Feline Lindhout

Quantitative mapping of transcriptome and proteome dynamics during polarization of human iPSC-derived neurons 2 55 loaded on a Gemini 3µm C18 110A 100 x 1.0 mm column (Phenomenex) using an Agilent 1100 pump equipped with a degasser and a photodiode array (PDA) detector. Peptides were concentrated on the column at 100µl/min using 100% buffer A (10mM ammonium hydroxide, pH 10) after which the fractionation gradient was applied as follow: 5% solvent B (10mM ammonium hydroxide in 90%ACN, pH 10) to 30% B in 53 mins, 70% B in 7 min and increased to 100% B in 3 min at a flow rate of 100µl/min. In total 60 fractions of 1 min were collected using an Agilent 1260 infinity fraction collector and then concatenated into 12 final fractions. Collected fractions were vacuum-dried, reconstituted in 5% formic acid/5% DMSO and stored at -80°C prior to mass spectrometry analysis. Mass spectrometry analysis We analyzed the samples on an Orbitrap Q-Exactive HF mass spectrometer (Thermo Fisher Scientific) coupled online to an Agilent UPLC 1290 system (Agilent Technologies). Peptides were loaded onto a trap column (Reprosil C18, 3 µm, 2 cm × 100 µm; Dr. Maisch) and separated on an analytical column (Poroshell 120 EC-C18, 2.7µm, 50cm x 75µm; Agilent Technologies). Peptides were trapped for 10 min at 5 µl/min in solvent A (0.1M acetic acid in H20) and then separated at a flow rate of approximately 300 nl/min (split flow from 0.2ml/min) by applying a 120 min linear gradient as follows: 13% up to 40% solvent B (0.1M acetic acid in 80% ACN) in 95 min, 40-100% in 3 min and finally 100% for 1 min. The mass spectrometer was operated in data-dependent acquisition mode. Full MS spectra from m/z 375-1600 were acquired at a resolution of 60.000 with an automatic gain control (AGC) target value of 3e6 and maximum injection time (IT) of 20 ms. The 15 most intense precursor ions were selected for HCD fragmentation. HCD fragmentation was performed at a normalized collision energy (NCE) of 32%. MS/MS spectra were obtained at a 30.000 resolution with an AGC target of 1e5 and maximum injection time (IT) of 50 ms. Isolation window was set at 1.0 m/z and dynamic exclusion to 16.0s. Data processing proteomics RawMS files were processed for data analysis with Proteome Discoverer 1.4 (Thermo Fisher Scientific). A database search was performed using the Swissprot homo sapiens database and Mascot (version 2.5.1, Matrix Science, UK) as search engine. Carbamidomethylation of cysteines was set as a fixed modification, and oxidation of methionine, acetylation at the N-termini, TMT-6plex of lysine residues and TMT-6plex at the peptide N-termini were set as variable modifications. Trypsin was set as cleavage specificity, allowing a maximum of two missed cleavages. Data filtering was performed using percolator, resulting in 1% false discovery rate (FDR). Additional filters were search engine rank 1 and mascot ion score > 20. Only unique peptides were included for quantification and the obtained TMT ratios were normalized to the median. Common contaminant proteins (such as keratins and albumin) were removed from the list.