| Metadata |
| datasetIdentifier | PASS00251 |
| datasetType | MSMS |
| submitter | Robert Moritz <rmoritz@systemsbiology.org> |
| submitter_organization | |
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| datasetTag | HIV-1_DCs |
| datasetTitle | HIV-1 infection of Dendritic cells is affected by microvesicles |
| publicReleaseDate | 2014-06-01 00:00:00 |
| finalizedDate | |
| summary | HIV-1 is taken up by immature monocyte derived dendritic cells (iMDDCs) into tetraspanin rich caves from which the virus can either be transferred to T lymphocytes or enter into endosomes resulting in degradation. HIV-1 binding and fusion with the DC membrane results in low level de novo infection that can also be transferred to T lymphocytes at a later stage. Analysis of the protein complement of these MVs and or, comparing HIV-1 inocula before and after MV depletion showed that Heat Shock Proteins (HSPs) and nef were the likely DC maturation candidates cells. Recombinant HSP90 α and β and nef both induced DC maturation and ICAM-1 expression, greater when combined. These results suggest that MVs contaminating HIV-1 released from infected T lymphocytes may be biologically important during uptake by DCs in vitro and in vivo, especially as MVs have been detected in the circulation of HIV-1 infected subjects. |
| contributors | Sarah K Mercier
Heather Donaghy
Rachel Botting
Stuart G Turville
Andrew N Harman
Najla Nasr
Hong Ji
Ulrike Kusebauch
Luis Mendoza
David Shteynberg
Kerrie Sandgren
Richard J Simpson
Robert L Moritz
Anthony L Cunningham |
| publication | The microvesicle component of HIV-1 inocula modulates dendritic cell infection maturation and adhesion to T Lymphocytes
1, 2Sarah K Mercier, 1*Heather Donaghy, 1, 2Rachel Botting, 1, 2Stuart G Turville, 1Andrew N Harman, 1Najla Nasr, 4 Hong Ji, 3Ulrike Kusebauch, 3Luis Mendoza, 3David Shteynberg, 1Kerrie Sandgren, 4Richard J Simpson, 3Robert L Moritz, 1, 2*Anthony L Cunningham.
1Centre for Virus Research, Westmead Millennium Institute and 2University of Sydney, Sydney, NSW, Australia
3Institute for Systems Biology, Seattle, WA, USA
4La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia
Plos (submitted)
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| growth | MDDCs were generated from CD14+ monocytes isolated from peripheral blood mononuclear cells (PBMC) of anonymous blood donors and matured for 48 hours in maturation mix consisting of (v/v) final concentration 50pg/mL IL-1B, 5U/mL IL-6, 50pg/mL TNF-a and 5ng/mL PGE2 in 0.1% (w/v) bovine serum albumin (BSA, Sigma) PBS. |
| treatment | Immature day 5 MDDCs were infected with pelleted (HIV-1BaL(pellet)) or CD45depleted (HIV-1BaL(CD45-)) virus in 200uL media with a MOI of 3 at 37°C for 2 hours before resuspending at 1x106/mL and incubating further as required. MVs were added at 20uL/mL to match the CD45 concentration to HIV-1BaL(pellet) virus stocks |
| extraction | Viral, MV and parent cell preparations were lysed for 1 hour at 4ºC in SDS lysis buffer (10mM HEPES, 150mM NaCl, 1% (v/v) Triton®-X-100, 1ug/mL protease inhibitor cocktail (Sigma) at pH of 7.5), followed by centrifugation for 10 minutes at 16,000xg at 4ºC. The protein concentration was determined using the DC Protein Assay (Bio-Rad) according to the manufacturer’s instructions. |
| separation | For Mass Spectrometry, 30ug of each sample was prepared in SDS lysis buffer, run on 4-12% bis-tris gradient SDS-PAGE gel and stained with Brilliant Blue G (Sigma).
