Ultraflex maldi tof tof mass spectrometer

Manually excised gel plugs were subjected to an automated platform for the identification of gel-separated proteins69 as described. An Ultraflex MALDI-TOF-TOF mass spectrometer (Bruker Daltonik) was used to acquire both PMF and fragment ion spectra, resulting in confident protein identifications based on peptide mass and sequence information. Database searches in the Swiss-Prot and NCBI primary sequence database restricted to the taxonomy rattus norvegicus were performed using the MASCOT Software 2.2 (Matrix Science). Carboxamidomethylation of Cys residues was specified as fixed and oxidation of Met as variable modifications. One trypsin missed cleavage was allowed. Mass tolerances were set to 100 ppm for PMF searches and to 100 ppm (precursor ions) and 0.7 Da (fragment ions) for MS/MS ion searches. The minimal requirement for accepting a protein as identified was at least one peptide sequence match above identity threshold in addition to at least 20% sequence coverage in the PMF.

The selected protein spots were manually excised from stained 2-DE gels for mass spectrometric analysis. All the samples were digested by trypsin and then were analyzed by Ultraflex MALDI-TOF/TOF mass spectrometer (Bruker, Bremen, Germany). The analysis was performed under the control of FlexControl™ 3.3 software (Bruker) with external mass calibration. The mass spectrometer was set to perform data acquisition with a selected mass range of 800–3500 m/z. Internal calibration of the standard spectra was performed after every 10 consecutive spectra using Pepmix peptide calibration standards (Bruker Daltonics). The obtained spectrum was analyzed with FlexAnalysis™ 3.3 (Bruker) and Biotool™ 2.2 (Bruker). Peptide mass fingerprinting was searched using the program Mascot (Matrix Science, London, UK) against the NCBI database. Zero–two peptide cleavage sites were set on the Mascot search engine. The mass tolerance was 100 ppm, and MS/MS tolerance was 0.6 Da. Protein scores more than 76 were considered statistically significant (p < 0.05) for peptide mass fingerprinting in MS/MS analysis.

Copper arecoline complex (1:1 molar ratio) mass spectra was recorded on Ultraflex MALDI TOF/TOF mass spectrometer (Bruker Daltonics, GmbH, Bremen, Germany) in the reflection mode using a 90 nsec. time delay, acceleration voltage was 25-kV in the positive ion mode. The system utilizes a 50 Hz pulsed nitrogen laser, emiting at 337 nm and α-Cyano-4-hydroxy cinnamic acid was used as a matrix.

The molecular weight of proteins discovered during RF-HPLC were evaluated by MALDI-TOF MS (Ultraflex MALDI-TOF-TOF mass spectrometer, Bruker Daltonics, Bremen, Germany). Calibration was performed using a ProteoMass peptide and protein MALDI-MS calibration kit (mass range 700–66 000 Da; Sigma–Aldrich). Molecular masses were determined in linear and reflector positive-ion mode. The samples prepared by the dried-droplet method with α-cyano-4-hydroxycinnamic acid (Sigma–Aldrich) as a matrix (10 mg/ml of 50% acetonitrile with 0.1% trifluoroacetic acid).
N-terminal amino acid sequences of the protein specific elicitor fraction V were determined by automated Edman degradation on a model 492 Procise sequencer (Applied Biosystems, Foster City, CA, USA).

The 2-D gel electrophoresis was performed by the EttanIPGphor3 system (GE Healthcare, Chicago, IL, USA). The first dimension was isoelectric focusing (IEF) by using the EthanIPHphor3 system and the range of pH was 3–10 while the second dimension was Ettan DALT Six System (GE Healthcare, USA) for SDS-PAGE. The experiments were performed in triplicates and the 2-D PAGE Naveena et al. protocol was followed [33 (link)]. Proteins spots with good reproducibility were selected for mass spectrometry analysis, excised manually from the gel, destined, washed, and digested with trypsin (Promega, USA). The samples were alkylated, mixed with an equivalent matrix solution (HCCA), and MALDI-TOF-MS/MS analysis was performed using a fuzzy logic feedback control system (Ultraflex MALDI-TOF-TOF mass spectrometer Bruker Karlsruhe, Germany) [29 (link)]. The identification of proteins was performed by searching the MASCOT program (http://www.matrixscience.com, accessed on 29 December 2020) in the non-redundant sequence database of NCBI, and those with identification score <30 were excluded.

To identify the phage structural proteins that intact with Gp255, purified phage virions were separated by 15% SDS-PAGE and transferred onto PVDF membrane (Millpore, Massachusetts, USA). Far western blotting was carried out as described previously (Javed et al., 2015 (link)) by using the biotin-labeled Gp255 at a hybrid concentration of 5 μg/ml. The protein band in the gel corresponding the hybridization band on the PVDF membrane was sliced and in-gel digested, followed by identification with MALDI-TOF-TOF MS (Matrix-assisted Laser Desorption Ionization Tandem Time of Flight Mass Spectrometry) on an Ultraflex MALDI-TOF/-TOF mass spectrometer (Bruker, Karlsruhe, Germany; Lavigne et al., 2006 (link)). To verify the interaction of Gp255 with the identified protein Gp287, purified Gp287 was separated by SDS-PAGE, and Far western blotting was carried out as described above. The Western blotting of Gp287 was performed by using the mouse monoclonal antibody anti-His₆ (Roche, Basel, Switzerland), and biotin-labeled Gp255.

Manually excised gel plugs were subjected to an automated platform for the identification of gel-separated proteins as described recently [20 (link)]. An Ultraflex MALDI-TOF-TOF mass spectrometer (Bruker Daltonik, Bremen, Germany) was used to acquire both peptide mass fingerprinting and fragment ion spectra, resulting in confident protein identifications based on peptide mass and sequence information. Database searches in the Swiss-Prot primary sequence database restricted to the taxonomy Homo sapiens were performed using the MASCOT Software 2.2 (Matrix Science, London, UK). Carboxamidomethylation of Cys residues was specified as fixed modification and oxidation of Met residues as variable modifications. One trypsin-missed cleavage was allowed. Mass tolerances were set to 100 ppm for peptide mass fingerprinting searches and to 100 ppm (precursor ions) and 0.7 Da (fragment ions) for MS/MS ion searches. The minimal requirement for accepting a protein as identified was at least one peptide sequence match above identity threshold in addition to at least 20% sequence coverage in the peptide mass fingerprinting.