Peptides were purified using a JASCO HPLC system with a reverse phase Luna® C18 column (Phenomenex, 5 μm particle size, 100 Å pore size, 150 x 10 mm). A linear gradient of 10 – 60% buffer B (0.1 vol% TFA in MeCN, VWR Chemicals) vs. buffer A (0.1 vol% TFA in H2O) was typically used. The identities of the peptides (with the exception of PPα-ϕNO2) was confirmed by MALDI-TOF mass spectrometry using a Bruker ultrafleXtreme II instrument in reflector mode. Peptides were co-crystallized with dihydroxybenzoic acid matrix (Sigma Aldrich) on a ground steel plate. The identity of PPα-NO2 was confirmed by infusing the sample from an Advion Nanomate Triverser (Nanospray source) at 1.4 kV into a Waters Synapt G2S IMS Q-TOF mass spectrometer. Peptide purities were determined to be >95% by a JASCO analytical HPLC system equipped with a reverse phase Kinetex® C18 analytical column (Phenomenex, 5 μm particles size, 100 Å pore size, 100 x 4.6 mm).
Dihydroxybenzoic acid matrix
Manufactured by Merck Group
Dihydroxybenzoic acid matrix is a laboratory reagent used as a sample preparation material in analytical techniques. It functions as a matrix for the analysis of various compounds using mass spectrometry methods.
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Lab products found in correlation
4 protocols using dihydroxybenzoic acid matrix
1
Peptide Purification and Characterization
2
Lipid A Characterization by MALDI-TOF MS
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Isolation of lipid A molecules and subsequent analysis by negative-ion MALDI-TOF mass spectrometry was performed as previously described (19 (link), 60 (link), 61 (link)). Briefly, Escherichia strains were grown in LB (Oxoid), and the lipid A was purified from stationary cultures using the ammonium hydroxide-isobutyric acid method described earlier (62 (link)). Mass spectrometry analyses were performed on a Bruker autoflex speed TOF/TOF mass spectrometer in negative reflective mode with delayed extraction using as matrix equal volumes of dihydroxybenzoic acid matrix (Sigma-Aldrich) dissolved in (1:2) acetonitrile-0.1% trifluoroacetic acid. The ion-accelerating voltage was set at 20 kV. Each spectrum was an average of 300 shots. A peptide calibration standard (Bruker) was used to calibrate the MALDI-TOF. Further calibration for lipid A analysis was performed externally using lipid A extracted from E. coli strain MG1655 grown in LB medium at 37°C.
3
Lipid A Characterization by MALDI-TOF
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Lipid A was extracted using the ammonium hydroxide/isobutyric acid method described earlier (El Hamidi et al, 2005 ). Negative ion MALDI‐TOF mass spectrometry analysis (Bruker Daltonics) of the samples was undertaken using an equal volume of dihydroxybenzoic acid matrix (Sigma‐Aldrich) dissolved in (1:2) acetonitrile‐0.1% trifluoroacetic acid.
4
Lipid A Profiling by MALDI-TOF Mass Spectrometry
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Isolation of lipid A molecules and subsequent analysis by negative-ion matrix-assisted laser desorption-ionisation time-of-flight (MALDI-TOF) mass spectrometry was performed as previously described [41, 65, 66] . Briefly, K. pneumoniae strains were grown in LB (Oxoid) and the lipid A was purified from stationary cultures using the ammonium hydroxide/isobutyric acid isolation method described earlier [67] . Mass spectrometry analysis were performed on a Bruker autoflex® speed TOF/TOF mass spectrometer in negative reflective mode with delayed extraction using as matrix an equal volume of dihydroxybenzoic acid matrix (Sigma-Aldrich) dissolved in (1:2) acetonitrile-0.1% trifluoroacetic acid. The ion-accelerating voltage was set at 20 kV. Each spectrum was an average of 300 shots. A peptide calibration standard (Bruker) was used to calibrate the MALDI-TOF. Further calibration for lipid A analysis was performed externally using lipid A extracted from Escherichia coli strain MG1655 grown in LB medium at 37°C.
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