MALDI mass spectra were recorded on the Axima Confidence time-of-flight spectrometer (Shimadzu Biotech, Columbia, MD, USA) in the linear mode with the nitrogen laser (λ = 337.1 nm; maximum firing rate, 60 Hz; maximum power, 150 μJ/pulse). Positive ions were recorded and the mass spectra were averaged for approximately 100 shots. The mass range of m/z 500–20,000 was scanned. CHCA (α-cyano-4-hydroxycinnamic acid) was used as the matrix for all measurements. The matrix solution was prepared by dissolving 1 mg of CHCA in 100 μL of a 1:1 (v/v) mixture of acetonitrile and water with 0.1% trifluoroacetic acid (TFA). On the MALDI target plate, 1 μL of sample followed by 2 μL of matrix solution was deposited and dried in air.
Axima confidence
Manufactured by Shimadzu
Sourced in Japan, United Kingdom
The Axima Confidence is a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer designed for analytical applications. It provides accurate mass measurement capabilities for a wide range of analytes, including proteins, peptides, polymers, and small molecules.
Automatically generated - may contain errors
Lab products found in correlation
Alpha cyano 4 hydroxycinnamic acid chca, by Merck Group (1 mentions)
Escherichia coli dh5α, by Thermo Fisher Scientific (1 mentions)
Acetonitrile solution, by Merck Group (1 mentions)
Bovine serum albumin fraction 5, by Carl Roth (1 mentions)
Centrifugal spin columns, by Avantor (1 mentions)
Roti nanoquant, by Carl Roth (1 mentions)
Bacterial 16s rdna pcr kit fast 800, by Takara Bio (1 mentions)
Axima qit, by Shimadzu (1 mentions)
Imaging ms solution analysis software, by Shimadzu (1 mentions)
Imlayer, by Shimadzu (1 mentions)
Dynapro nanostar 2, by Wyatt Technology (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Jem 1400flash microscope, by JEOL (1 mentions)
Rink amide chemmatrix resin, by Merck Group (1 mentions)
6120 quadrupole lc ms system, by Agilent Technologies (1 mentions)
Avance 300, by JEOL (1 mentions)
Ir 660 apparatus, by Agilent Technologies (1 mentions)
Model 341 polarimeter, by PerkinElmer (1 mentions)
Maxis 4g, by Bruker (1 mentions)
1200 series hplc system, by Agilent Technologies (1 mentions)
16 protocols using axima confidence
1
MALDI-TOF Mass Spectrometry Protocol
2
Chemical Synthesis and Purification of Mouse EMRE
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The chemical synthesis of mouse mature EMRE (Ser53‐Asp107) and the purification of the synthesized protein were carried out by the Sapporo Laboratory of Cosmo Bio Co., Ltd. (Tokyo, Japan). Briefly, the Ser53‐Asp107 sequence was synthesized by the Fmoc solid‐phase peptide synthesis method using Liberty Blue (CEM); and the synthesized product was purified by reverse‐phase protein column chromatography. The monoisotopic mass of the purified protein was determined by mass spectrometry (Shimadzu AXIMA Confidence). The observed monoisotopic mass was 6111.28 (theoretical monoisotopic mass, 6109.04). The purified EMRE pellet was resolved in 2% SDS solution, and the concentrations of the EMRE protein solution were determined by the same procedure as that used for the MCU protein solution.
3
MALDI-TOF MS Identification of Bacteria and Yeasts
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Freshly cultured isolates were spotted in duplicates directly onto MALDI TOF target plates. The spots were then overlaid with 1 ul of 25% formic acid and allowed to air dry. They were then overlaid with 1 ul of matrix solution consisting of 40 g of Alpha–cyano-4-hydroxycinnamic acid (CHCA; Sigma-Aldrich, Buchs, Switzerland) in 33% ethanol, 33% deionized water, 33% acetonitrile (ACN) (Sigma-Aldrich) and 3% trifluoroacetic acid (TFA). For the preparation of yeast, a formic acid suspension protocol was used instead of direct smear. A colony of yeast was picked using a 1 ul plastic inoculation loop and suspended in 20 ul of 25% formic acid. One microliter of this suspension was then spotted onto the MALDI plate, allowed to dry and then overlaid with the matrix. The matrix was then allowed to dry in room air.
