Ltq orbitrap velos mass spectrometer

The 60 fractions from the two experiments were sequentially reconstituted in 0.1% FA and subjected to low‐pH RPLC‐MS. A trap column (75 μm × 2 cm) (Acclaim® PepMap100 C18 column, 3 μm, 100 Å; Thermo Fisher Scientific) and an analytical column (75 μm × 25 cm) (Acclaim® PepMap RSLC C18 column, 2 μm, 100 Å; Thermo Fisher Scientific) with a flow rate of 300 nl/min were operated on an Easy‐nLC 1000 system, which was coupled to a LTQ Orbitrap Velos mass spectrometer (Thermo Finnigan, San Jose, CA). MS acquisition was performed in a data‐dependent mode. The parameter settings for MS can be found in previously published papers (Castaneda et al., 2017 (link)).

The C-terminal ATTO 565-biotin-labeled Tat was separated by SDS-PAGE, identified by staining with Coomassie Blue, and in-gel digested. For multistage MS/MS experiments, the LC–MS analysis was performed using an LTQ Orbitrap Velos mass spectrometer (Thermo Finnigan, San Jose, CA, USA) equipped with an nESI source to allow simultaneous recording of full-scan mass and collision-induced dissociation (CID) spectra. Data were acquired in data-dependent mode. For peptide mapping of the Tat protein, the CID spectra were compared to the sequence of HIV-1 Tat using Sequest (Bioworks; Thermo Electron Corp., Waltham, MA, USA). PE (control group) is an accessory photosynthetic pigment found in red algae. It exists in vitro as a 240 kDa protein with 23 phycoerythrobilin chromophores per molecule (554061 BD Biosciences).

LC-MS/MS analysis of ITRAQ-labeled peptides was carried out on an LTQ-OrbitrapVelos mass spectrometer (Thermo Electron, Bremen, Germany) interfaced with Agilent’s 1100 series nanoflow liquid chromatography system (Agilent Technologies, Santa Clara, CA). Peptides from each fraction were enriched and washed on a trap column (75 µm × 2 cm, 5 µm, 120Å, Magic C₁₈ AQ; Michrom Bioresource), at a flow rate of 3 µL/min and then resolved on an analytical column (75 µm × 10 cm, 5 µm, 120Å, Magic C₁₈ AQ; Michrom Bioresource) at a flow rate of 300 nL/min using a linear gradient of 5–40% solvent B (90% acetonitrile in 0.1% formic acid) over a period of 65 min. The total run time per sample was 85 min. The resolved peptides from analytical column were delivered to mass spectrometer through an emitter tip (8 µm, New Objective, Woburn, MA). LC-MS/MS data were acquired in a data-dependent manner in FT mode. MS spectra were acquired with a window of m/z 350 to 1800. The 20 most-abundant precursor ions were selected for fragmentation from each MS scan. Data were acquired at MS resolution of 60,000 (m/z 400) and MS/MS resolution of 15,000. Precursor ion fragmentation was carried out using higher-energy collision mode with normalized collision energy of 41%. Monoisotopic precursor selection was enabled and the precursor ions that were selected for fragmentation was dynamically excluded for 50 sec.

All data were collected on a LTQ Orbitrap Velos mass spectrometer (Thermo Electron, Waltham, MA) coupled to a Next-Gen 3 high performance liquid chromatography system (Agilent Corporation, Santa Clara, CA) through 75 um × 70 cm columns packed with Phenomenex Jupiter C-18 derivatized 3 um silica beads (Phenomenex, Torrance, CA). Samples were loaded onto columns with 0.05% formic acid in water and eluted with 0.05% formic acid in Acetonitrile over 99 min. Ten data-dependent MS/MS scans were recorded for each survey MS scan (70 K nominal resolution) using normalized collision energy of 35, isolation width of 2.0 m/z, and rolling exclusion window lasting 30 s before previously fragmented signals are eligible for re-analysis.

Peptides were eluted into 96-well microtitre plates with 20 μl 40% MeCN, 0.5% AcOH followed by 20 μl 50% MeCN, 0.5% AcOH. Organic solvents were removed by vacuum centrifugation in a speed-vac and dried to ∼2 μl. The peptides were reconstituted with 10 μl of 2% MeCN, 0.5% AcOH, 0.1% TFA. Five microlitres of this eluate was analysed by online reversed-phase C18 nanoscale LC-MS/MS on an LTQ-Orbitrap Velos mass spectrometer (Thermo Electron) using a top10 higher-energy collisional dissociation (HCD) fragmentation method as described previously34 (link). The LC-MS analysis was performed with a nanoflow Easy –nLC system (Proxeon Biosystems) connected through a nano-electrospray ion source to the MS. Peptides were separated by a linear MeCN gradient for 220 min in a 15-cm fused-silica emitter in house packed with reversed-phase ReproSil-Pur C18-AQ 3 μm resin (Dr Maisch GmbH). Full-scan MS spectra were acquired from 350 to 1,750 m/z at a target value of 1e6 and a resolution of 30,000, and the HCD-MS/MS spectra were recorded at a target value of 5e4 and with resolution of 7,500 using a normalized collision energy of 40%.