The 1D SDS-Gel lanes were sliced into 31 1mm x 5 mm bands using a disposable grid cutter (The Gel Company, USA) and in-gel digested with trypsin using an automated liquid handling procedure with a TECAN Freedom Evo liquid handling system (Männedorf, Switzerland). The 31 fractions were pooled (2 gel fractions per pool) and analysed by LC-MS/MS in duplicate. |
| digestion | Bands were in-gel digested with trypsin using an automated liquid handling procedure with a TECAN Freedom Evo liquid handling system (Männedorf, Switzerland). The 31 fractions were pooled (2 gel fractions per pool) and analysed by LC-MS/MS in duplicate. |
| acquisition | The in-gel digested samples were analysed with a Thermo-Fisher LTQ-Velos Orbitrap. MS1 data were collected over the range of 300 - 2000 m/z in the Orbitrap set at resolution 30,000. FTMS preview scan and predictive automatic gain control (pAGC) were enabled. The full scan FTMS target ion volume was 1x106 with a max fill time of 500 ms. MS2 data were collected in the LTQ-Velos with a target ion volume of 1x104 and a max fill time of 100 ms. The 20 most intense peaks from a preview scan of each full Orbitrap scan were selected (with a selection window of 2.0 Da) for collision-induced dissociation (CID) with wide-band activation. Dynamic exclusion was enabled to exclude an observed precursor for 180 seconds after two observations. The dynamic exclusion list size was set at the maximum 500 and the exclusion width was set at ±5 ppm based on precursor mass. Monoisotopic precursor selection and charge state rejection were enabled to reject precursors with z = +1 or unassigned charge state. |
| informatics | For Mass Spectrometry analysis, Thermo .RAW files were converted to mzXML format using MSConvert (ProteoWizard, ) and searched with X!Tandem version 2010.10.01.1. Spectra were searched against the SwissProt/Uniprot database (Ver 2012_05), common lab protein contaminants, and decoys. The contaminant database was a modified version of the common Repository of Adventitious Proteins (cRAP, www.thegpm.org/crap) with the Sigma Universal Standard Proteins removed and human angiotensin II and [Glu-1] fibrinopeptide B (MS test peptides) added, for a total of 66 entries. Decoys were generated with Mimic (www.kaell.org), which randomly shuffles peptide sequences between tryptic residues but also retains peptide sequence homology in decoy entries. Decoy protein sequences were interleaved among real entries, randomly alternating between listing the real entry or the decoy entry first.
Search criteria used for X!Tandem was modified for wide precursor mass tolerance of 0.1 Da to improve the performance of the accurate mass binning tool available in Peptide Prophet. Peptides were assumed to be semi-tryptic (cleavage after K or R except when followed by P). Semi-tryptic peptides with up to 2 missed cleavages were allowed. The search parameters included a static modification of + 57.021464 Da at C for carbamidomethylation by iodoacetamide and a potential modifications of + 15.994915 for oxidation at M for oxidation, -17.026549 Da for deamidation at N-terminal Q and -18.010565 Da for loss of water at N-terminal E from formation of pyro-Glu as well as -17.026549 Da at N-terminal carbamidomethylated C for deamidation from formation of S-carbamoylmethylcysteine. MS/MS data were analysed using the Trans Proteomic Pipeline [3] version 4.5 Rev.2. Peptide spectrum matches (PSM) generated were analysed with Peptide Prophet to assign each PSM a probability of being correct. Accurate mass binning was employed to promote PSMs whose theoretical mass closely matched the observed mass of the precursor ion, and to correct for any systematic mass error. Decoys and the non-parametric model option were used to improve PSM scoring. The Peptide Prophet scores for a given analysis were then combined in iProphet [4], which assigns a probability for each unique peptide sequence based on how often it is observed at different charge states with different modifications and by different search engines, as well as whether other peptides from the same protein are also observed. Protein identifications were inferred with Protein Prophet [3]. The false discovery rate for a given Protein Prophet probability was calculated by the number of decoy protein inferences at that probability. Only proteins identified at Protein Prophet probabilities corresponding to a false discovery rate less than 1.0% were reported. Quantitative analysis was performed by spectral counting and proteins were assigned a spectrum share for abundance within the sample and total spectrum values for each SDS 1D-lane was compared to provide a quantitative value between samples. The identified peptides and their inferred proteins are published as a supplemental file |
| instruments | Thermo Scientific LTQ-Velos Orbitrap |
| species | Human |
| massModifications | static: C+57.021464
variable: M+15.994915
N+Q deamidation -17.026549 Da
Loss of water at N-terminal E-18.010565 Da for loss of water at N-terminal E-18.010565 |
