buffer. Finally, we spotted 1 μl eluted peptide and 1μl alpha-cyano-4-hydroxycinnamic acid (Bruker Daltonics) in 50% acetonitrile onto MALDI-TOF MS targets, and added 0.5% trifluoroacetic acid twice to the MALDI AnchorChip surface. All samples were spotted in triplicate to evaluate reproducibility.
5
Extracting Intact Plasmodium Parasites from RBCs
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To extract parasites from RBC, 250 µl blood was vortex mixed for 15 s with 250 µl 0.7% saponin in 140 mM phosphate-buffered saline pH 7.4 (PBS) and incubated 5 min at room temperature. After incubation 1500 µl PBS was added and samples were vortex mixed for 15 s and centrifuged for 2 min at 2579 g (6200 RPM, Minispin F-45-12-11, Eppendorf AG, Hamborg, Germany). After centrifugation, the supernatant was removed, and the lid and edges of the sample tube were carefully cleaned with a sterile cotton swap without touching the pellet. The pellet was then resuspended in 2000 µl PBS and samples were centrifuged for 1 min at 12,045 g (13.400 RPM, Minispin F-45-12-11, Eppendorf AG). The resulting pellet was spotted in a thin layer on clean steel target plates using a wooden toothpick and 1 µl matrix (alpha-cyano-4-hydroxycinnamic acid, Bruker Daltonics, Billerica, MA, USA) was applied to each sample spot. Samples were dried at room temperature before further processing. The sample preparation protocol was based on a method described by Baumeister et al. to separate intact Plasmodium parasites from erythrocytes [11 ].
6
Serum Peptide Profiling using MB-WCX
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Serum samples were separated using MB-WCX (ClinProt purification reagent sets; Bruker Daltonics, Bremen, Germany) and a magnetic separator, with the magnet lowered, 5 μL serum samples were diluted in 10 μL binding solution in a standard thin wall PCR tube, added to 10 μL of MB-WCX beads and then carefully mixed using the mixing feature of the robot. After thorough stirring, samples were incubated at room temperature for 5 min, then the tubes were placed into the magnetic separator to collect the beads on the wall of the tube until the supernatant was clear (~1 min). The supernatant was then removed and the magnet was lowered again. Following the stepwise application of sample and MB-WCX separation, we eluted the peptide fraction from the magnetic beads with 5 μL of elution solution and 5 μL of stabilization buffer [6 (link)]. Eluted peptides were spotted onto the MALDI AnchorChip with 1 μL alpha-cyano-4-hydroxycinnamic acid (Bruker Daltonics) in 50% acetonitrile, and 0.5% trifluoroacetic acid was added twice to the MALDI AnchorChip surface. Each sample was spotted in triplicate in order to evaluate reproducibility.
7
MALDI-TOF MS Analysis of Peptides
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MALDI-TOF MS analysis was performed by the Proteomics Service facility at the University of Oslo, Norway, as described previously [25 (link)]. Briefly, the peptides were analyzed using an ULTRAFLEX II (Bruker Daltonics, Bremen, Germany) MALDI-TOF/TOF mass spectrometer in positive ion reflector mode. 0.4 µL sample was mixed with 0.4 µL alpha-cyano-4-hydroxycinnamic acid (Bruker Daltonics) matrix solution (10 mg/mL in 0.1% trifluoroacetic acid/acetonitrile) in a 1:1 ratio and spotted onto a MALDI plate. The mass spectra were internally calculated from the raw mass spectra using the SNAP algorithm in FlexAnalysis 2.4 (Bruker Daltonics) and compared to theoretical masses.
8
Reagents and Materials for Proteomics
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Unless otherwise stated, all chemicals used in this study were obtained from Sigma (St. Louis, MO). Locostatin was purchased from Acros Organics. Ammonium acetate and calcium chloride (CaCl2) were procured from Merck (Darmstadt, Germany). Glycine was purchased from Eurobio (Courtaboeuf, France). Sequencing grade endoproteinases Asp-N, chymotrypsin, and trypsin were from Roche. Alpha-cyano-4hydroxycinnamic acid was obtained from Bruker Daltonics (Bremen, Germany). Acetonitrile and isopropanol were procured from Carlo Erba (Milan, Italy). Formic acid 90% (FA) and trifluoroacetic acid (TFA) were purchased from Fisher Scientific (Illkirch, France). All solvents and buffers were prepared using 18 M ultrapure water (MilliQ reagent grade system, Millipore).
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