The MALDI plates were loaded onto the Axima™ Confidence (Shimadzu-Biotech Corp., Kyoto, Japan) mass spectrometer and mass spectra obtained in positive linear mode at a frequency of 50 Hz and within mass range of 3000 Da to 20,000 Da. Each MALDI plate was externally calibrated using a spectra of reference strain of Escherichia coli DH5α (Invitrogen, Carlsbad, USA) that was also spotted onto the plates.
The MALDI plates were loaded onto the Axima™ Confidence (Shimadzu-Biotech Corp., Kyoto, Japan) mass spectrometer and mass spectra obtained in positive linear mode at a frequency of 50 Hz and within mass range of 3000 Da to 20,000 Da. Each MALDI plate was externally calibrated using a spectra of reference strain of Escherichia coli DH5α (Invitrogen, Carlsbad, USA) that was also spotted onto the plates.
4
MALDI-TOF MS Analysis of C5a Peptide Cleavage
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The human C5a peptide substrate (0.6 μg) was dissolved in 50 mM Tris–HCl buffer (pH 7.5, 20 μL) containing 100 mM of NaCl and 5 mM of CaCl2, and different amounts of each recombinant protein (0.02, 0.06, 0.6, 6.0 μg) were added. After incubating at 20 °C for 30 min, 1 μL of the reaction mixture was spotted onto a stainless-steel MALDI-TOF MS plate and then covered with sinapinic acid matrix. The sample plate was dried and then subjected to MS analysis with a MALDI-TOF mass spectrometer (AXIMA Confidence, Shimadzu Co., Manchester, UK) with linear-positive mode ionization.
5
MALDI-TOF MS Analysis of Isolated SBE
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Isolated SBE products were analyzed using the Axima Confidence (Shimadzu Biotech) MALDI-TOF MS instrument in linear negative mode. First, they are dissolved in 1 μL of deionized water, mixed with 1 μL matrix solution, and hand-spotted on an MS sample plate to air-dry. Matrix was composed of 27 mg of 3-hydroxypicolinic acid (Fluka) and 5 mg of ammonium citrate (Fluka) dissolved in 500 μL of 50% acetonitrile solution (Sigma). MS measurement parameters were set to 92~97 unit laser intensity with the ion gate off at the ion chamber voltage 20kV and pulsed extraction at 5700 Da. An accumulated spectrum was generated by measuring five areas of a sample spot with 30 laser shots taken for each area, on average.
6
MALDI-TOF-MS Profiling of Bacterial Isolates
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Samples were analysed in batches using an AXIMA-Confidence (Shimadzu Biotech, Manchester, UK) mass spectrometer. This MALDI-TOF-MS device contained a nitrogen pulsed UV laser with a wavelength of 337 nm as described previously [40] (link). The power of the laser at the laser head used was set to 140 mV. Each profile contained 20 shots, and 100 profiles were collected using a circular raster pattern. The MS was operated in positive ion source and linear TOF was used over the range from 1000 to 80,000 m/z. The collection time for each sample was ∼3 min and each biological sample was analysed four times (technical replicates). A single biological replicate for each of the 34 bacteria was analysed each day, and the analysis time took 5 days of machine time during a 2 week period. The result of this analysis generated 680 MALDI-TOF-MS spectra: 34 bacteria × 5 biological replicates × 4 technical replicates. The MALDI device was calibrated using the protein mixture mentioned above.
7
Protein Characterization by Mass Spectrometry and Gel Electrophoresis
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Protein concentrations were determined according to Bradford (1976 (link)) using Roti®Nanoquant (Carl Roth, Germany) and bovine serum albumin fraction V (Carl Roth, Germany) as a standard and measuring the absorbance ratio 590 nm/450 nm. Matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF-MS) was performed with the Axima confidence instrument (Shimadzu Europe, Duisburg, Germany) as previously described (Falkenberg et al. 2022b (link)). SDS-PAGE analysis was performed as described by Miller et al. (2016 (link)) using an 8–20% (v/v) resolving gel and a 6% (v/v) stacking gel. The sample preparation and electrophoresis were performed as described before (Falkenberg et al. 2022b (link)). For isoelectric focusing–polyacrylamide gel electrophoresis (IEF-PAGE), purified proteases were re-buffered in 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)-NaOH pH 7.0 using centrifugal spin columns (VWR, Radnor, USA) with a molecular mass cut-off of 3 kDa. The SERVALYT™ PRECOTES™ 3-10 gel (SERVA, Heidelberg, Germany) was used according to the manufacturer’s recommendations.
