| Metadata |
| datasetIdentifier | PASS00278 |
| datasetType | SRM |
| submitter | Aleksey Filimonov <filimonov.a.d@gmail.com> |
| submitter_organization | |
| lab_head_full_name | Alexander Archakov |
| lab_head_email | alexander.archakov@ibmc.msk.ru |
| lab_head_organization | the Russian Academy of Medical Sciences |
| lab_head_country | Russia |
| datasetTag | IBMC-JPR-2013-bio |
| datasetTitle | HPP Chr18: depleted plasma and HepG2 cells, 2013 |
| publicReleaseDate | 2013-08-01 00:00:00 |
| finalizedDate | |
| summary | The workflow for targeted proteome survey of proteins encoded by chromosome 18 was performed in three steps. SRM scouting enabled to acquire the preliminary data about the presence of proteotypic peptides in the depleted plasma (HPL) and HepG2 cells (HG2). For the observed signals from target peptides the developed acquisition and qualitative analysis methods were checked and confirmed by comparison of purified non-labeled analogues for the endogenous peptides. The scheduled MRM methods were developed from the confirmed MRM methods for distinct peptides and the final data were acquired from biosamples into this dataset named "bio". |
| contributors |
Victor Zgoda
Arthur Kopylov
Alexander Moisa
Olga Tikhonova
Andrey Lisitsa
Aleksey Filimonov
Ekaterina Ilgisonis
Alexander Archakov |
| publication |
Ponomarenko EA, Kopylov AT, Lisitsa AV, et al,
Chromosome 18 TranscriptoProteome of Liver Tissue and HepG2 Cells and Targeted Proteome Mapping in Depleted Plasma: Update 2013,
J. of Proteome Res., 2013 |
| growth | |
| treatment | |
| extraction |  - Human blood
- in amount of 4 to 5 ml
- was collected from 50 individuals in ETDA tubes
- following centrifugation at 5,000 rpm for 10 minutes to obtain plasma.
- The resulting plasma specimens
- were depleted on MARS (Agilent) Hu-14 immunoaffinity column (10 x 100 mm)
- and fraction of minor proteins were collected.
- The obtained fractions were desalted and concentrated using 3,000 KWCO Amicon Ultra-4 spin columns with regenerated celluloce-acetate memebrane.
- Proteins concentration in the final volume (from 100 to 200 μl) of th depleted plasma samples was measured using BCA assay.
- HepG2:
- 20 millioncells of HepG2 culture were lysed in 500 μl of hypotonic buffer,
- contained 10 mM of Na2HPO4, 0.3% SDS and 10 mM of beta-octyl glucopyranoside
- for 3-5 minutes
- at 4 degrees celsius.
- The obtained crude homogenated was sonicated for
- 2 minutes
- at 40% power
- with 5 cycles for 15 seconds.
- The resulting solution was
- centrifuged at 10,000 rpm
- for 10 minutes
- at 4 degrees celsius to decant cells debris.
- Diluted proteins extract was desalted and concentrated using
- Amicon Ultra-4 filter units
- with regenerated cellulose (3,000 KWCO)
- at 7,000g
- for 20 minutes
- at 4 degrees celsius,
- and the proteins extract buffer was exchanged
- to 10 mM Na2HPO4 pH 7.8
- in 200 μl final volume.
- The proteins concentration was measured
- using BCA assay
- with linear calibration of HSA concentration from 2.5 to 70 mg/ml.
- The measured total proteins concentration in the extract was 15.1 mg/ml.
|
| separation | - Peptides separation RP:
Separation of peptides after enzymatic digestion with trypsin was performed- on Eclipse-XDB-C18 column (3.0 x 100 mm, 3.5 um particle size)
in gradient of- mobile phase A (0.1% formic acid, 0.015% trufluoacetic acid) and
- mobile phase B (80% acetonitrile, 0.1% formic acid, 0.015% trifluoracetic acid)
- over 52 minutes.
- One μl of peptides was loaded
- on the column in 95% / 5% (A/B)
- for 3 minutes
- at flow rate of 100 μl/min
- following application of elution gradient
- for 42 minutes
- from 5% to 68% of mobile phase B.
- Column was washed
- at 100% of mobile phase B
- for 5 minutes
- at flow rate of 50 μl/min
- and following the amount of acetonitrile rapidly decreased for 2 minutes.
- The post-acquisition column reconstitution
- in an initial conditions (95% / 5%, A/B) was lasted
- for 6 minutes
- at constant flow rate of 100 μl/min.
- Plasma depletion HU-14:
- 1 ml of whole normal human plasma was diluted
- four times with sodium-phosphate buffer
- supplied by column manufacturer (mobile phase A).
- The diluted plasma was filtered through 0.22 μm syringe filters (Whatman)
- and a portion of 200 μl was loaded
- on Hu-14 column
- in isocratic mode of mobile phase A
- for 12 minutes
- at flow rate 1 ml/min,
- then
- increasing the flow rate to 1.5 ml/min
- and collecting the fraction of minor plasma proteins from 13 to 15 minutes.
