HIV-Tat treatment of human neurons in vitro over time: towards a model for the molecular pathology of HIV-associated neurocognitive disorders (~90% cell culture confluence dataset)
We have used LC-MSMS discovery proteomics to identify the quantitative, cell-wide changes that occur when non-transformed, differentiated human neurons are treated with HIV-Tat over time. This may represent a novel cell culture model for initial HIV-associated neurocognitive disorder progression.
Sample Processing Protocol
Cells were lifted using tryp-LE (Gibco), washed with phosphate buffered saline (PBS) and pelleted at 600g. Supernatants were aspirated and cell pellets flash frozen in liquid nitrogen. Cell pellets were lysed in the presence of 1x protease inhibitors (Sigma Aldrich) and 1x phosphatase inhibitors (PhosStop, Roche) using a 1x Tris/ HCl pH7.6 radioimmunoprecipitation assay (RIPA) buffer excluding dithiothreitol (DTT), which otherwise interferes with bicinchoninic acid (BCA) protein quantitation. Cell lysates were incubated at 4°C for 1 hour with 1μl Benzonase nuclease ultrapure (Sigma Aldrich). Subsequently, lysates were centrifuged at 14 000g for 10 minutes and supernatants containing the cell protein content were transferred to new 1.5ml plastic tubes. Protein quantitation was then performed using the Pierce BCA Protein Assay Kit microplate procedure (ThermoScientific) with BSA as the standard. Tryptic peptides for MS analysis were generated on 30kDa molecular weight cutoff (MWCO) filters (Millipore) using the Filter-Aided Sample Preparation (FASP) protocol (Wiśniewski et al. 2009). Samples were desalted using reverse-phase C18 chromatography (C18 columns manufactured in-house from C18 discs, Millipore) prior to MS analysis. Briefly, C18 columns were equilibrated with 80% mass spectrometry (MS) grade acetonitrile (ACN, Sigma Aldrich) acidified with 0.1% formic acid (FA). 10μg peptide from each sample was loaded on separate C18 columns - one column per sample – and desalted by the addition of 2% ACN acidified with 0.1% FA. Finally, peptides were eluted in to glass vials with 60% ACN acidified with 0.1 % FA. Desalted peptides were dried at room temperature using a SpeedyVac (Savant) and resuspended in 0.1% FA (Sigma Aldrich) in ultrapure dH2O to a final concentration of 250ng/μl, which was estimated from BCA quantitation at the protein level on the assumption of no protein loss during FASP. Liquid chromatography tandem MS (LC-MS2) analysis was performed on a Q Exactive MS instrument coupled to a Dionex Ultimate 3500 RSLCnano ultra high-pressure nano liquid chromatography system (both Thermo Fischer Scientific). All samples were injected into the sample loop at a flow rate of 5μl/min in 2% buffer B (0.1%FA/ACN) and 98% buffer A (0.1% FA/ultrapure dH2O). Samples were then loaded onto a 40cm, 75μm diameter, C18 reverse phase analytical column (3.6μm diameter Aeris Peptide C18 packing material, packed in house; Phenomenex 04A-4507) and subsequently eluted during a 120-minute segmented gradient (4-30% buffer B) at a flow rate of 400nl/min. At the end of each sample run, buffer B was ramped to 80% for 10min, which was then followed by a 20-minute wash cycle, from 2% to 50%, and a 20-minute column equilibration. After every fourth sample, a longer wash (80 minutes) was run. Mass spectra were collected with settings essentially as described by Nel et al. (2015) with the following amendments: intensity threshold for ion selection was set at 6.3e3; isolation window for tandem mass spectrometry (MS2) was set at 2.0 m/z; and MS2 spectra were acquired at a target value of 5e4.
Data Processing Protocol
Raw mass spectra were processed for peptide peak identification and quantitation by MaxQuant software (version 1.5.03) (Cox et al. 2009). Default MaxQuant settings were used with the following modifications: labeling was set to singlet, label free quantitation (LFQ) was selected for relative label free quantitation, ‘match between runs’ was selected, and only peptides unique to a protein group were used for quantitation. MaxQuant’s inbuilt search engine, Andromeda (Cox et al. 2011), was used for protein group identification using the UniProt human proteome database as the reference (FASTA, downloaded on 12/01/15, containing 121996 sequence entries). The datasets generated by MaxQuant was filtered to exclude protein groups that mapped to a sequence in the decoy database or that mapped to a known contaminant. In addition, only protein groups that had an LFQ measurement in each experimental sample, and had a satisfactory posterior error probability (PEP) score (< 0.01), were taken forward for downstream data analyses.
Kim Gurwitz, University of Cape Town
Prof. Jonathan Blackburn, Department of Integrative Biomedical Science, Blackburn Group, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, South Africa ( lab head )
Gurwitz KT, Burman RJ, Murugan BD, Garnett S, Ganief T, Soares NC, Raimondo JV, Blackburn JM. Time-Dependent, HIV-Tat-Induced Perturbation of Human Neurons In Vitro: Towards a Model for the Molecular Pathology of HIV-Associated Neurocognitive Disorders. Front Mol Neurosci. 2017 May 29;10:163. eCollection 2017 PubMed: 28611588