Proteinscape 3

All MS/MS data were analyzed using Compass Data Analysis software 4.1 (Bruker, USA) and Proteinscape 3.0 with a glycan search engine (Bruker, USA). GlycomeDB (https://glytoucan.org) was used to identify glycans. The search parameters were as follows: Charge: 1+, 2+, 3+ and 4+; Taxonomy: Homo sapiens; Reducing end: 2AB; H+ up to 5, Na + up to 1 and K+ up to 1; MS tolerance: 0.05 Da; MS/MS Tolerance: 0.1 Da; Score > 20.0; fragmentation coverage> 15.0%; and intensity coverage> 15.0%.

All MS/MS data were analyzed using Compass 1.4, Data Analysis 4.1 bulid 335 (Bruker, USA), and Proteinscape 3.0 (Bruker, USA) for protein search. Ensembl lamprey protein database (www.ensembl.org) and NCBI protein database (www.ncbi.nlm.nih.gov) were used separately to replenish each other for the peptide searching. The search parameters are listed as follows: Fixed Modifications: Carboxymethyl (C) Vari; Modifications: Oxidation (M); Missed Cleavages: 1; Peptide Charge: 1+, 2+ and 3+; Taxonomy: All entries; Peptide Tolerance: 0.1 Da; MS/MS Tolerance: 0.2 Da; Mass: Monoisotopic; Min. Ion Score: 15; Significance: 0.05; Min. Peptide Length: 5. Mascot was used to give every peptide we identified a test of independence, to make sure every peptide’s significance threshold P < 0.05. And the distribution of mass error is near zero and most of them are less than 20 ppm (Additional file 1: Figure S1). The data were gathered in the area-proportional Venn diagrams which depicted the variation in number of shared and distinct buccal gland secretion proteins of lampreys which fed for different times.

Relevant protein bands were cut out and digested in gel. S-alkylation with iodoacetamide and digestion with sequencing grade modified trypsin (Promega) were performed. The peptide mixture was analysed using a Dionex Ultimate 3000 system directly linked to a QTOF instrument (maXis 4G ETD, Bruker) equipped with the standard ESI source in the positive ion, DDA mode (= switching to MSMS mode for eluting peaks). MS-scans were recorded (range: 150–2200 Da) and the 6 highest peaks were selected for fragmentation. Instrument calibration was performed using ESIcalibration mixture (Agilent). For separation of the peptides a Thermo BioBasic C18 separation column (5 μm particle size, 150°0.360 mm) was used. A gradient from 95% solvent A and 5% solvent B (Solvent A: 65 mM ammonium formiate buffer, B: 100% ACCN) to 32% B in 45 min was applied, followed by a 15 min gradient from 32% B to 75% B, at a flow rate of 6 μL∙min⁻¹. The analysis files were converted using Data Analysis 4.0 (Bruker) to XML files, which are suitable to perform MS/MS ion searches with MASCOT (embedded in ProteinScape 3.0, Bruker) for protein identification. Only proteins identified with at least 2 peptides with a protein score higher than 80 were accepted. For searches the SwissProt database was used.