| Metadata |
| datasetIdentifier | PASS00456 |
| datasetType | SRM |
| submitter | Sandra Goetze <sgoetze@ethz.ch> |
| submitter_organization | University of Zurich |
| lab_head_full_name | Erich Brunner |
| lab_head_email | erich.brunner@imls.uzh.ch |
| lab_head_organization | University of Zurich |
| lab_head_country | Switzerland |
| datasetTag | Ariadne-data |
| datasetTitle | Ariadne: Automated Absolute and Relative Quantification of SRM Data |
| publicReleaseDate | 2014-09-01 00:00:00 |
| finalizedDate | 2014-04-04 08:16:50 |
| summary | Selected reaction monitoring (SRM) MS is a highly selective and , sensitive technique to quantify protein abundances in complex biological samples. SRM analyses are well supported by state-of-the-art software for both interactive data analysis (e.g., Skyline) and statistical validation of acquired data (e.g., mProphet). However, at present, there is no validated approach to automate the estimation of absolute protein quantities, and manual evaluation, which is widely used, is inherently biased and error-prone. Here, we present Ariadne, a Matlab® software solution supporting automated absolute and relative quantification of SRM data.
Ariadne quantifies the targeted peptides listed in the transition lists. If an mProphet output is available for the current experiment, targets can also be filtered according to its statistical validation. Signal processing and statistical learning approaches are combined to provide relative – and, if requested, absolute – peptide quantifications. In order to robustly estimate absolute abundances, the external calibration curve method is applied ensuring linearity over the dynamic range.
|
| contributors | Sandra Goetze |
| publication | unpublished |
| growth | Drosophila KC cells were grown in Schneider's Medium. |
| treatment | |
| extraction | Total KC cell lysate was prepared in 8 volumes of RIPA Buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 500 µM EDTA, 100 µM EGTA, 1.0% Triton X-100, 1% sodium deoxycholate, protease inhibitors)and douncing with a tight pistle |
| separation | |
| digestion | Proteins were reduced with 5 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and treated with 10 mM iodoacetamide to modify cysteine residues prior to digestion with trypsin. Resulting peptides were cleaned up by reverse phase C18 chromatography (Finisterre SPE columns, WICOM). For mass spectrometry analysis, samples were diluted in buffer containing 5% acetonitrile, 0.2% formic acid. |
| acquisition | Triplicates SRM measurements were performed on a triple quadrupole mass spectrometer (TSQ Quantum Ultra EMR, Thermo Fisher Scientific) operated with Xcalibur® 2.0.7 (Thermo Fisher Scientific). 2 µl of each sample were injected via a coupled Eksigent nano-LC system. Samples were automatically injected into a 10-µl sample loop and loaded onto an analytical column, which was packed in house with Magic C18 AQ beads 5 µm, 100 Ĺ (Microm) (8 cm length×75 µm (internal diameter)). Peptide mixtures were delivered to the analytical column at a flow rate of 500 nl/min of buffer A (5% acetonitrile, 0.2% formic acid) for 18 min and then eluted using a gradient (10%–35%; 0.36%/min) of buffer B (acetonitrile, 0.2% formic acid) at a flow rate of 250 nl/min.
SRM transitions were generated using Skyline. Collision energies (CE) were calculated according to the following formulas: CE = 0.034 × m/z + 3.314 (2+) and CE = 0.044 × m/z + 3.314 (3+). Measurements were carried out with an isolation width of 0.4 Da for Q1 and 0.7 Da for Q3. Scan speed was set to obtain a maximum cycle time of 1 s and minimum dwell time of 20 ms. The scheduling window was set to 6 minutes and the maximum number of overlapping transitions to 50. Assays consisted of 4 transitions for each target peptide and 3 transitions for each iRT -kit peptide. |
| informatics | The performance of the Ariadne software is described in the manuscript:
Ariadne’s thread: a robust software solution leading to automated absolute and relative quantification of SRM data |
| instruments | Thermo Scientific Quantum Ultra EMR |
| species | Drosophila melanogaster |
| massModifications | variable: K+8.014199, R+10.008269 |
