Analytical protocols

1. Chromosome 1

2. Chromosome 2

3. Chromosome 3

4. Chromosome 4

5. Chromosome 5

  • We dispose of 2D-LC-MS/MS platform including nanoLC and an Agilent 6510 qTOF mass spectrometer.

6. Chromosome 6

7. Chromosome 7

8. Chromosome 8

9. Chromosome 9

10. Chromosome 10

11. Chromosome 11

Offgel fractionation of peptides that were obtained by trypsin digestion of proteins
CID/ETD dissociation and Orbitrap MS/MS analysis
Mascot (v. 2.3) search

12. Chromosome 12

13. Chromosome 13

  • For proteomic profiling, tryptic-digested placental tissue proteins were fractionated using three different methods, including hydrophilic interaction chromatography, strong cationic exchange chromatography, and OFFGEL electrophoresis according to the manufacturers’ protocols (Agilent Technology). Moreover, the membrane fraction method for identifying membrane proteins in placental tissue was conducted as previously described. LTQ-Orbitrap mass spectrometry (Thermo Fisher San Jose, CA) was used for acquiring mass spectra to protein identification..

14. Chromosome 14

nano-LC MS-based workflows (Q-exactive plus, Orbitrap XL, Orbitrap Velos-pro, Triple-TOF 5600 and QTrap 4000, 5500 and 6500) for:
  • proteins identification
  • quantitative proteomics
  • targeted analysis
  • top-down analysis
Contact: jerome.garin at cea.fr

15. Chromosome 15

16. Chromosome 16

17. Chromosome 17

Provided by Emma (Yue) Zhang.
Twenty microliters of lysis buffer (2% SDS in 50 mM NH4CO3) was added to 10 μL of cell lysate. Cells were solubilized by sonication using 20 s bursts, followed by cooling on ice for 20 s, in a process that was repeated for 10 times. The entire extract was concentrated down to 15 μL in a speed vacuum and loaded onto a gel (SDS-PAGE, 4−12% gradient) to separate proteins by molecular weight. After staining with Coomassie blue, each gel lane was cut into five individual slices as shown in Figure S1 (Supporting Information). Each slice was further minced into smaller pieces (approximately 0.5 mm2). The gel slices were washed with 600 mL of water for 15 min and centrifuged, supernatant was removed, and 50% ACN was added (1 mL), followed by shaking until no visible Coomassie stain remained. Proteins were then reduced with dithiothreitol (DTT) by adding 250 μL of 10 mMDTT in 0.1M NH4CO3 and incubated for 30 min at 56 °C. Samples were subsequently alkylated at room temperature and in the dark for 80 min with 250 μL of 55 mM iodoacetamide (IAA) in 0.1 M NH4CO3. Trypsin digestion reagent (200 μL; 10 ng/mL of trypsin in 50 mM NH4CO3, pH 8.0) was added, and samples were incubated for 30 min at 4 °C. The trypsin concentration was based upon an estimate of approximately 0.1−0.5 mg of protein per gel slice and adjusted as necessary. The solution was then replaced with 50 mM NH4CO3 to cover the gel pieces (50 μL) and incubated overnight at 37 °C to elute peptides from the gel. Following this step, supernatant was removed and stored. Gel pieces were further extracted with 5% formic acid (30 μL) and acetonitrile (ACN, 400 μL) at 37 °C for 10 min and then twice with 5% formic acid (30 μL) and ACN (200 μL). The formic acid solution containing tryptic peptides was combined with the previous supernatant and concentrated to 5−10 μL. The concentrated solution (trypsin-digested peptides) was subjected to LC−MS analysis.

18. Chromosome 18

Instrument / parameterQ-exactiveAgilent 6490 QQQTSQ-Vantage QQQ
LC flow rateNanoflow-Dionex 300 nL/minMicroflow Agilent 1200 300 μL/minNanoflow-Dionex 300 nL/min
Trap15 cm х 0.75 mm RP C18 Thermo (2 μM, 100 Å)Nanobore RP C18 5 cm x 2.1 mm (2 μM, 100 Å)15 cm х 0.75 mm RP C18 Thermo (2 μM, 100 Å)
Sample injected Mass / Max volume1 μg / 1 μL10 μg / 20 μL1 μg / 1 μL
Sample injected (trapping column)1 µg (С18 0.05*20mm)10 µg (none)1 µg (С18 0.05*20mm)
Gradient length for data acquisition120 min45 min120 min
Peak width at half height20 sec5 sec20 sec
МАХ ejected volume1 µL20 µL1 µL
Sensitivity (by BSA as a standard)10-11 M (10 amole/μL)10-14 M* (0.1 amole/μL)10-12 M (1 amole/μL)
Survey scan resolution140 00020002000
Protein identificationMASCOT (score>50+decoy)Coincidence RT ± 10 s; ≥3 transitions with S/N > 7; identical transitional profilesCoincidence RT ± 60 s; ≥3 transitions with S/N > 7; identical transitional profiles
* Sensitivity for the irreversible binding 10-18М (0.1 ymole/μL) by BSA and CYP102 as the standard Kopylov et al., 2013(external link)

19. Chromosome 19

20. Chromosome 20

21. Chromosome 21

22. Chromosome 22

23. Chromosome X

24. Chromosome Y

25. Mitochondrial Chromosome