| Metadata |
| datasetIdentifier | PASS00770 |
| datasetType | SRM |
| submitter | Thierry Schmidlin <t.t.schmidlin@uu.nl> |
| submitter_organization | Utrecht University |
| lab_head_full_name | A. F. Maarten Altelaar |
| lab_head_email | m.altelaar@uu.nl |
| lab_head_organization | Utrecht University |
| lab_head_country | Netherlands |
| datasetTag | AssessmentSRMDIAMRM3 |
| datasetTitle | Assessment of SRM, MRM3 and DIA for the Targeted Analysis of Phosphorylation Dynamics in Non-small Cell Lung Cancer |
| publicReleaseDate | 2015-11-16 00:00:00 |
| finalizedDate | 2016-09-19 02:42:09 |
| summary | Performance assessment of DIA/SRM/MRM3 for phosphoproteomics |
| contributors | Thierry Schmidlin, Luc Garrigues, Catherine S. Lane, T. Celine Mulder, Sander van Doorn, Harm Post, Erik L. de Graaf, Simone Lemeer, Albert J. R. Heck, A. F. Maarten Altelaar
|
| publication | PMID: 27219855 |
| growth | PC9 and H1975 cells were grown in standard RPMI medium at 37degC in humidified atmosphere containing 5 % CO2. Cells were detached from the culture surface using trypsin (Lonza), and washed three times with PBS before lysis. |
| treatment | N/A |
| extraction | (1) Cell lysis in urea based lysis buffer using sonication
(2) Protein reduction and alkylation by DTT and iodoacetamide
(3) LysC/Trypsin double digestion
(4) SepPak desalting
(5) Phosphopeptide enrichment by Ti(4+)-IMAC |
| separation | DIA: chromatographic separation was performed using an Agilent 1290 Infinity System (Agilent Technologies) coupled to in-house packed trap column (Dr Maisch Reprosil C18, 3 μm, 2 cm x 100 μm) and analytical column (Agilent Poroshell 120 EC-C18, 2.7 μm, 50 cm x 75 μm) using a 155 min gradient running from 7% to 23% solvent B (0.1% FA in 80% ACN) in the first 77.5 min and from 23% to 35% solvent B in the second 77.5 min at a flow rate of 350 nL/min with a subsequent washing step of 100% B for 2 min.
SRM : Chromatographic separation was performed using an EASY-spray system containing an Easy-nLC 1000 coupled to a 25 cm, 75 µm ID PepMap RLSC, C18, 100 Å, 2 µm particle size column (Thermo Scientific, Odense, DK). Phosphopeptides were separated on a gradient going from 0 % to 25 % B in 170 minutes.
MRM3: All MRM3 measurements were performed on an ekspert™ nanoLC 425 (Eeksigent) coupled to a QTRAP® 6500 (SCIEX). LC separation was carried out on using a nanoAcquity (75µm x 25cm, 1.8mm, HSS T3) column with a nanoAcquity (180µm x 20mm, 5µm Symmetry C18) trap column (Waters) in trap-elute configuration. Peptide separation was performed on a gradient running from 2 % solvent B to 25 % solvent B in 90 minutes, from 25 % solvent B to 40 % solvent B in 10 minutes and from 40 % solvent B to 90 % solvent B in 3 minutes followed by a 29 min washing and re-equilibration step. |
| digestion | LysC/Trypsin double digestion |
| acquisition | SRM: scheduled SRM experiments on a TSQ Vantage, 10 min time windows, Q1/Q3 at resolution of 0.7 m/z half max peak width, cycle time 4 sec
MRM3: Optimized MRM3 parameters for three target peptides
DIA: 64 variable DIA windows were used, each with 1 amu overlap spanning an m/z range of 350–1250. The collision energy for each window was determined based on the collision energy for a 2+ ion with a collision energy spread of 0 eV. An accumulation time of 50 ms was used for each fragment ion scan which in combination with a 100 ms survey scans results resulted in a total cycle time of 3.4 s. |
| informatics | SRM/DIA: AUC quantification using skyline daily and MSStats for significance analysis (normalization based on internal standards (SRM) and equalizing medians (DIA))
MRM3: Multiquant quantification. Two-tailed t-test assuming equal variances was used to test for significant changes in abundance between the two cell lines, using three biological replica with three phosphopeptide enrichment replica each. |
| instruments | TripleTOF 5600, TSQ Vantage, QTRAP 6500 |
| species | Human cell lines (PC9 and H1975) |
| massModifications | static: C+57.021464
variable: M(oxidation)+15.994915, S/T/Y(phosphorylation)+79.966331, K(label)+8.014199, R(label)+10.008269 |
