Easy nlc1000 nanoflow hplc system

The LC-MS/MS measurement was performed with the Q-Exactive hybrid mass spectrometer and the Easy-nLC1000 nanoflow HPLC system (Thermo Fisher Scientific). The trap column used for the nano HPLC was a 2 cm × 75 μm capillary column packed with 3 μm C18-silica particles (Thermo Fisher Scientific), while the separation column was a 12.5 cm × 75 μm capillary column packed with 3 μm C18-silica particles (Nikkyo Technos, Tokyo, Japan). The flow rate of the nano HPLC was 300 nL/min. The separation was conducted using a 10–40% linear acetonitrile gradient for 70 min in the presence of 0.1% formic acid. Each sample was measured three times as technical replicates. The LC-MS/MS data were acquired in the data-dependent acquisition mode controlled using Xcalibur 4.0 (Thermo Fisher Scientific). The settings of the data-dependent acquisition were as follows: the resolutions were 70,000 for the full MS scan and 17,500 for the MS2 scan; the AGC targets were 3.0E6 for the full MS scan and 5.0E5 for the MS2 scan; the maximum IT was 60 msec for both the full MS and MS2 scans; the scan range was 310–1500 m/z for the full MS scan, with the top 10 signals being selected for the MS2 scan per one full MS scan; and the dynamic exclusion was 15 s.

Cleaned peptides from each fraction were analyzed using a Q Exactive Orbitrap mass spectrometer (MS; Thermo Scientific) coupled to an EASY-nLC1000 nanoflow HPLC system (Thermo Scientific). Reversed-phase chromatographic separation was performed on an in-house packed reverse-phase column (75 μm × 10 cm Halo 2.7-μm 160 Å ES-C18, Advanced Materials Technology). Labeled peptides were separated for 2 h using a gradient of 1%–30% solvent B (99.9% acetonitrile/0.1% formic acid) and Solvent A (97.9% water/2% acetonitrile/0.1% formic acid). The Q Exactive MS was operated in the data-dependent acquisition mode to automatically switch between full MS and MS/MS acquisition. Following the full MS scan from m/z 350–1850, MS/MS spectra were acquired at a resolution of 70,000 at m/z 400 and an automatic gain control target value of 10⁶ ions. The top ten most abundant ions were selected with a precursor isolation width of 0.7 m/z for higher energy collisional dissociation (HCD) fragmentation. HCD-normalized collision energy was set to 35%, and fragmentation ions were detected in the Orbitrap at a resolution of 70,000. Target ions that had been selected for MS/MS were dynamically excluded for 90 s. After quality control of protein homogenates for MS analysis, six AD and six NC retinas and ten AD and eight NC brains were included for further analyses.

Tryptic peptides were analyzed by nanoflow liquid chromatography–tandem mass spectrometry (nLC–MS/MS) using a Q Exactive Orbitrap mass spectrometer coupled to an EASY-nLC1000 nanoflow HPLC system (Thermo Scientific, San Jose, CA, USA). Reversed phase columns (75 µm internal diameter) were packed in-house to 10 cm with Halo C18 packing material (2.7 µm beads, 160 Å pore size, Advanced Materials Technology). Peptides were eluted from the column using a 2 h linear solvent gradient, starting with 100% Buffer A [0.1% formic acid], with steps from 0 to 40% of buffer B [99.9% (v/v) ACN, 0.1% (v/v) formic acid] over 110 min and 40% to 85% of buffer B over 10 min. Tandem mass spectrometry was performed in the data-dependent acquisition (DDA) mode with MS/MS of the top 10 most abundance precursor ions at HCD normalized collision energy of 35%. Xcalibur software (version 2.06) (Thermo, Fremont, CA) was used to perform spectral acquisition over the mass range of 400 to 1500m/z, automated peak recognition, detection of ions in the Orbitrap at a resolution of 70,000, HCD fragmentation of target ions, and dynamic exclusion of fragmented ions for 90 s.