DOI: 10.25345/c5cz32j35
title: MassIVE MSV000099362 - Proteomic analysis of human adipose tissue samples from amyloidosis patients and respective controls.
title: Proteomic analysis of human adipose tissue samples was performed with the FASP method. A total protein content of 200 ug for each sample were mixed with 200 uL of urea buffer (8M Urea in 0.1M Tris-HCl pH 8.5, UA buffer) and placed in Amicon filter units (0.5mL, 30kDa MW cutoff). The units were centrifuged at 14,000 rcf for 15 minutes twice and the flow-through was discarded. Afterwards, 100 uL of 0.05M Iodoacetamide in UA buffer were added and incubated for 20 minutes in the dark at room temperature. The filter units were centrifuged at 14,000 rcf for 10 minutes, the flow through was discarded, washed with equivalent volume of UA buffer and centrifuged again. Afterwards, 100 uL of ammonium bicarbonate buffer (50mM NH4HCO3 pH 8.5, in ultrapure water) were added to the filter unit and centrifuged at 14,000 rcf for 10 minutes twice. Finally, the samples were trypsinized in a humidified container overnight at room temperature in the dark using a solution of 50 mM NH4HCO3 pH 8.5, and trypsin to protein ratio 1:100 (w/w). The elution of peptides was performed after centrifugation of the filter units at 14,000 rcf for 10 minutes. Centrifugation was repeated after another addition of 40 uL 50mM NH4HCO3 pH 8.5 into the filter units. Eluted peptides were lyophilized and stored at -80C until use. LC/MS-MS experiments were performed on the Dionex Ultimate 3000 UHPLC system coupled with the high-resolution nano-ESI Q Exactive mass spectrometer (Thermo Scientific) in DDA mode. Each sample was reconstituted in 200 uL loading solution composed of 0.1 % v/v formic acid. A 5 uL volume was injected and loaded on the Acclaim PepMap 100, 100 um-2 cm C18, 5 um, 100 Angstrom trapping column with the ul PickUp Injection mode with the loading pump operating at flow rate 5 uL/min. For the peptide separation the Acclaim PepMap RSLC, 75 um-50 cm, nanoViper, C18, 2 um, 100 Angstrom column retrofitted to a PicoTip emitter was used for multi-step gradient elution. Mobile phase (A) was composed of 0.1 % formic acid and mobile phase (B) was composed of 100% acetonitrile, 0.1% formic acid. The peptides were eluted under a 240-minute gradient from 2% (B) to 80% (B). Flow rate and column temperature were set at 300 nL/min and 35C respectively. Gaseous phase transition of the separated peptides was achieved with positive ion electrospray ionization applying a voltage of 2.5 kV. For every MS survey scan, the top 10 most abundant multiply charged precursor ions between m/z ratio 300 and 2200 and intensity threshold 500 counts were selected with FT mass resolution of 60,000 and subjected to HCD fragmentation. Tandem mass spectra were acquired with FT resolution of 15,000. Normalized collision energy was set to 33 and already targeted precursors were dynamically excluded for further isolation and activation for 45 sec with 5 ppm mass tolerance. Raw files were analyzed with Proteome Discoverer 1.4 software package (Thermo Finnigan), using the Sequest search engine and the Uniprot human (Homo sapiens) reviewed database, downloaded on 2017 , including 20243 canonical entries. The search was performed using carbamidomethylation of cysteine as static and oxidation of methionine as dynamic modifications. Two missed cleavage sites, a precursor mass tolerance of 10 ppm and fragment mass tolerance of 0.05 Da were allowed while FDR was 1%. Label free quantification was performed by utilizing the precursor area values of each sample as defined by the Proteome Discoverer 1.4 software package. Samples were analyzed individually (not pooled) and assigned to distinct groups. Statistical analysis was performed using ProtE library in R language. Functional analysis was performed with the statistically significant and differentially regulated proteins using the default settings of Webgestaltweb interface.