8
MALDI-MS Peptide Profiling Protocol
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Untargeted MALDI‐MS peptide profiling was performed on a Shimadzu Axima Confidence (Kratos, Manchester, UK) mass spectrometer in reflectron‐positive ion mode. For each sample spot, 36 profiles with 50 laser shots/profile and a laser power between 50 and 56 were carried out. The laser repetition rate was set to 50 Hz that together with an ion gate of 800 Da allowed the detection of peptide signals over a mass range of 100–4000 m/z. For peak processing, the peak clean up settings were based on a peak width of five, a Gaussian smoothing filter width of two and a baseline subtraction filter width of six. The peak detection method utilizes a 25%‐centroid arched threshold, an offset of 0.2 mV and a onefold response factor. Monoisotopic peak picking was performed by the Poisson peptide method 29 with a peak picking minimum and maximum mass of 800 and 3900 m/z, respectively. External mass calibration using bovine serum albumin peptides (Life Technologies) as standards was performed every four spots. For the eight BSA peptides (mass range: 927.493–2045.028 m/z) the maximum allowed mass accuracy variation was adjusted to 10 ppm.
9
Bacterial Identification by MALDI-TOF MS
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Each bacterial colony identified from the culture was placed in duplicate on the MALDI-TOF plate target. The direct smear plus formic acid sample preparation method was utilized. Briefly, a colony was transferred from the agar surface to the target plate and was overlaid with 0.5 μl of 25% formic acid solution. After drying, 0.5 ul of a saturated solution of alpha-cyano-4-hydroxycinnamic acid (10mg/ml) diluted in 250 ul solutions comprising of acetonitrile, HPLC grade water, and ethanol in a ratio of 3:3:3 and containing a final concentration of 3% of trifluoroacetic acid was added. The target plate was then analyzed using the MALDI-TOF spectrometry for bacterial identification against the SARAMIS database. Spectra range recording was done in linear mode with the mass ranging between 2000 to 20000 Daltons. Genotyping of the colonies was done by MALDI-TOF MS and the plate was then read using the Shimadzu software (Axima Confidence, Shimadzu, Japan).
10
Bovine Mastitis: Milk Analysis Protocol
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Milk from the affected quarters was collected daily from day 0 to day 3. The somatic cell
count (SCC) was measured immediately after milk collection using the simple somatic cell
analyzer ADAM SCC, (NanoEnTek Inc.Co., Seoul, Korea) and expressed as a linear score
(LS)(LS=log2 (somatic cell count/100) +3). A 100 µL sample of day 0 milk was
applied to a 5% sheep blood agar medium (Trypcase soy 5% sheep blood agar medium (TSS),
Sysmex Biomérieux Co., Ltd., Tokyo, Japan) and incubated at 37°C for 24–48 hr. Bacterial
species were identified either by 16SrDNA sequence analysis or mass spectrometry, and
partial 16S rDNA sequences (approximately 0.8 kbp) were determined using the Bacterial 16S
rDNA PCR Kit Fast (800; Takara Bio Inc., Tokyo, Japan). The sequences were analyzed based
on their homogeneity using the Basic Local Alignment Search Tool (BLAST) program. AXIMA
confidence (Shimadzu, Co., Kyoto, Japan) was used to identify bacteria using
matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF-MS). Data analysis was performed by Saramis software. Whey was centrifuged
(4°C, RCF 3,426 × g, 10 min) to remove milk fat and stored at −80°C until
measurement.
count (SCC) was measured immediately after milk collection using the simple somatic cell
analyzer ADAM SCC, (NanoEnTek Inc.Co., Seoul, Korea) and expressed as a linear score
(LS)(LS=log2 (somatic cell count/100) +3). A 100 µL sample of day 0 milk was
applied to a 5% sheep blood agar medium (Trypcase soy 5% sheep blood agar medium (TSS),
Sysmex Biomérieux Co., Ltd., Tokyo, Japan) and incubated at 37°C for 24–48 hr. Bacterial
species were identified either by 16SrDNA sequence analysis or mass spectrometry, and
partial 16S rDNA sequences (approximately 0.8 kbp) were determined using the Bacterial 16S
rDNA PCR Kit Fast (800; Takara Bio Inc., Tokyo, Japan). The sequences were analyzed based
on their homogeneity using the Basic Local Alignment Search Tool (BLAST) program. AXIMA
confidence (Shimadzu, Co., Kyoto, Japan) was used to identify bacteria using
matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF-MS). Data analysis was performed by Saramis software. Whey was centrifuged
(4°C, RCF 3,426 × g, 10 min) to remove milk fat and stored at −80°C until
measurement.
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