Computational protocol: Effects of individually silenced N-glycosylation sites and non-synonymous single-nucleotide polymorphisms on the fusogenic function of human syncytin-2

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Protocol publication

[…] Immunoprecipitation of the concentrated proteins was carried out with protein A sepharose beads (10–1141, Invitrogen) essentially as described by the manufacturer. 293T cells were transfected with phCMV empty vector, phCMV-syncyntin-2 WT, phCMV-syncytin-2 N312Q and phCMV-syncytin-2 T522M plasmids, respectively. After transfection for 48 h, the cells were lysed by suspension in ice-cold lysis buffer for 1 h. Lysates were collected after centrifugation at 12,000 g for 20 min and immunoprecipitated with 3 μg anti-syncytin-2 antibody. Following an overnight incubation, samples were added to 30 μl of protein A bead slurry and rotated for 4 hours at 4°C. After centrifuging and washing 3 times, samples were separated on an SDS-PAGE resolving gel and subjected to direct mass spectrometry (MS) analysis. Using the LTQ-orbitrap XL mass spectrometer (Thermo Fisher Scientific), 4 samples were run and analyzed by Scaffold (Proteome Software). The peptide identities were accepted when the Peptide Prophet algorithm specified probabilities were at >95.0%. Argot2 (Annotation Retrieval of Gene Ontology Terms; http://www.medcomp.medicina.unipd.it/Argot2) was used to identify the functional classifications of the total and specific expressed proteins. DAVID (DAVID Bioinformatics Resources 6.7; http://david.abcc.ncifcrf.gov/tools.jsp) was used to perform functional classification of the differentially expressed proteins (the fold change was greater than or equal to 1.5 and the spectrum count number was not less than 4). Cytoscape was used to draw programs. […]

Pipeline specifications

Software tools Argot, DAVID
Application Protein sequence analysis
Organisms Homo sapiens
Chemicals Amino Acids, Nucleotides