Nanoacquity uhplc system

Samples were thawed, centrifuged, and peptides fractionated off-line using high-pH reversed-phase chromatography on a 3 μm Extend-C18 column (4.6 × 100mm; Agilent, UK) on an Agilent 1200 series LC system at 45 °C using a 30 min gradient from 3% to 40% acetonitrile in 0.1% ammonium hydroxide at 0.75 ml/min 30-s fractions were collected, dried, and stored at −20 °C until analysis.
For analysis by low-pH reversed-phase LC-tandem MS analysis, dried fractions were resuspended in 10 μL 3% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid, with 1 μL analyzed by low-pH reversed-phase chromatography using a nanoACQUITY UHPLC system (Waters) online to a Triple-Tof 6600 mass spectrometer (AB Sciex) as previously described [21] (link).

Purified NPC1–EGFP-containing membranes were subjected to reduction and alkylation, digestion with trypsin or endoproteinase GluC (New England Biolabs) similar to that described previously (Champion et al., 2012 (link); Williams et al., 2017 (link)). Samples were desalted by ZipTips (Millipore, Billerica, MA) according to the manufacturer's instructions and separated on a nanoAcquity UHPLC system (Waters Billerica, MA) prior to MS/MS analysis. Liquid chromatography (LC)/MS-MS analysis was performed on an amaZon ion trap instrument (Bruker Daltronics, Billerica, MA) running in data-dependent acquisition mode. Acquisition and processing were performed in the Mass Spectrometry and Proteomics Facility (MSPF) at the University of Notre Dame. Spectra were converted to .mgf files (MS/MS peak lists) using Data Analysis (Bruker) and subjected to database search with Mascot against the human UniProt database (downloaded 15 October 2009). MS tolerances were set to ±1.5 Da for MS1 and 1.7 Da for MS2. Owing to the small search space, traditional false discovery rate was not employed as it is not accurate for small-n datasets. A Mascot score of 40 and two peptides uniquely matching were used for ID cutoff.

For LC/SRM analysis, samples were injected onto a NanoAcquity UHPLC system with a 2G‐V/M Symmetry C18 trap column (180 μm × 20 mm, 5 μm) for desalting and chromatographic focusing before elution onto an Acquity HSS T3 analytical UHPLC column (75 μm × 250 mm, 1.8 μm) (all from Waters, Milford, MA, USA). The analytical column temperature was set at 40°C and the auto sampler temperature was maintained at 6°C. Trapping occurred for 3 min with 99.9% solvent A and 0.1% solvent B at a flow rate of 5 μL/min. A 60 min liquid chromatography gradient was initiated on elution from the trap column. The following gradient was used: 0 min, 3% B; 40 min, 50% B; 40.33 min, 85% B; 51.60 min, 85% B; and 52 min, 3% B. The flow rate was set at 0.3 μL/min. Solvent A was LC/MS‐grade water containing 0.1% formic acid. Solvent B was acetonitrile containing 0.1% formic acid.