Agilent 6540 mass spectrometer

Selected protein spots were excised from 2-DE gels and digested with trypsin, and the peptides were extracted based on standard techniques [79 (link)] at Proteomics International, Nedlands, Western Australia. Protein spots were identified using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) with an Agilent 1260 Infinity HPLC system (Agilent) coupled to an Agilent 1260 Chipcube Nanospray interface (Agilent) on an Agilent 6540 mass spectrometer (Agilent). Peptides were loaded onto a ProtID-Chip-150 C18 column (Agilent) and separated with a linear gradient of water/acetonitrile/0.1% formic acid (v/v). To identify proteins of interest, the raw spectra were submitted to the MSPnr100 database search using MASCOT sequence matching software (Matrix Science, London, UK; http://www.matrixscience.com). Database interrogation parameters for LC-MS/MS analysis on the Agilent 6540 mass spectrometer were as follows: taxonomy (Viridiplantae: green plants), variable modifications (carbamidomethyl, oxidation of methionine residues), mass values (monoisotopic), protein mass (unrestricted), peptide mass tolerance (±0.2 Da), MS/MS tolerance (± 0.2 Da), peptide charge state (2+, 3+ and 4+), enzyme (trypsin), and maximum missed cleavages (1). Only significant hits, as defined by the MASCOT probability analysis (p ≤ 0.05) were accepted.

Prominent bands in the polyacrylamide gel were excised and submitted to Proteomics International (Perth, Western Australia, Australia) for identification. Protein samples were trypsin digested and peptides extracted according to standard techniques32 (link). Peptides were analysed by electrospray ionisation mass spectrometry using the Agilent 1260 Infinity HPLC system (Agilent Technologies, Santa Clara, CA, USA) coupled to an Agilent 6540 mass spectrometer (Agilent). Tryptic peptides were loaded onto a C18 column 300 SB, 5 μm (Agilent) and separated with a linear gradient of water/acetonitrile/0.1% formic acid (v/v). Spectra were analysed to identify proteins of interest using Mascot sequence matching software (Matrix Science Inc., Boston, MA, USA) with Ludwig NR database. A match was considered positive if two or more peptides map to the same protein and were within the predicted size parameters as estimated by the standard on the acrylamide gel.

Whole cell protein lysate extracted from a NPC cell line (HK1) was incubated with RPeL27 or RPeL43 primary antibody immobilized onto Dynabeads Protein G (Life Technologies) for 10 minutes with rotation at room temperature. The beads were vortexed for 30 seconds, and the vial was placed on DynaMag Magnet (Life Technologies) to separate them from the solution. Following removal of supernatant, sample buffer was added to the vial and this mixture was boiled for 10 minutes. The resulting immunoprecipitates were resolved on SDS-PAGE and stained with Coomassie Blue (Bio-Rad). Protein bands of interest were excised and sent to a service provider for protein identification. Identification analysis was via the Electrospray Ionization-Quadrupole-Time-of-Flight (ESI-QUAD-TOF) method using the Agilent 1260 Infinity HPLC system coupled to Agilent 6540 mass spectrometer (Agilent) and the Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) method using the 5800 Protein Analyzer (AB Sciex). Identities of proteins were extracted using Mascot software (Matrix Science) on Swiss-Prot database. The search parameters utilized trypsin as the proteolytic enzyme with one missed cleavage permitted and the carbamidomethylation of cysteines and oxidation of methionines as variable modifications. Mass tolerance was 50 ppm and 0.4 Da for precursor and fragment ions, respectively.