Proteinscape software

SDS-PAGE was carried out with the rPbDrk1 protein. The assumed recombinant protein band was cut out from the gel and subjected to sample preparation as described by Marcos et al. [29 (link)] to then analyzed by LC-MS/MS to confirm the protein identity. Extracted peptides were analyzed with amaZon SL ion trap mass spectrometer (Bruker Daltonics, Billerica, MA, USA) connected to a UFLC system (SHIMADZU-Nexera XR, Kioto, Japan) under control of HyStar software (Bruker Daltonics, Billerica, MA, USA) data acquisition. LC-MS/MS data files were converted to a mascot generic file format using DataAnalysis software (Bruker Daltonics, Billerica, MA, USA). MS data processing was performed with Proteinscape software (version 3.1, Bruker Daltonics, Billerica, MA, USA) using the Mascot algorithm (v2.3, Matrix Science, London, UK) for database searching configured with UniProtKB/Swiss-Protdatabase (https://web.expasy.org/docs/swiss-prot_guideline.html) 28 February 2018.

The acquired raw MS/MS data were processed using ProteinScape software (Bruker) and exported to the mzXML file. PEAKS Studio-X software platform was used for peptide mass fingerprint data search with precursor ion tolerance of 15 ppm and fragment mass tolerance of 0.05 Daltons [36 ]. The data generated for CP and GFC peaks were searched against a database containing all protein entries for Cuscuta in UniProtKB. The score threshold (-10lgP) of > 20, the false discovery rate of < 1%, and carbamidomethyl as fixed while oxidation as the variable modification was selected as database searching parameters in PEAKS-X studio. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE⁵⁷ partner repository with the dataset identifier PXD044708 and 10.6019/PXD044708.

ProteinScape software (Bruker) supported by the Mascot search engine (Matrix Science) was used with the following parameters: SWISS-PROT non-redundant database filtered by Homo sapiens taxonomy, two missed cleavage permission, 50-ppm measurement tolerance.
Carbamidomethylation of cysteines was set as a fixed modification and methionine oxidation was set as a variable modification. Positive identifications were accepted with a Mascot score higher than that corresponding to a P value of 0.05. The quantification of the ratio MetOx-148/Met-148 was performed using the intensity of the peptide K.LSPLGEEMS.D (Flex Analysis, Bruker).

All the MS and MS/MS experiments for peptide identification were performed using a maXis impact™ high resolution qTOF mass spectrometer (Bruker Daltonics) equipped with a Captive Spray (Bruker Michrom) electrospray ionization platform. Separation was performed on a Dionex PepMap C18 column (250mmx75μm, 3 μm particle). The flow rate was set at 300 nL/min. The mobile phases A and B were 0.1% formic acid in water and 0.1% formic acid in 80% ACN, respectively. Positive ions (charge state +1, +2, +3) were generated by the electrospray ionization captive source. The following source settings were used for all subsequent data collection: Drying gas (nitrogen): 3 L/min, Dry temperature: 150°C, Capillary voltage: start at 1500–1700 V, decrease in 50 V steps until signal drops and add 300 V, End plate offset: 0 V.
Survey scans were acquired within a range from 200 to 2000 m/z. The spectrometer sequentially conducted MS/MS (in CID chamber) on the precursor ions (+2 and +3 charge state, excluding +1) detected in the full scan in data independent manner. MS/MS scans were conducted within a range from 25 to 2000 m/z. All MS and MS/MS raw data were acquired in.mgf format using Bruker’s ProteinScape™ software (version 3.0).

Protein identifications were made processing the raw files with the DataAnalysis-otof-default script from the Bruker Compass DataAnalysis software (version 4.4 SR1, Bruker), the Protein Scape software (version 3.1.3 461, Bruker) using Mascot 2.4.1 (Matrix Science): trypsin as the digestion enzyme, two missed cleavages allowed, carbamidomethyl Cys as a fixed modification and oxidation on Met as variable modification. Monoisotopic peptide masses were searched with 7.0 ppm peptide mass tolerance and 0.05 Da fragment mass tolerance. FDR was set to 1% with the peptide decoy and percolator options active. The SwissProt database for Homo sapiens was used. Proteins with Mascot scores > 13 were considered as successful identifications [16 ].

Protein identifications were made processing the raw files with the DataAnalysis-otof-default script from the Bruker Compass DataAnalysis software (version 4.4 SR1, Bruker, Billerica, MA, USA), the Protein Scape software (version 3.1.3 461, Bruker) using Mascot 2.4.1 (Matrix Science, City of Industry, CA, USA): trypsin as the digestion enzyme, two missed cleavages were allowed, carbamidomethyl Cys as a fixed modification and oxidation on Met as variable modification. Monoisotopic peptide masses were searched with 7.0 ppm peptide mass tolerance and 0.05 Da fragment mass tolerance. False discovery rate (FDR) was set to 1% with the peptide decoy and percolator options active. The SwissProt database for Homo sapiens was used. Proteins with Mascot scores >13 were considered as successful identifications [9 (link)].