Nano acquity uplc system

The Functional Genomics Center of the University of Zurich (FGCZ) was commissioned to perform the mass spectrometry analysis. HA immunoprecipitation was performed as described above. After the last wash, precipitated material was resuspended in PBS buffer pH 7.45 and subjected to on-beads trypsin digestion according to the following protocol. Beads were washed twice with digestion buffer (10 mM Tris, 2 mM CaCl₂, pH 8.2). After the last wash, the buffer was discarded and monoclonal anti-HA agarose beads were resuspended in 10 mM Tris, 2 mM CaCl₂, pH 8.2 buffer supplemented with trypsin (100 ng/μL in 10 mM HCl). The pH was adjusted to 8.2 by the addition of 1 M Tris pH 8.2. Digestion was performed at 60 °C for 30 min. Supernatants were collected, and peptides were extracted from beads using 150 μL of 0.1% trifluoroacetic acid (TFA). Digested samples were dried and reconstituted in 20 μL ddH2O + 0.1% formic acid before performing liquid chromatography-mass spectrometry analysis (LC MS/MS). For the analysis 1 μL were injected on a nanoACQUITY UPLC system coupled to a Q-Exactive mass spectrometer (Thermo Scientific). MS data were processed for identification using the Mascot search engine (Matrixscience) and the spectra was searched against the Swissprot protein database.

Mass spectrometry analysis was done as previously described [21 (link)]. The labeled peptides were analyzed on the LTQ Orbitrap-Velos instrument (Thermo Fisher, USA) connecting to a Nano ACQUITY UPLC system via a nanospray source. The reverse-phase separation of peptides was performed using the buffer A(2% ACN, 0.5% acetic acid) and buffer B (80% ACN, 0.5% acetic acid); the gradient was set as following: 4% to 9% buffer B for 3 min, 9% to 33% buffer B for 170 min, 33% to 50% buffer B for 10 min, 50% to 100% buffer B for 1 min, 100% buffer B for 8 min, 100% to 4% buffer B for 1 min. For analysis of plasma membrane proteins, one full scan was followed by the selection of the eight most intense ions for collision-induced dissociation (CID) fragmentation (collision energy 35%). The most intense product ion from the MS2 step was selected for higher energy collision-induced dissociation (HCD)-MS3 fragmentation.