Bacterial materials and growth conditions

S.agalactiae ZQ0910, a virulent strain isolated from intensive tilapia farm with a typical streptococcicosis outbreak in the Guangdong province of China in 2009 [14]. It belongs to serotype Ia and used for challenge study. The strain was grown aerobically overnight at 28 °C in a shaker bath, and then overnight cultured cells were diluted into 1:100 in BHI medium. Bacteria were then harvested by centrifugation (5000 g, 5 min). Cell pellets were washed and resuspended in PBS to obtain the desired concentrations for the infection doses. The final bacterial concentrations were confirmed by plating ten-fold serial dilutions onto blood-agar plates.

Protein extraction

Sample was sonicated three times on ice using a high intensity ultrasonic processor (Scientz) in lysis buffer (8 M urea, 3 μM TSA and 50 mM NAM and 1% Protease Inhibitor Cocktail, Millipore). The remaining debris was removed by centrifugation at 12,000 g at 4 °C for 10 min. Finally, the supernatant was collected and the protein concentration was determined with BCA kit according to the manufacturer’s instructions.

Trypsin digestion

For digestion, the protein solution was reduced with 5 mM dithiothreitol (final concentration) for 30 min at 56 °C and alkylated with 11 mM iodoacetamide (final concentration) for 45 min at room temperature in darkness. The protein sample was then diluted by adding 100 mM TEAB to urea concentration less than 2 M. Finally, trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1:100 trypsin-to-protein mass ratio for a second 4 h-digestion [15].

Affinity enrichment

To enrich Kcro peptides, tryptic peptides dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) were incubated with pre-washed antibody beads (PTM Biolabs) at 4 °C overnight with gentle shaking. Then the beads were washed four times with NETN buffer and twice with H2O. The bound peptides were eluted from the beads with 0.1% trifluoroacetic acid. Finally, the eluted fractions were combined and vacuum-dried. For LC-MS/MS analysis, the resulting peptides were desalted with C18 ZipTips (Millipore) according to the manufacturer’s instructions, followed by LC-MS/MS analysis.

Quantitative proteomic analysis by LC-MS/MS

The tryptic peptides were dissolved in 0.1% formic acid (solvent A), directly loaded onto a reversed-phase pre-column (Acclaim PepMap 100; Thermo Fisher Scientific, Inc.). Peptide separation was performed using a reversed-phase analytical column (Acclaim PepMap RSLC; Thermo Fisher Scientific, Inc.) with gradient that comprised of an increase from 6 to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, 23 to 35% in 8 min and climbing to 80% in 3 min then holding at 80% for the last 3 min, all at a constant flow rate of 400 nL/min on an EASY-nLC 1000 UPLC system [16].

The peptides were subjected to nanospray ion source (NSI) source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using normalized collision energy (NCE) setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0 s dynamic exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the Qrbitrap; 50,000 ions were accunulated to generate MS/MS spectra. For MS scans, the m/z scan range was 350 to 1800. The fixed first mass was set as 100 m/z.

Database search

The resulting MS/MS data were processed using Maxquant search engine (v.1.5.2.8). Tandem mass spectra were searched against 1123 database concatenated with reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to 4 missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in First search and 5 ppm in Main search, and the mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on Cys was specified as fixed modification and crotonylation on lysine were specified as variable modifications. FDR was adjusted to < 1% and minimum score for modified peptides was set > 40. For selected specific Kcr sites, site localization probability was set to > 0.75. All other parameters in MaxQuant were used as default.

Bioinformatics analysis for gene ontology annotation

The Gene Ontology, or GO, is a major bioinformatics initiative to unify the representation of gene and gene product attributes across all species. Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database (www. http://www.ebi.ac.uk/GOA/) [17]. Firstly, Converting identified protein ID to UniProt ID and then mapping to GO IDs by protein ID. If some identified proteins were not annotated by UniProt-GOA database, the InterProScan soft would be used to annotated protein’s GO functional based on protein sequence alignment method. Then proteins were classified by Gene Ontology annotation based on three categories: biological process, cellular component and molecular function.

