Computational protocol: Low Bioavailability and High Immunogenicity of a New Brand of E. colil-Asparaginase with Active Host Contaminating Proteins

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Protocol publication

[…] The LC-MS/MS method adopted for the analysis conducted at LNBio (Campinas, Brazil) is described here. The corresponding method used at MS Bioworks (Ann Arbor, MI, USA) can be provided on request. l-asparaginase samples were reconstituted in saline (0.9% NaCl), 30 μg of protein were aliquoted and 10 μL Ureia 8 M and 0.4 μL 250 mM DTT were added. This mix was then incubated at 56 °C for 25 min, followed by addition of 0.57 μL 500 mM iodoacetamide and incubation for 30 min at room temperature in the dark. After alkylation, 0.4 μL 250 mM DTT was added again and incubated for 15 min. After these steps of reduction and alkylation, samples were digested by addition of 53.25 μL 50 mM ammonium bicarbonate, 0.74 μL 100 mM CaCl2 and 1 μg of trypsin or chymotrypsin (Sequence Grade Modified, Sigma Aldrich) and incubated at 37 °C for 13 h. The reaction was stopped by addition of trifluoroacetic acid to a final concentration of 1%. Samples were then de-salted by the method of Stage Tips (). The samples were dried in a vacuum concentrator and reconstituted in 135 μL of 0.1% of formic acid. Two μL containing 0.44 μg of the resulting peptide mixture was analyzed on an ETD enabled LTQ Velos Orbitrap mass spectrometer (Thermo Fisher Scientific) coupled with LC-MS/MS by an EASY-nLC system (Proxeon Biosystem) through a Proxeon nanoelectrospray ion source.Peptides were separated by a 2–30% acetonitrile gradient in 0.1% formic acid using a C18 PicoFrit Column (20 cm × ID75 μm, 5-μm particle size; New Objective) and an EASY-nLC at a flow rate of 300 nL/min over 30 min. The nanoelectrospray voltage was set to 2.2 kV, and the source temperature was 275 °C. The scan MS spectra (m/z 300–1600) were acquired in the Orbitrap analyzer after accumulation to a target value of 1 × 106 (). Resolution in the Orbitrap was set to r = 60,000 (m/z 400). Peptide ions were sequentially isolated to a target value of 80,000 and fragmented in the HCD (high collisional dissociation) energy (normalized collision energy of 40%). The signal threshold for triggering an MS/MS event was set to 7500 counts. An activation time of 0.1 ms was used.The raw files were processed using Proteome Discoverer 1.4 (Thermo Scientific), and the MS/MS spectra were searched using the Sequest software against the Uniprot SwissProt E. coli database (Release: March 31th, 2017; 10,082 entries), with a tolerance of 10 ppm for precursor ions, 0.02 Da for fragment ions, and a maximum of 1 missed cleavage for protein identification. Carbamidomethylation of cysteine was set as a fixed modification, and oxidation of methionine was chosen as a variable modification. Both peptide and protein identifications were filtered at a maximum of 1% false discovery rate. Raw data for LNBio and MS Bioworks may be provided on request. Results from Butantan Institute can be found by entering the code 157051214204026500000001247715 in the following link: [...] The raw data from MS Bioworks were processed using Mascot Distiller 2.3 and the resulting MGF file was used in Mascot server 2.3 (Matrix Science Ltd) to search for non-specific cleavages using the same parameters described above. The scoring function model was not specific to any particular digestion enzyme, so that all peptides (including but not restricted to those generated by trypsin) were accessible. […]

Pipeline specifications

Software tools Proteome Discoverer, Comet, Mascot Distiller, Mascot Server
Application MS-based untargeted proteomics
Organisms Homo sapiens, Mus musculus
Diseases Carcinoma, Renal Cell, Precursor Cell Lymphoblastic Leukemia-Lymphoma