| Metadata |
| datasetIdentifier | PASS00277 |
| 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-QC |
| 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 synthesized analogues for the endogenous peptides with the data set named Scout. The second dataset is acquired from synthesized peptides and named QC. |
| 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 | |
| separation | |
| digestion |
|
| 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 900-1200 V,
- drying gas (nitrigen) flow 12 L/min,
- sheath gas flow (nitrogen) 8 L/min,
- drying gas temperature 280 degrees celsius,
- sheath gas temperature 250 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,
- dwell time fixed at 12 ms.
- 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. - Peptides mixed in single vial were analyzed using two alternating LC approach.
- The synthetic peptides were analyzed
- either in isocratic mode
- during 3.2 minutes per analysis
- in 68% acetonitrile with 0.1% formic acid,
- or in short rapid gradient of elution.
- In case of gradient of elution following composition changes of mobile phase A (0.1% formic acid) and mobile phase B (80% acetonitrile in 0.1% fromic acid) were used:
- loading of injected sample (1 μl of peptides mixter) onto the column (Zorbax C18)
- for 2 minutes
- at flow rate 100 μl/min (5% B),
- rapid gradient elution to 15 minutes (5 - 80% of B),
- the increasing content of B to 100% for 1 minute
- and washing the column for 5 minutes (16 - 21 miutes)
- following column reconstitution for 6 minutes.
- Transitions derived from different precursor's charge states of single peptides were applyed in one method.
- The total single scan cycle time
- for rapid short LC gradient method was made of 0.4 - 0.8 seconds,
- for isocratic LC mode was made of 0.2 - 0.5 seconds.
- The aquired transitions
- with correspoing MassHunter method files (*.m)
- was stored in the MassHunter data files (*.d)
- named with metanotation [0-9][0-9][A-H]*-r00[1-3].d (FreeBSD glob routine) where "[0-9][0-9][A-H]*" is the mask for local peptide identifier which is mapped to a Uniprot Accession Number for a protein in the assay's "Comment" column.
|
| informatics | |
| instruments | Agilent Triple Quadrupole LC-MS/MS 6490 w/ Jet-Stream |
| species | Human |
| massModifications | none |
