Microorganisms
The G. lamblia WB strain was cultivated in TYI-S-33 medium [24] supplemented with 10% inactivated adult bovine serum, at 37°C for 48 to 72 h.
Fractionation of the ventral disc
The protocol to obtain the ventral disc fraction was adapted from Holberton et al. (1981) [16]. Briefly, trophozoites were cultivated for 48 h (107 cells). After being placed on ice for 15 min, the cultures were centrifuged (Sorvall RC-5B) at 12,000 g for 20 min. Subsequently, cells were resuspended in 0.1 M PHEM buffer (60 mM Pipes, 20 mM Hepes, 10 mM EGTA and 2 mM MgCl2), pH 7.2, supplemented with 1% Triton X-100, at 37°C for 10 min. Parasites were disrupted by sonication on ice; an ultrasonic apparatus (Sigma, GEX 600 Model) with a standard probe for periods of 10-15 s with amplitude of 8% was used. Samples were then centrifuged at 10,000 g for 20 min, the pellets were washed with PBS buffer (0.02 M Na2HPO4 and 0.9% NaCl) and used in all the analysis described below.
Light microscopy
Isolated ventral disc fractions were mounted between a glass slide and a coverslip and observed using a Zeiss Axio Observer D1 microscope.
Transmission electron microscopy
For ammonium molybdate staining, sample preparations of isolated discs were placed on copper grids previously coated with 5% FORMVAR in chloroform for 2 min. The materials were then washed in 0.1 M PHEM buffer, pH 7.2 and stained with 1% ammonium molybdate for 3 min prior to observation with a Zeiss EM900 electron microscope.
For routine transmission electron microscopy preparations fractions were fixed in a solution containing 2.5% glutaraldehyde, 4% freshly prepared formaldehyde, 4% sucrose in 0.1 M PHEM buffer for 1 h at room temperature, and post-fixed in 0.1 M cacodylate buffer with 1% OsO4, 0.8% potassium ferrocyanide and 5 mM calcium chloride for 40 min. Samples were dehydrated in an acetone series and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and observed using a Zeiss EM900 transmission electron microscope.
1D SDS-PAGE
A total of 30 μg of ventral disc proteins were separated by both 7.5% and 12% SDS-PAGE. Each lane was arbitrarily divided into approximately 2-5 mm slices that were then subjected to in-gel proteolysis with trypsin.
2D SDS-PAGE
To remove salts and detergents from the samples, the samples were precipitated using a 2-D Clean-Up Kit (GE, USA). The first dimension used 18 cm strips with an immobilized pH gradient from 3 to 10. The analyzed proteins (100 μg) were dissolved in 300 μL of rehydration buffer (8 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris, 1% IPG, 0.002% bromophenol blue, and 60 mM dithiothreitol). Isoeletric protein focusing was performed using an IPGPhor instrument (GE-Health Care) with the following voltages: 30 V for 12 h, 200 V for 1 h, 500 V for 1 h, 1000 V for 1 h, 3500 V in gradient step for 30 min and 35000/18000 Vh. Between the first and second dimension, the focused strips were equilibrated for 15 min in a solution containing 50 mM Tris-HCl pH 8.8; 6 M urea; 30% glycerol; 2% SDS; 0.002% blue bromophenol (equilibrium buffer) and 6 mM dithiothreitol. Next, the strips were submitted to a second equilibrium step with an alkylation solution (2.5% w/v of iodoacetamide in equilibrium buffer) for 30 min. After equilibrium, strips were directly applied onto 12% polyacrylamide gels and electrophoresis was performed as described by the manufacturer. A Dalt six GE-Healh Care apparatus was used for 18 cm strips, and electrophoresis was conducted at 25 W per gel. Gels were then fixed and stained with Comassie Blue G 250 (Sigma-Aldrich).
