Computational protocol: Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology

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Protocol publication

[…] DRMs were prepared for MS by two consecutive step-gradient purifications as described above. Trypomastigotes (800×106) were lysed and centrifuged as above. The 35%/5% OptiPrep interface was collected in 1ml into an ultracentrifuge tube, mixed with 60% OptiPrep to a final 40% OptiPrep concentration and purification continued as before. After the second centrifugation step, the 35%-5% OptiPrep interface was collected in 300μl to avoid contamination and precipitated with 3 volumes of cold acetone. Samples were analyzed at ITSI-Biosciences (Johnstown, PA) by in solution digestion and tandem mass spectrometry analysis using nano-LC/MS/MS. After digestion, peptides were dried and reconstituted in 2% Acetonitrile with 0.1% Formic acid. After salt removing, samples were loaded onto a PicoFrit C18 nanospray column (New Objective) using a Thermo Scientific Surveyor Autosampler operated in the no waste injection mode. Peptides were eluted with a 2–32% linear Acetonitrile gradient over 230 minutes followed by high and low organic washes for another 5 min into an LTQ XL mass spectrometer (Thermo Scientific) via a nanospray source with the spray voltage set to 1.8kV and the ion transfer capillary set at 180°C. A full MS scan from m/z 350–1500 was followed by MS/MS scans on the seven most abundant ions. Raw data files were searched using Proteome Discoverer 1.3 (Thermo Scientific) and the SEQUEST algorithm against the most recent species-specific database for T. cruzi from NCBI. Trypsin was the selected enzyme allowing for up to two missed cleavages per peptide, Carbamidomethyl Cysteine was used as a static modification and Oxidation of Methionine as a variable modification. Proteins were identified when two or more unique peptides had X-correlation scores greater than 1.5, 2.0, and 2.5 for respective charge states of +1, +2, and +3. A total of 152 proteins were identified. […]

Pipeline specifications

Software tools Proteome Discoverer, Comet
Application MS-based untargeted proteomics
Organisms Trypanosoma cruzi
Diseases Chagas Disease
Chemicals Phosphatidylinositols, Sialic Acids, Glycosylphosphatidylinositols, N-Acetylneuraminic Acid