Collection of human blood and extraction of plasma
Human volunteers (male; mean weight: 65 kg; mean age: 24 y) were exposed to high altitude (4267 m; 14,000 ft; pO2 12.81 kPa) for 7 days (HAD7), 30 days (HAD30) and 120 days (HAD120) subsequent to their stay in normoxic conditions (Baseline). All were reported to be acclimatized and free of altitude illnesses. Obesity, hypertension, addiction, lung diseases and other diseases resulted in exclusion from study. Blood samples (2 ml) were collected from the antecubital vein while the volunteers (n=3 per group) lay prone. The sample collected was subsequently centrifuged (3500 rpm; 15 mins) and plasma collected in fresh tubes. Protease inhibitor (PI) cocktail (Cat. no. P8340, Sigma, USA) was immediately added to all collected samples and tubes stored at -80 ºC.
Quantitative proteomics using LC-MS/MS
Sample preparation
The plasma/lung tissue homogenate were pelleted using lyophilizer. The lyophilized sample was then re-suspended in lysis buffer. Samples were again centrifuged and both supernatant and lysate separately retained. Pellet was further treated with ToPI protein isolation buffer (K-0011) and centrifuged. The lysate was transferred back into tube containing the retained supernatant. Protein estimation of this total lysate was carried out using ToPA Bradford Protein Assay kit. 100 µg protein per sample were processed to remove reducing, alkylating and interfering substances from the sample before MS analysis. Overnight trypsin digestion was then performed on the sample. iTRAQ reagents were added to the digested peptides for labeling of samples. SCX fractionation of each sample was done. Elution was performed at 75 mM, 150 mM, 450 mM ammonium acetate for each sample. The individual samples were collected and analyzed by nano-LC-MS/MS and combined for MudPIT.
LC-MS/MS
Samples were desalted using ZipTip. Then, samples were lyophilized and subsequently re-suspended in appropriate mobile phase for LC-MS/MS. Elution of peptides was done using linear acetonitrile gradient (5 to 45%) over 180 minutes and immediately followed by high and low organic washes lasting 20 minutes. Samples were injected into an LTQ XL mass spectrometer (Thermo Scientific) using a nano-spray source with 1.8kV spray voltage and temperature of 180 ºC in ion transfer capillary. The Top 5 data dependent method was used for a full MS scan (m/z 400-1500) followed by MS/MS scan (5 most abundant ions). Ratio >1.5 was considered up-regulated, ratio <0.67 was considered downregulated and ratio between 1.5 – 0.67 were considered non-significant. 100-fold was the maximal allowed expression ratio.