In gel digestion
1D gel sections or 2D gels spot were excised and cut into smaller pieces (approximately 2 mm). Gel pieces were destained with 25 mM ammonium bicarbonate in 50% acetonitrile for 12 h. The slices obtained from the non-reduced gels were previously reduced in a 10 mM dithiothreitol, 25 mM NH4HCO3 solution for 1 h at 56°C and then alkylated in a 55 mM iodoacetamide, 25 mM NH4HCO3 solution for 45 min in the dark. The solution was removed, and the spots were washed with 25 mM NH4HCO3 in 50% acetonitrile and then dehydrated with 100% acetonitrile. Finally, the gel spots were air-dried and rehydrated with a solution of NH4HCO3 containing 100 ng of trypsin. Gel spots were digested overnight at 37°C. The tryptic peptides were then collected in 10 μL of a solution containing 0.1% TFA and 50% acetonitrile.
Nano LC-MS/MS mass spectrometry
The extracted peptides from the 1D SDS-PAGE gels spots were loaded into a Waters Nano Acquity system (Waters, Milford, MA). The peptides were desalted on-line using a Waters Opti-Pak C18 trap column. The sample injection volume was typically 10 μL, and the LC was performed using a nanoEase C18 150 mm × 75 μm column (Waters, Milford, MA) eluting (0.4 μL/min) with a linear gradient (10-50%) of acetonitrile containing 0.1% formic acid. Electrospray tandem mass spectra were recorded using a Q-Tof quadrupole/orthogonal acceleration Time-of-Flight spectrometer (Waters, Milford, MA) interfaced to the Nano Acquity system capillary chromatograph. The ESI voltage was set at 3500 V using a metal needle, the source temperature was 100°C and the cone voltage was 100 V. Instrument control and data acquisition were conducted by the MassLynx data system (Version 4.1, Waters); experiments were performed by scanning from a mass-to-charge ratio (m/z) of 200-2000 using a scan time of 1 s, applied during the whole chromatographic process. The mass spectra corresponding to each signal from the total ion current (TIC) chromatogram were averaged, allowing for an accurate molecular mass measurement. The exact mass was determined automatically using the Q-Tof's LockSpray™ (Waters, Milford, MA) and the Q-Tof Ultima API (Waters, Milford, MA). Data-dependent MS/MS acquisitions were performed on precursors with charge states of 2, 3 or 4 over a range of 50-2000 m/z and under a 2 m/z window. A maximum of three ions were selected for MS/MS from a single MS survey. Adduct masses of Na+ and K+ were automatically excluded. Collision-induced dissociation (CID) MS/MS spectra were obtained using argon as the collision gas at a pressure of 13 psi; the collision voltage varied between 18 and 45 V depending on the mass of the precursor. The scan rate was 1 scan/s. All data were processed using the ProteinLynx Global server (version 2.0, Waters). The processing automatically lock mass corrected the m/z scale of both the MS and MS/MS data utilizing the lock spray reference ion. The MS/MS data were also charge-state deconvoluted and deisotoped with a maximum entropy algorithm (MaxEnt 3, Waters).
MALDI-TOF/TOF mass spectrometry
Roughly 0.3 μL of the extracted peptide solutions were mixed with an equal volume of a saturated matrix solution (α-cyano-4-hydroxycinnamic acid (Aldrich, Milwaukee, WI) 10 mg/mL in 50% acetonitrile, 0.1% trifluoroacetic acid) on the target plate and allowed to dry at room temperature. Raw data for protein identification were obtained on the 4700 Proteomics Analyzer (Applied Biosystems, Foster City, CA).
Data analysis
Proteins were identified by correlation of tandem mass spectra and the NCBInr database of proteins (Version 050623, 2,564,994 sequences) using the MASCOT software (Matrix Science, version 2.1). One missed cleavage per peptide was allowed and an initial mass tolerance of 0.05 Da was used in all searches. Cysteines were assumed to be carbamidomethylated, and a variable modification of methionine (oxidation) was allowed. Identification was considered positive when at least two peptides matched with a mass accuracy of less than 0.2 Da.