Ultraflex 3 maldi tof instrument

Peptide mass mapping spectra were acquired on an Ultraflex III MALDI-TOF instrument in the mass range of 700–4000 Da and calibrated externally using a peptide standard PepMix II (Bruker Daltonics). At least two peptides per sample were selected for MS/MS sequencing using LIFT technology. MS/MS data were searched against the SwissProt20171124 database subset of vertebrate proteins using in-house MASCOT search engine (Matrix Science) with the following search settings; enzyme: trypsin, variable modification: oxidation (M), MS mass tolerance: 20 ppm, MS/MS mass tolerance: 0.6 Da, number of missed cleavages: 1. MS/MS spectra with a MOWSE score over the threshold of 25 (calculated for the settings used) were considered as reliably identified. Peptide sequences of Neumann’s grass rat hemoglobin were derived by de novo MS/MS sequencing. One MALDI peptide mass map (sample EME8) was also measured on a Solarix FT-ICR mass spectrometer (Bruker Daltonics) with mass accuracy below 1 ppm.

The pET28b plasmid was used as expression vector with the cDNA of either DNJ-13, CDC-37 and the respective fragments of CDC-37 (ΔN & ΔC), subcloned after the N-terminal His₆ tag. For expression, transformed E. coli BL21-CodonPlus(DE3)RIL cells were grown to an OD₆₀₀ of 0.6 at 37 °C. Protein production was induced by adding 1 mM isopropyl 1-thio-β-D-galactopyranoside and further incubation at 20 °C. Cells were harvested and subsequently disrupted in a TS 0.75 cell disruption instrument (Constant Systems Ltd., Northants, UK). The His₆-tagged proteins were trapped on a HisTrap FF 5-mL affinity column (GE Healthcare) in 40 mM HEPES/KOH, pH7.5, 20 mM KCl, 1 mM DTT and eluted with buffer containing 300 mM imidazole. ResourceS ion-exchange chromatography and size exclusion chromatography on a 16/60 Superdex 75 HiLoad column (GE Healthcare) were subsequently performed. Proteins were stored and measured in a buffer containing 40 mM HEPES/KOH, pH7.5, 20 mM KCl, 1 mM DTT. The quality of each purified protein was confirmed by SDS-PAGE and mass spectroscopy on a Bruker UltraFlex III MALDI-TOF instrument (Bruker, Massachusetts, USA).

PCR was performed in volumes of 50 μl as described above. Primer pairs PNOC-F/PNOC-R and PCR cycles were performed as described by Haouas et al. [25 (link)] and sequencing was performed as described above. Obtained sequences were submitted to GenBank. A male Ph. mascittii specimen and filtered H₂O were used as negative controls.
MALDI-TOF peptide mass mapping analysis of host-specific hemoglobin peptides was performed according to a protocol by Hlavackova et al. [26 (link)]. Blood from engorged abdomens was digested using trypsin (Promega) and the resulting peptides were mixed with an α-cyano-4-hydroxycinnamic acid matrix (Bruker Daltonics). Peptide mass maps were acquired with an Ultraflex III MALDI-TOF instrument (Bruker Daltonics) and at least two peptides per female were selected for MS/MS sequencing. MS/MS spectra were searched against the SwissProt 2019_05 database subset of vertebrate proteins using an in-house MASCOT search engine (Matrix Science).