| Metadata |
| datasetIdentifier | PASS00710 |
| datasetType | SRM |
| submitter | Tommaso De Marchi <t.demarchi@erasmusmc.nl> |
| submitter_organization | Erasmus MC |
| lab_head_full_name | Arzu Umar |
| lab_head_email | a.umar@erasmusmc.nl |
| lab_head_organization | Erasmus University Medical Center |
| lab_head_country | Netherlands |
| datasetTag | BRCTAM |
| datasetTitle | immuno-MRM analysis of a 4 protein based classifier predicting outcome to tamoxifen resistance |
| publicReleaseDate | 2015-11-30 00:00:00 |
| finalizedDate | 2017-07-03 10:28:48 |
| summary | We selected a total of 47 breast cancer tissues which displayed at least (≥) 50% tumor area from patients that manifested either good or poor outcome to tamoxifen treatment, which was defined as whether patients manifested disease progression before (≤; poor) or after (>; good) 6 months. This subset comprised 28 and 19 patients who manifested good and poor outcome, respectively (Table 1). An independent set of 26 breast cancer patient derived sera was selected as verification cohort. Sera were collected both prior start of first line tamoxifen therapy (Supplemental Table 1), and comprised 14 good and 12 poor outcome patients. Both cohorts were analyzed in singlicate as biological replicates.
In order to assess the efficacy of immuno-capture coupled to MRM MS, a set of 8 ER positive tumor specimens (≥ 50% tumor area) was analyzed in two triplicated series (i.e. 8 x 3 replicates by standard MRM, 8 x 3 replicates by iMRM).
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| contributors | De Marchi T,Kuhn E, Dekker L, Stingl S, Braakman RB, Opdam M, Linn SC, Sweep FCGJ, Span PN, Martens JWM, Luider TM, Foekens JA, Carr S and Umar A. |
| publication | De Marchi T,Kuhn E, Dekker L, Stingl S, Braakman RB, Opdam M, Linn SC, Sweep FCGJ, Span PN, Martens JWM, Luider TM, Foekens JA, Carr S and Umar A.Targeted MS assay for accurate quantitation of proteins predicting tamoxifen resistance in Estrogen Receptor positive breast cancer. Manuscript in preparation |
| growth | none |
| treatment | none |
| extraction | All selected tumor tissue specimens were sectioned into 10 x 10 µm sections and processed according to our tissue proteomic workflow. A total of 50uL of serum were collected and digested out of serum specimens. Collected material was disrupted in a horn sonifier bath using an Ultrasonic Disruptor Sonifier II (Bransons Utrasonics, Danbury, CT, USA) at 70% amplitude. |
| separation | Tissue
The 47 tumor tissue samples were analyzed on a 4000 Q-Trap MS system, which was coupled online to a Tempo liquid chromatography (LC) system (Applied Biosystems, Foster City, CA). Peptides were eluted with a binary gradient (flow: 300 nl/min; mobile phase A: 0.1% formic acid in H2O; mobile phase B: 90% acetonitrile and 0.1% formic acid). Sample injection was performed on PicoFrit columns (inner diameter: 75 µm; New Objective, Woburn, MA) packed in-house with ReproSil reversed phase resin (C18-AQ; diameter: 3 µm; Dr. Maisch, GmbH) for a total column length of 10-12 cm. Gradient was run as follows: 3 to 20% of solvent B for 3 min, 20 to 55% solvent B for 35 min, and 55 to 80% solvent B for 3 min. Ion spray voltage was set at 2200 V, curtain and nebulizer gasses were set at 20 and 3 p.s.i. respectively.
Sera
A total of 10µL of each captured serum sample were injected into a Symmetry 2-cm C18 nano-ACQUITY column (particle size: 5 µm, internal diameter: 180 µm; Waters, Milford, MA, USA). Solvent A: 0.1% formic acid in water; solvent B: 0.1% formic acid in acetonitrile. All samples were washed for 5 mins prior injection (flow rate: 8 µL/min; 99% A and 1% B) and injected on a 20-cm BEH 300 C18 column (particle size: 1.7 µm, internal diameter: 75 µm; Waters) at a 300 nL/min flow rate. Gradient was run as follows: 30 mins of B (1% to 40%), 5 mins of B (80%), 25 mins of A (99%). Column was connected to a Z-spray nano-source Xevo TQ-S MS. |
| digestion | Proteins were denatured at 95°C, reduced with 100 mM DTT for 30 min at room temperature, and alkylated in the dark with 300 mM iodoacetamide for 30 min at room temperature. Samples were then digested for 4 h at 37°C after addition of MS grade trypsin at a 1:4 enzyme-protein ratio (i.e. 100 ng/µl). Samples were acidified with TFA, and spun down at 14,000 RPM. Supernatants were collected and transferred to HPLC vials (Sigma-Aldrich Corporation, St. Louis, MO, USA).
Healthy donor plasma and serum proteins were denatured and reduced in a 7 M urea and a 100mM DTT solution respectively, and alkylated in the dark with a 500mM of iodoacetamide solution for 30 min at room temperature. Samples were then digested overnight at 37°C by adding Trypsin in a 1:50 enzyme-protein ratio after dilution of Urea with Trizma® (Sigma-Aldrich, Steinheim, Germany). Samples were acidified with formic acid, spun down at 14,000 RPM, de-salted through OASIS® (Waters) cartridges and vacuum-dried.
Breast cancer trypsin digested whole tissue lysates and sera were captured with a constant amount of heavy peptide mix (tissue: 200 fmoles; serum: 10 fmoles). Peptides captured by antibody-beads were washed with PBS + 0.03% CHAPS (5 mins for three times), eluted in 25 µL of a 3% acetonitrile + 5% AcOH and stored at 4°C until targeted MS analysis. |
| acquisition | Tissue
The same de-clustering potential and collision energy were used for each ligh/heavy peptide pair. Of each target peptides, three transitions were monitored and analyzed by MRM-MS in unscheduled mode.
Sera
Capillary voltage was 3.00 kV, cone voltage was 50 V, source offset was 50V and source temperature was 70°C. CID based dissociation was obtained by injection of 0.15mL/min Argon gas. Of each target peptides, three transitions were monitored and analyzed by MRM-MS in unscheduled mode. |
| informatics | Analyst and MassLynx derived MRM data were imported and analyzed in Skyline free software. Areas under the curve (AUC) of extracted ion chromatograms (XIC) of each light and heavy peptide transition were used to assess peptide abundances in antibody-beads titration experiments. AUC values for each transition were plotted in Microsoft Excel for each antibody-beads concentration point. Peak area ratio (PAR) between the AUC of light and heavy peptides were used for accurate quantitation in the analysis of breast cancer tissue samples. Isotope dilutions measurements were analyzed in QuaSAR in Skyline environment: limit of detection (LOD), lower limit of quantitation (LLOQ) and coefficient of variation (CV) for each measured transition were then calculated in QuaSAR (https://skyline.gs.washington.edu/labkey/skyts/home/software/Skyline/tools/details.view?name=QuaSAR). |
| instruments | ABI Sciex Qtrap 4000; Waters Xevo TQ-S |
| species | Homo Sapiens |
| massModifications | none |
