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.