| Metadata |
| datasetIdentifier | PASS00094 |
| datasetType | SRM |
| submitter | Aleksey Filimonov <filimonov.a.d@gmail.com> |
| submitter_organization | IMSB |
| lab_head_full_name | Alexander Archakov |
| lab_head_email | alexander.archakov@ibmc.msk.ru |
| lab_head_organization | IMSB |
| lab_head_country | Russia |
| datasetTag | IBMC-JPR-2012-bio1 |
| datasetTitle | Human Chr18. Transition groups for 23 light endogenous peptides corresponding to 19 proteins in biosample. |
| publicReleaseDate | 2012-11-01 00:00:00 |
| finalizedDate | 2012-12-11 18:19:25 |
| summary |
Dataset bio1 was acquired to detect and quantitate proteotypic peptides of 19 human proteins encoded by chromosome 18. SRM-data was acquired from 23 light endogenous peptides of Human Depleted Plasma (HPL), Human Liver (HLV) and Hep G2 cell line (HG2). |
| contributors |
Victor Zgoda
Arthur Kopylov
Alexander Moisa
Olga Tikhonova
Andrey Lisitsa
Aleksey Filimonov
Ekaterina Ilgisonis
Alexander Archakov |
| publication |
Zgoda, VG, Kopylov, AT, Archakov, AI, et al, "Chromosome 18 transcriptome profiling and targeted proteome mapping in depleted plasma, liver tissue and HepG2 cells", Journal of Proteome Research, submitted 2012-08-31 |
| growth | |
| treatment | |
| extraction | |
| separation |
Peptides of biosample (depleted human plasma, human liver tissue, Hep G2 cell line) proteins were reconstituted in 100 μL of 5% FA after digestion with trypsin to get final concentration of 1 μg/μL. The peptides mixture was analyzed on an Agilent G6410A triple quadrupole (QQQ) mass spectrometer equipped with an HPLC-Chip nano-ESI interface. For LC-MS/MS analysis 1 μL of sample (human plasma or liver tissue) was loaded onto a 40 nL trapping column (4 mm length, 5 μm particle size) and then separated on a Zorbax 300-SB C-18 graphitized 150 mm x 75 μm analytical column (5μm particle size). Peptides were loaded at a flow rate of 2 μL/min in isocratic solution C (5% acetonitrile with 0.1% FA and 0.05% trifluoracetic acid) for 3.5 minutes. Mobile phase A was 0.1% FA and mobile phase B was 0.1% FA in 80% acetonitrile, and a linear gradient of 5-100% B in 57 minutes was used in following conditions: 0 – 2 minutes 5% of mobile phase B, 2 – 41 minutes from 5 to 65 % of mobile phase B, 41 - 43 minutes from 65 to 100% of mobile phase B, them remaining 100% of B for 5 minutes until 48 minute, and 48 – 51 minutes from 100 to 5% of B. Column reconstitution to initial gradient conditions was provided for 6 minutes.
See also the detailed general Separation and Acquisition Protocol at
http://msr.ibmc.msk.ru/jpr2012/SepAndAcqProt
|
| digestion |
Proteins were dissolved in denaturating buffer contained acid-labile detergent to 10 mg/ml final concentration and disulfide bonds of cysteines were reduced with mixter of DTT and TCEP. The reduced thiolg roups were protected by alkylation with 4-vinylpyridine. Solution of reduced and alkylated proteins was diluted to 1 mg/ml concentration and trypsin was added in two consequential portions in two hours each. Reaction of proteolytic digestion was incubated at 44oC initially following decreasing the temperature to 37oC. After digestion with trypsin completed, the resulting peptides mixture was acidified with formic acid and centrifuged to remove pellet of sedimented acid-labile detergent. Estimated concentration of final peptide mixture was 1 mg/ml.
See also the detailed digestion protocol at
http://msr.ibmc.msk.ru/jpr2012/DigProt
|
| acquisition | - For each biosample type {HPL,HLV,HG2} the MassHunter method files composed for aquiring QC1 were used. The result multiplex protein assays extracted from QC1 were used. Target (T) and decoy (F) transition groups were in the seperate MRM-methods.
- Data acquisition
- Data acquisition for each assay for target transitions was conducted using each prepared biosample in three technical runs on the triple-quadrupole mass-spectrometer.
- Data acquisition for each assay for decoy transitions was conducted using each prepared biosample in two technical runs on the triple-quadrupole mass-spectrometer.
- Triple-quadrupole mass-spectrometer for each data acquisitions was operating under the conditions specified below.
- Fragmentor voltage was varied from 107 to 135 V
- and the capillary voltage to -1970 - 2050 V,
- drying gas flow 5 L/min, drying gas temperature 280°C.
- Cell accelerating voltage was set to -6.0 V.
- 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. - The dwell time per fragment ion was varied from 30 to 60 ms depending on number of concurrent transitions in acquisition method while the full cycle time was within 1.2 seconds.
- The aquired transitions with correspoing MassHunter method files (*.m) was stored in the MassHunter data files (*.d) named with metanotation
{HPL,HLV,HG2}-[A-E]L{T,F}-r00[1-3].d
(FreeBSD glob routine).
See also the detailed general Separation and Acquisition Protocol at
http://msr.ibmc.msk.ru/jpr2012/SepAndAcqProt
|
| informatics | - Peak groups for light endogenous peptides and corresponding profiles of relative intensities of transitions were extracted using manual inspection of the acquired raw spectra in MassHunter Qualitative Analysis (Agilent Technologies). Peak apexes were used to estimate the transitions relative intensity. Corresponding profile of transition groups extracted from QC1 were used as a reference.
- Alternatively candidate peak groups were extracted via processing the same raw spectra by in-house extended version of mQuest\mProphet which generates ms2prot-reports with traces and detected peptides.
- Manual inspection found candidate peak groups and separation of them onto the true and false peak group categories by comparing with corresponding profile of transition groups extracted from QC0 and the peak groups proposed by expert on step 1.
- Creating for each biosample type the result table for light endogenous proteotypic peptides by marking detected and extracting RT-bounds of true peak group that detects peptide.
See the result page with links to the online ms2prot-reports which contain dataset descriptions, traces, peak groups, detected peptides and mProphet output histograms and files, protocols and measurements statistics at
http://msr.ibmc.msk.ru/jpr2012
|
| instruments | Agilent Triple Quadrupole LC-MS/MS 6410 |
| species | Human |
| massModifications | none |