- The elution of bound proteins was perfomed
- by applying mobile phase B
(sodium phasphate buffer supplied with 2M urea, pH 3.0) - for 7 minutes
- at flow rate of 3 ml/min.
- Post-analysis column eqiulibration
- in mobile phase A
- at flow rate of 1 ml/ml
- was lasted for 10 minutes.
- The proteins were collected
using on-line UV diod array detector at 280 nm wave length.
|
| digestion | - Proteins were dissolved to 5-10 mg/ml final concentration
- in lysis buffer contained
- 2.5 mM EDTA,
- 12 mM deoxycolic acid sodium salt,
- 2 M thiourea,
- 50 mM beta-octyl glucopyranoside and
- 75 mM triethylammonium hydrogencarbonate,
- pH 8.5.
- Before lysis,
- 6.7 mM of TCEP and
- 80 mM of DTT
- were added in buffer to reduce cysteins amimoacid residue.
- The reaction of cysteins reduction was incubated
- at 44 degrees celsius
- for 1 hour
- following addition of 4-vinylpyridine
- in 50% N,N-dimethylformamide
- to 37 mM final concentration for alkylation.
- The reaction was incubated
- for 45 miutes
- at ambient temperature
- and kept away from light.
- After reducing and alkylation completed, the proteins were dissolved
- to 1 mg/ml final concentration
- by 35 mM triethylammoniun hydrogencarbonate and
- the pH value was checked.
- Trypsin (200 ng/ml) modified (Promega) was added to proteins
- at 1 :100 ration (w/w) and
- incubated for 2 hours
- at 42 degrees celsius.
- Then additional aliquote of trypsinat 1 : 50 ration was added and reaction was incubated for 2 hours in a rest.
- The reaction was quenched
- by fomic acid
- to 3% finally
- and centrifuged
- at 14,000 rpm
- for 15 minutes
- at ambient temperature
- to sediment acid-labile detergent (deoxycolic acid).
- The resultingsupernatant was moved to glass vials and used for LC-MS-SRM analysis.
|
| acquisition | - LC-MS-SRM analysis was performed
- on QQQ6490 triple quadrupole mass spectrometer (Agilent)
- equipped Jet-Stream ion source in positive mode.
- Following parameters were set:
- capillary voltage was set at 4000 V,
- nozzle voltage 1200 V,
- drying gas (nitrigen) flow 15 L/min,
- sheath gas flow (nitrogen) 9 L/min,
- drying gas temperature 300 degrees celsius,
- sheath gas temperature 270 degrees celsuis,
- fragmentor voltage constant at 380 V,
- cell accelerator voltage varied from 3.8 to 6.2 V depending on m/z value and chagrge state of a precursor ion.
- The collision energies for precursor ions were calculated according the formula
CE(eV) = (([m/z]/100*3.6) - 4.8)+lg(m/z)*(m/z*0.00062)+(L/2.58),
where
m/z - is mass-to-charge value of precursor ion,
L - is length of peptide in number of amino acid residues,
and 0.00062 - is correction coefficient. - Measuring of the proteins concentration was
- accomplished in time-scheduled SRM analysis
- with retention time tolerance window set from +1.25 to +1.5 minutes depending on location of the certain peptide over gradinet of elution.
- The retention time was predicted previuosly using 113 in-house synthesized peptides deffered by hydrophobicity, pI value and length.
- The defined retention time for the peptides was plotted against gradinet of elution and refined using estimated prediction formula.
- The accuracy of prediction was estimated as 1.4minutes with SD+0.47 minutes.
- Calibration was performed
- using in-house synthesized peptides
- which were confidently registered during scouting of chromosome 18th proteins in human plasma and HepG2 cultivating cells.
- The synthetic peptides were combined in 16 sets according their predicted retention time and hydrophobocity to avoid overlapping retention times and eliminate concurrent transitions.
- Each set contains
- at least 30 peptides
- with 3-5 transitions per single peptide.
- The combined peptides were
- diluted in step-wise manner
- with one order of magnitude step
- giving a series of concentrations from 10e-14 M to 10e-9 M.
- Each calibration point was measured in five technical replicated.
- Calibration was successful if accuracy of 80-125% and SD+20% over at least three replications were observed.
- Measuring of proteins concentration in human plasma and HepG2 cells was accomplished using the same method as it was applied for calibration.
- The measured concentrations were further recalculated
- either in proteins copy number per one μl in case of human plasma
- or in proteins copy number per one cell in case of HepG2 culture
- taking into account known amount of the cells for measurements.
- The aquired transitions
- with correspoing MassHunter method files (*.m)
- was stored in the MassHunter data files (*.d)
- named with metanotation {HepG2|HPL_[A-F]}_CM*-r00[1-4].d (FreeBSD glob routine).
|
| informatics | See srm2prot reports from msr.ibmc.msk.ru/jpr2013 page. |
| instruments | Agilent Triple Quadrupole LC-MS/MS 6490 w/ Jet-Stream |
| species | Human |
| massModifications | none |
