Computational protocol: A covalent and cleavable antibody-DNA conjugation strategy for sensitive protein detection via immuno-PCR

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Protocol publication

[…] To determine the localization of tetrazine modifications, 1 μg of functionalized antibody in 1 ul was diluted in 15 μL of 8 M Urea in 100 mM Tris, pH 8. Disulphide bonds were reduced by adding 2 ul 10 mM dithiothreitol and subsequently alkylated by adding 2 ul 50 mM iodoacetamide, for 15 minutes in the dark. Subsequently, the antibody was digested using 0.5 ul of Trypsin/Lys-C mix (0.04 μg/ul, Promega) overnight at rt. The digestion was stopped by acidifying with trifluoroacetic acid and the peptides were purified on StageTips. Thirty percent of the peptides were loaded onto a pulled fused silica column (New Objectives) packed in house with 1.8 μm Reprosil-Pur C18-AQ (Dr. Maisch, 9852). Using the Easy-nLC 1000 (Thermo Fisher Scientific), peptides were separated in a 60 min. gradient and directly injected into a QExactive mass spectrometer (Thermo Fisher Scientific). The mass spectrometer was operated in TOP10 data dependent acquisition. Full MS were recorded at a resolution of 70,000 at m/z = 400 and a scan range of 300–1,650 m/z. MS/MS spectra were recorded at a resolution of 17,500. Raw mass spectrometry data was analyzed using the MaxQuant software package, version 1.5.0.0, with standard settings if not further specified. The following variable modifications were allowed: Oxidation of methionines, acetylation of protein N-termini, and carbamylation of cysteines. Furthermore, a modification of lysines and protein N-termini corresponding to the reduced and alkylated linker (Δm = 145.01975) was allowed. This modification was only allowed for peptide internal lysines due to the missed trypsin cleavage that is caused by the linker modification. Three missed cleavages were allowed and the maximum peptide mass was set to 8000 Dalton. Data was searched against the mouse Uniprot database (downloaded 13.06.2014) using the integrated Andromeda search engine. The search was performed with a mass tolerance of 4.5 ppm mass accuracy for the precursor ion and 20 ppm for fragment ions. Peptides, modified peptides and proteins were accepted at an FDR of 0.01. […]

Pipeline specifications

Software tools MaxQuant, Andromeda
Application MS-based untargeted proteomics
Organisms Homo sapiens