Nanoacquity uhplc

In Banner and BLSA, protein identification and quantification were performed using Liquid Chromatography Coupled to Tandem Mass Spectrometry (LC-MS/MS) as described in (Seyfried et al., 2017 (link); Swarup et al., 2020 (link)). Briefly, tissue homogenization and protein digestion were performed. Resulting peptides were desalted with a Sep-Pak C18 column (Waters) and dried under vacuum. Peptide mixtures were separated on a self-packed C18 (1.9 um Dr. Maisch, Germany) fused silica column (25 cm × 75 μM internal diameter; New Objective, Woburn, MA) by a NanoAcquity UHPLC (Waters, Milford, FA) and monitored on a Q-Exactive Plus mass spectrometer (ThermoFisher Scientific, San Jose, CA). MaxQuant (v1.5.2.) with Thermo Foundation 2.0 for RAW file reading capability was used to generate label-free quantification. The quantitation method only considered razor plus unique peptides for protein level quantitation. The protein abundances were log2 transformed and regressed for age at death and post-mortem interval.

The peptides were reconstituted in 0.1% formic acid and peptide estimation was carried out using a NanoDrop. Peptides (1 μg) were analysed on LTQ‐Orbitrap Fusion mass spectrometer (Thermo Scientific, Bremen, Germany) interfaced with a nanoAcquity UHPLC (Waters, MA, USA). The peptide samples were first loaded onto a trap column (Waters, Milford, MA, USA) at a flow rate of 5 μl/min and then resolved on a BEH C18 nanoAcquity column (Waters, Milford, MA, USA). The peptides were resolved using a gradient of 8% to 70% solvent B (0.1% formic acid in acetonitrile) for 45 min using a flow rate of 300 nl/min. The total run time for each sample was 60 min. The mass spectrometry settings were as follows: MS1 Resolution – 60,000; Mass Range – 350–1800 m/z; AGC Target – 1e6, Maximum injection time 22 ms. Include charge state – 2–6; Dynamic exclusion – 30 s. MS2: Isolation mode – Quadrupole; Isolation window – 1.2; Activation type – HCD, Collision energy – 30%; AGC target – 50,000, Maximum injection time – 40 ms.

LC-MS/MS for iodoTMT and the total proteome samples were performed using a nanoAcquity UHPLC (Waters, Milford, MA, USA) system connected to a Orbitrap Fusion Tribrid mass spectrometer (Thermo-Fisher Scientific, Waltham, MA, USA). Peptides were reconstituted in 0.1% formic acid and were loaded onto an Acquity UPLC M-Class V/M symmetry trap column (Waters) at 5 μL/min before resolving with the analytical column (Acquity UPLC M-Class Peptide BEH C18 nanoAcquity column; Waters) over a run time of 120 min. Separation of peptides was performed at 300 nL/min using a linear ACN gradient of buffer A (0.1% formic acid in water) and buffer B (0.1% formic acid in ACN). The column compartment was held at 45°C for the entire analysis.
Mass spectra were collected in data-dependent acquisition (DDA) mode. Both MS1 and HCD-MS2 spectra were collected in the Orbitrap. MS1 scan parameters were scan range of 350–1800 m/z, 60,000 resolution, maximum (max) injection time of 22 ms, and Automatic Gain Control (AGC) target 1e6. Dynamic exclusion parameters were set as follows: exclude isotope true, duration 30 s and using the peptide monoisotopic peak determination mode, charge states of 2–6 were included. Peptides were fragmented using stepped collision energy of 25, 30 and 35. MS2 spectra were collected at a resolution of 15K with an AGC target of 5e4, maximum ion injection time (IT) of 40 ms.