| Metadata |
| datasetIdentifier | PASS00875 |
| datasetType | SRM |
| submitter | Ekaterina Ilgisonis <ilgisonis.ev@gmail.com> |
| submitter_organization | Institute of Biomedical Chemistry, Moscow |
| lab_head_full_name | Zgoda V.G. |
| lab_head_email | victor.zgoda@gmail.com |
| lab_head_organization | Institute of Biomedical Chemistry |
| lab_head_country | Russian Federation |
| datasetTag | astronaut_plasma |
| datasetTitle | Quantitative proteomics of astronaut candidates plasma |
| publicReleaseDate | 2016-05-04 00:00:00 |
| finalizedDate | 2016-05-05 02:36:37 |
| summary | This work was aimed at estimating the concentrations of proteins encoded by human
chromosome 18 (Chr 18) in blood plasma of 54 healthy male volunteers. These young male persons are certified by the medical evaluation board as healthy subjects ready to space flight training. Over 260 stable isotope-labeled peptide standard (SIS) as well as corresponding non-labeled proteotypic peptides were synthesized to perform the measurements of the proteins encoded by Chr 18. Selected reaction monitoring (SRM) with SIS allowed to estimate the levels of 84 out of the 276 proteins encoded by Chr 18 in the samples from 54 donors. |
| contributors | Arthur T. Kopylov,
Ekaterina V. Ilgisonis,
Alexander A. Moysa,
Olga V. Tikhonova,
Maria G. Zavialova,
Svetlana E. Novikova,
Victor G. Zgoda,
Andrey V.Lisitsa,
Elena A. Ponomarenko,
Alexander I. Archakov |
| publication | Kopylov et al.,Targeted quantitative screening of Chromosome 18 encoded proteome in the blood plasma of astronaut candidates, J Proteome Res., submitted 2016-05-04 |
| growth | |
| treatment | |
| extraction | |
| separation | |
| digestion | Venous blood was collected from the volunteers to the EDTA Vacutainer plasma tubes (BD, the USA). The blood samples were processed according to the manufacturer’s instructions. The plasma supernatant was filtered through 0.22 µm cellulose-acetate filters (Whatman, NJ, USA) and stored at -80°C. The plasma samples obtained were depleted using two MARS (Multi-Affinity Removal System) Hu-14 columns (10 x 100 mm) according to the manufacturer’s protocol (Agilent, USA). The collected fractions containing unbound proteins were desalted using cellulose-acetate 5K MWCO (Agilent, USA) spin columns and concentrated to 50 μL final volume. The protein concentration was determined using the Micro BCA protein assay (Thermo Scientific, Rockford, IL, USA).
The aliquot of plasma protein (100 µg) was supplemented with a lysis buffer (4% SDS in 0.1 M Tris-HCl pH 8.5) to a volume of 30 µL and placed on the Microcon YM-10 filters (Millipore, USA) for the tryptic digestion according to the FASP protocol9. Briefly, the samples were supplemented with 100 μl of a reducing solution (0.1 M 1,4-dithiothreitol (DTT) in 0,1 M Tris-HCl pH 8,5), incubated for 40 min at 56°C, and centrifuged at 10 000 g for 15 min at 20°C. The samples were then washed 2 times with 200 μl of solution of 8M urea in 0,1 M Tris-HCl, pH 8,5, followed by centrifugation at 10 000 g at 20°C for 15 min. The process of carbamidomethylation was carried out in 100 μl of 50 mM iodoacetamide (Sigma-Aldrich) in 0.1 M Tris-HCl, pH 8.5, for 30 min at room temperature. The samples were then washed 2 times with 200 μl of solution of 8M urea in 0,1 M Tris-HCl, pH 8,5, followed by centrifugation at 10 000 g at 20°C for 15 min and 3 times with buffer for tryptic cleavage (50 mM tetraethylammonium bicarbonate pH 8.5). To carry out the enzymatic hydrolytic cleavage the sample was supplemented with 50 μl of buffer for tryptic cleavage containing trypsin (20 ng/μl) (Promega; Madison, WI, USA). After 16 hours of incubation at 37°C, the samples were supplemented with 50 μl of 30% formic acid, containing all isotope-labeled standards at various concentrations and centrifuged at 10 000 g for 10 minutes. Pass through peptide fractions were used for further SRM analysis.
After tryptic digestion all samples were dissolved in 100 μL 3% (v/v) formic acid. |
| acquisition | |
| informatics | Targeted quantitative screening for 267 proteins encoded by chromosome 18 was performed in the digested plasma samples. Manual selection of the unique proteotypic peptides and the most intense transitions was performed on the basis of the SRM scouting of chromosome 18 results 6. A final list of peptides for SRM assay was complied according the criteria described in Table 1.
For each protein, one standard peptide with three transitions was used. The peptides selected were arranged into one SRM assay. The information (m/z of precursors, m/z of transition ions, CE values, b, y ions transition ions and MS platform were used for the analysis) on SIS and the target peptides is provided in Supplementary Table 2.
Each SRM experiment was repeated in 3 technical runs. The results were manually inspected using the Skyline software12 to find transitions that were similar to those in the target peptides. For interference screening we applied the criteria described in Percy at al13. Briefly, the peptide was considered to be detected in the run if the differences between relative intensities for three transitions of endogenous and isotopically labeled peptide did not exceed 25% in the run and the transition chromatographic profiles of endogenous peptide were identical to the corresponding transitions of stable-isotope labeled peptide.
Calibration curves were obtained for each of the desired peptides using the mixtures of purified synthetic native peptides in the concentration range of 10-8 – 10-13 М and its isotopically labeled analogues were added at the concentration of 10-9 M – 10-12M. All calibration curves were linear in the range of 10-8 – 10-13 М and showed the coefficient of linear regression equal to 0.95.
Prior to the sample processing, the performance of the LC-SRM platforms used was validated by obtaining the calibration curves of the corresponding set of SIS and synthetic natural peptides. Moreover, after five LC-SRM runs we verified the relevance of calibration by analyzing one of the calibration peptide solution at 10-10M.
The detection limit was defined as the lowest concentration determined on the linear part of calibration curve. It varies for different peptides in the range from 10-13 M to 10-11 M.
Labeled/unlabeled peptide peak area ratios were used to calculate the concentration of the targeted peptide in a sample.
Cpept = Clab * Spept/Slab ,
Where:
Cpept – target peptide concentration,
Clab – labeled peptide concentration,
Spept – area of target peptide peak,
Slab - area of labeled peptide peak. |
| instruments | Agilent Triple Quadrupole LC-MS/MS 6490 w/ Jet-Stream |
| species | Human |
| massModifications | 13C(6)15N(2) (C-term Leu) |
