Tmt reagent

TMT labeling of peptides was conducted according to manufacturer instructions. In brief, 100 μg of peptides were resuspended in 50 mM HEPES buffer (pH 8.5) at the final concentration of 5 µg/µl. TMT reagent (Thermo Fisher Scientific, 10 plex, 1 × 0.8 mg) was dissolved in anhydrous acetonitrile (Sigma-Aldrich) and added to the peptide solution. The mixture was incubated at room temperature with a vortex (1000 rpm) for 1 hour. The reaction was quenched by adding 5% hydroxylamine to the solution. The TMT-labeled peptides were mixed together for glycopeptide enrichment.

The TCA/acetone precipitation and SDT lysis method was employed to extract proteins. Protein quantitation was performed by a BCA assay (CWBIO, Beijing, China), and separation detected by SDS-PAGE. The C. albicans protein samples were digested according to the FASP procedure and labeled using TMT reagent according to the manufacturer’s instructions (Thermo Fisher Scientific). The subsequent procedures included peptide fractionation with reversed phase (RP) chromatography, mass spectrometry analysis, data analysis by Proteome Discoverer 2.2 software (Thermo Fisher Scientific), and bioinformatic analysis including Gene Ontology (GO) and KEGG Pathway annotations. Three biological replicates were prepared in each group. The final proteins that were deemed to be differentially expressed were screened by the following criteria: 1.2-fold changes (upregulation or downregulation) relative to the control group, and P value <0.05.

Buffer composed of 4% SDS, 100 mM Tris-HCl, and 1 mM dithiothreitol (DTT), (pH7.6) was used for sample lysis and protein extraction. An aliquot (200 μg) of proteins from each sample was digested using trypsin (Promega, Madison, WI, USA). After SDS-PAGE, 100 μg peptide mixture of each sample was labeled using TMT reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the instructions of the manufacturer. The TMT tags 129,130, and 131 were used to label BrYV-carrying aphids; the control aphids were labeled as 126,127, and 128. Then, labeled peptides were fractionated using the High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis was performed on a Q Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) that was coupled to Easy nLC (Thermo Fisher Scientific, Waltham, MA, USA). Finally, the MS raw reads for each sample were searched using the MASCOT engine “matrixscience.com (accessed on 25 July 2021)” embedded into Proteome Discoverer 1.4 software for identification and quantitation analysis.

After performing the abovementioned procedures, the shrimp samples were processed as follows. The TMT reagent (Thermo Fisher Scientific No. a44522) was taken out at 20°C and restored to room temperature. The reagent was centrifuged, acetonitrile was added, and centrifuge was vortex mixed. A tube of TMT reagent was added for every 100 μg polypeptide and incubated at room temperature for 2 h. Then, hydroxylamine was added, and the samples were kept at room temperature for 30 min. Each group of moderately labeled products was mixed into a tube and drained using a vacuum concentrator.
The polypeptide samples were redissolved with UPLC loading buffer (2% acetonitrile with ammonia adjusted to pH 10) and separated by a reversed-phase C18 column. Column information: ACQUITY UPLC BEH C18 Column 1.7 μm, 2.1 mm × 150 mm (Waters, United States); Chromatographic instrument: Thermo Scientific Vanquish Flex Binary UHPLC system; phase A: 2% acetonitrile (ammonia adjusted to pH 10); phase B: 80% acetonitrile (adjust ammonia to pH 10); UV detection wavelength: 214 nm; flow rate: 200 μl/min; and gradient: 48 min. According to the peak type and time, 20 fractions were collected and combined into 10 fractions. After vacuum centrifugation and concentration, the fractions were dissolved in mass spectrometry loading buffer (2% acetonitrile and 0.1% formic acid) for the second-dimensional analysis.

For TMT labeling, 15 μg peptides of each sample (pre-dried in a vacuum concentrator) were reconstituted in 100 mM TEAB (pH 8.5) to a concentration of 0.2 μg/μL. TMT-10 reagent (Thermo Scientific, Waltham, MA, USA, cat# 90406) was resuspended in anhydrous ACN to a concentration of 19.5 μg/μL. The appropriate TMT reagent was added to each sample at the 1:35 reagent/peptide (wt/wt) ratio, and incubated for 1 h at room temperature. The labeling design is shown in Table S1 and in Figure S1 (“TMT labeling design”). To quench the reaction, 5 % hydroxylamine was added and samples were incubated for 15 minutes at room temperature. Labeled samples that corresponded to the time points (0, 3, 6, 9, 12, and 72 h) were combined within each biological replicate, resulting in 3 samples for MS/MS analysis. The three pooled samples were dried via vacuum centrifugation, and desalted using a C18 Stage-Tip method. The peptides were dissolved in 0.1% formic acid to a final concentration of 3 µg/µL.

About 600 μg peptide mixture of each sample was labeled using TMT reagent [80 (link)] according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). The lyophilized samples of the FASP digested peptides were redissolved in 500 µL 1×DHB buffer. Then, TiO₂ beads were added and agitated for 2 h. The centrifugation was carried out for 1 min at 5000× g, resulting in the beads. Additionally, they were washed with 50 µL of washing buffer I three times and then 50 µL of washing buffer II three times. Finally, the phosphopeptides were eluted with 50 µL of elution buffer three times, followed by lyophilization and MS analysis.