For subcelluar localization, we used wolfpsort a subcellular localization predication soft to predict subcellular localization. Wolfpsort is an updated version of PSORT/PSORT II for the prediction of eukaryotic sequences. Special for protokaryon species, Subcellular localization prediction soft CELLO was used.

Kyoto Encyclopedia of Genes and Genomes (KEGG ) database was used to annotate protein pathway. Firstly, using KEGG online service tools KAAS to annotated protein’s KEGG database description. Then mapping the annotation result on the KEGG pathway database using KEGG online service tools KEGG mapper.

Bioinformatics analysis for enrichment GO and KEGG pathway analysis

Proteins were classified by GO annotation into three categories: biological process, cellular compartment and molecular function. For each category, a two-tailed Fisher’s exact test was employed to test the enrichment of the identified modified protein against all proteins of the species database. The GO with a corrected p-value < 0.05 is considered significant.

Encyclopedia of Genes and Genomes (KEGG) database was used to identify enriched pathways by a two-tailed Fisher’s exact test to test the enrichment of the identified modified protein against all proteins of the species database. The pathway with a corrected p-value < 0.05 was considered significant. These pathways were classified into hierarchical categories according to the KEGG website.

Motif analysis

Soft MoMo (motif-x algorithm) was used to analysis the model of sequences constituted with amino acids in specific positions of modify-21-mers (10 amino acids upstream and downstream of the site, but phosphorylation with modify-13-mers that 6 amino acids upstream and downstream of the site) in all protein sequences. And all the database protein sequences were used as background database parameter. Minimum number of occurrences was set to 20. Emulate original motif-x was ticked, and other parameters with default.

Enrichment-based clustering

For further hierarchical clustering based on differentially modified protein functional classification (such as: GO, Domain, Pathway, Complex). We first collated all the categories obtained after enrichment along with their P values, and then filtered for those categories which were at least enriched in one of the clusters with P value < 0.05. This filtered P value matrix was transformed by the function x = −log10 (P value). Finally these x values were z-transformed for each functional category. These z scores were then clustered by one-way hierarchical clustering (Euclidean distance, average linkage clustering) in Genesis. Cluster membership were visualized by a heat map using the “heatmap.2” function from the “gplots” R-package.

Protein-protein interaction network

All differentially expressed modified protein database accession or sequence were searched against the STRING database version 10.5 for protein-protein interactions [18]. Only interactions between the proteins belonging to the searched data set were selected, thereby excluding external candidates. STRING defines a metric called “confidence score” to define interaction confidence; we fetched all interactions that had a confidence score > 0.7 (high confidence). Interaction network form STRING was visualized in R package “networkD3”.

Western blot

Proteins were run 12% SDS-PAGE gels, then adjust the voltage to the 100 V and electrophoresis for 15 min. After the bromophenol blue migrated out of the concentrated gel, the voltage was switched to 200 V for 40 min. And transferred to a polyvinylidene fluoride (PVDF) membrane by semi-dry technique and set the power to 100 V (constant voltage) for 1 h at 4 °C. The membranes were blocked in TBS (Tris buffered saline) containing 0.05% (v/v) Tween 20 with 5% (w/v) skim milk and incubated 1 h at room temperature. The primary antibodies used in the western blot were anti-Kcr (1:2000), anti-YebC (1:5000), anti-CsbD (1:4000), anti-Sal (1:4000), anti-Coldshock (1:4000) and incubated overnight at 4 °C. After washing in 10 mM PBS (pH 7.4) containing 0.1% Tween-20, the membranes were incubated in horseradish peroxidase (HRP) conjugated goat anti-rat IgG (CWBIO, China) diluted 1:5000 at room temperature for 3 h. Finally, the membrane was visualized using the ECL system (Bio-Rad, Hercules, CA, USA), and recorded by the ChemiDoc™ MP (Bio-Rad, Hercules, CA, USA) imaging system [19].