# The Heat Shock Protein 60 and Pap1 Participate in the _Sporothrix schenckii_-Host Interaction
## 2 Materials and Methods
### Peptidormannoman Analysis by Liquid Chromatography  
The PRM fraction was deglycosylated as follows: aliquots containing 400 ug freeze-dried PRM were resuspended in 900 \(\mu\)L sterile deionized water, 100 \(\mu\)L 3 M sodium acetate, and 25 U endoglycosidase H (New England Biolabs, Ipswich, MA, USA) and incubated overnight at 37 \({}^{\circ}\)C [45]. Then, pH was neutralized and samples lyophilized, then resuspended in 1 mL 100 mM NaOH, and incubated overnight at room temperature and gentle shaking [46]. The pH was neutralized with 100 mM HCl and lyophilized again. Then, 200 \(\mu\)g were solubilized in 100 \(\mu\)L of 100 mM ammonium bicarbonate buffer, pH 8.0, and reduced with 20 \(\mu\)L of 50 mM DTT for 1 h at room temperature [47]. Then, samples were alkylated with 20 \(\mu\)L of 100 mM iodoacetamide at room temperature in darkness and reduced again with 20 \(\mu\)L of 50 mM DTT. For hydrolysis, 20 \(\mu\)L of trypsin (0.25 \(\mu\)L\({}^{-1}\)) was used and incubation at 37 \({}^{\circ}\)C was carried out overnight to then stopthe reaction with 20 \(\mu\)L of 50% (\(v/v\)) formic acid [47]. The samples were saved at \(-\)20 \({}^{\circ}\)C and centrifuged at 18,000\(\times\)\(g\) for 20 min at 4 \({}^{\circ}\)C before being analyzed by capillary liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (capLC-ESI-QTOF-MS), using an UltiMate 3000 RSLC (Thermo Fisher Scientific) coupled to a maXis impact mass spectrometer (Bruker Daltonics, Billerica, MA, USA), as previously described [47]. Briefly, aliquots of 5 \(\mu\)L PRM were loaded on a C18 capillary trap (5 \(\mu\)m, 100 A, 300 \(\mu\)m i.d. \(\times\) 5 mm, Agilent Technologies, Santa Clara, CA, USA) using 0.1% (\(v/v\)) formic acid and 2% (\(v/v\)) acetonitrile at a flux rate of 15 \(\mu\)L min\({}^{-1}\) for 2 min. Then, the flux was changed to the reversed phase capillary column (Halo C18, 150 \(\times\) 0.3 mm, 2.7 \(\mu\)m), and the separation was carried out at 40 \({}^{\circ}\)C and a flux rate of 3 \(\mu\)L min\({}^{-1}\) using two mobile phases (A-aquos 0.1% (\(v/v\)) formic acid; B-0.1%(\(v/v\)) formic acid in acetonitrile) with a linear gradient of 2 to 80% (\(v/v\)) B for 20 min. The column was connected to an electrospray ionization source via 50 cm capillary (i.d. 50 \(\mu\)m), introducing the lock-mass standard \(m/z\) 1221.9907 in the ion source. ESI was operated in a positive mode with ion spray voltage 4500 V, endplate offset 500 V, dry gas 4 L min\({}^{-1}\), drying temperature 180 \({}^{\circ}\)C, and nebulizing gas pressure 0.4 bar. MS data were obtained with an acquisition rate of 2 Hz within the 300-2200 \(m/z\) range. For auto-MS/MS mode, filtration for amino acids was applied with an acquisition rate of 2 Hz and 20 Hz for low (<25,000) and high (>25,000) ion counts, respectively. Line spectra calculated as a sum of intensities were always registered. For protein identification and for label-free quantification, LC-MS and MS/MS data were analyzed in MaxQuant (version 1.5.5.1) [48] using the _S. schanckii_ taxonomy database from UniProtKB ([[https://www.uniprot.org/proteomes/UP000033710](https://www.uniprot.org/proteomes/UP000033710)]([https://www.uniprot.org/proteomes/UP000033710](https://www.uniprot.org/proteomes/UP000033710)); accessed on 2 March 2018). The following parameters were applied: trypsin as specific digestion enzyme, methionine oxidation and acetylation of N-terminals as variable modification, and carbamidomethylylation of cysteines as fix modification. Minimum and maximum peptide charge states were set at +2 and +5, respectively. The initial precursor mass tolerance of 6 ppm and fragment mass tolerance of 0.01 Da were applied. The maximum false discovery rate for peptides and proteins was 1% based on comparison to a reverse database; seven amino acids was the minimum length of peptide. A time window of 0.7 min with an alignment time window of 15 min were selected for time matching between LC-MS/MS runs.