DOI: 10.25345/c5rv0db9r
title: MassIVE MSV000095104 - Giardia duodenalis secretes heterogenous extra-cellular vesicle sub-populations with distinct proteome signatures and assemblage-specific variations between assemblages A and B.
title: Proteins from the different preparations were separated on a 1D gradient gel NuPAGE and stained with Coomassie blue. The gels were run for half the length when microvesicles and exosomes were loaded, and each lane was cut into three bands. For secretome proteins, the gels were run entirely, bands containing highly abundant bovine serum proteins were cut out, whereas the rest of each lane was cut into eight bands. Similar amounts (about 15 ug) for each sample and biological replicate were loaded on the gel. All the bands were digested by trypsin, and the peptides were injected into the mass spectrometer Orbitrap Fusion Tribrid (Thermo Fisher Scientific, CA, United States) equipped with an Ultimate 3000 HPLC. Biological triplicates were prepared and analyzed for each sample. A desalting step was performed on a trap column (Acclaim PepMap 100 C18, Thermo Fisher Scientific), and the separation occurred on a 20 cm long silica capillary (Silica Tips FS 360 75 8, New Objective, MA, United States) packed in a C18, 5um, 100 A resin (Michrom BioResources, CA, United States). A 91 min long gradient was adopted using buffer A (95 percent water, 5 percent acetonitrile, and 0.1 percent formic acid) and buffer B (95 percent acetonitrile, 5 percent water, and 0.1 percent formic acid): buffer B was set at 5 percent at the start, then linearly increased to 30 percent in 60 min, to 80 percent in 10 min, stayed at 80 percent for 5 min to finally decrease at 5 percent for 10 min column re-equilibration step (Federici, Shahaj et al. 2020). Full scan MS data were acquired in the 350 550 m over z range in the Orbitrap at 60 k resolution, MS MS spectra were acquired in data dependent top-speed mode (3 s long maximum total cycle): the most intense precursors were selected through a monoisotopic precursor selection filter and with charge greater than 1, quadrupole isolated and fragmented by higher energy collision dissociation (HCD) (32 percent normalized collision energy). Fragment ions were analyzed in the ion trap with a rapid scan rate. Maximum injection times of 50 and 100ms were used for MS and MS MS, respectively. Raw mass spectra were analyzed with Proteome Discoverer 2.4 software (Thermo Fisher Scientific) using the Giardia duodenalis protein databases (GiardiaDB rel. 56, www.giardiadb.org) Giardia GS DB43 G intestinalis Assemblage BGS (4466 sequences), Giardia WB DB43 Gintestinalis Assemblage AWB (9295 sequences)(Aurrecoechea, Brestelli et al. 2008) and, the UniProtKBm Swiss Prot bovine database (UP000009136, 6909 sequences). Proteins were finally identified with 1 percent FDR using the Percolator node. For protein identification, complete cysteine carbamidomethylation and possible methionine oxidation and N acetylation on the protein terminus were required. Precursor and fragment tolerance were set to 10 ppm and 0.6 Da, respectively. Proteins were quantified by label free quantification approach through summed peptide (intensities) abundance measurement, considering unique and razor peptides. The normalized summed intensities of each protein for each sample were finally collected and analyzed using Perseus software (Tyanova, Temu et al. 2016). Bovine proteins and keratins were considered contaminants and excluded from the statistical analysis.