4.3. LC-MS/MS Identification and Bioinformatics

For further analysis, only bands and spots present in both SDS-PAGE and immunoblot were chosen. Selected fragments were manually excised from the silver-stained gels and subjected to LC-MS/MS in the Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences (Warsaw, Poland). Samples were subjected to the standard “in-gel digestion” procedure, in which they were first dehydrated with acetonitrile (ACN) and then reduced, alkylated, and digested with trypsin, as previously described by Kordan et al. [91]. Briefly, the gel pieces were first treated with 10 mM DTT in 100 mM NH₄HCO₃ for 30 min at 57 °C and then with 0.5 M iodoacetamide in 100 mM NH₄HCO₃ (45 min in the dark at room temperature). Proteins were digested overnight with 10 ng/μL trypsin in 25 mM NH₄HCO₃ at pH 8.5 (Promega, Madison, WI, USA) at 37 °C. The resulting tryptic peptides were extracted in a solution containing 0.1% formic acid and 2% ACN. Samples were concentrated and desalted on an RP-C18 pre-column (Waters Corporation, Milford, MA, USA), and further peptide separation was achieved on a nano-ultra performance liquid chromatography (UPLC) RP-C18 column (Waters, BEH130 C18 column, 75 μm i.d., 250 mm long) of a nano ACQUITY UPLC system, using a 45-min linear acetonitrile gradient. The column outlet was directly coupled to the electrospray ionization (ESI) ion source of the Orbitrap Velos type mass spectrometer (Thermo Fisher Scientific), working in the regime of data-dependent MS to MS/MS switch with high-energy collision dissociation (HCD) type peptide fragmentation. An electrospray voltage of 1.5 kV was used. Raw data files were pre-processed with Mascot Distiller software (version 2.5, Matrix Science Inc, Boston, MA, USA).

The obtained peptide masses and fragmentation spectra were matched to the National Center Biotechnology Information (NCBI) non-redundant database, with a Filarioidea filter using the Mascot search engine (Mascot Server v. 2.4.1, Matrix Science). The following search parameters were applied: enzyme specificity was set to trypsin, peptide mass tolerance to ±30 ppm, and fragment mass tolerance to ±0.1 Da. The protein mass was left as unrestricted, and mass values as monoisotopic with one missed cleavage was allowed. Alkylation of cysteine by carbamidomethylation as fixed and oxidation of methionine was set as a variable modification.

Multidimensional Protein Identification Technology–type (MudPIT-type) and/or the highest number of peptide sequences were selected. The expected value threshold of 0.05 was used for analysis, which means that all peptide identifications had a <1 in 20 chance of being a random match. Spectra derived from silver-stained gel pieces usually do not contain enough MS/MS fragmentations to calculate a meaningful FDR; therefore, a Mascot score threshold of 30 or above (p < 0.05) was used.

Mascot data analysis resulted in a list of identified proteins for every analyzed sample (gel fragment). Each identified protein was described by multiple parameters: score, matches, sequences, emPAI, and protein sequence coverage. Proteins selected for analysis were with matches and sequences above or equal to 4%, and protein sequence coverage was above or equal to 5%.

The identified proteins were classified according to their predicted molecular function, biological process, and cellular component using the UniProtKB database (http://www.uniprot.org/ (accessed on 20 December 2020), The UniProt Consortium, 2018). In a part of our analysis, we selected the molecules identified by both the 1-DE and 2-DE methods in each of the parasite stages (Table 1).