Wollscheid - MSGUIDE

Mass Spectrometry-GUided Immunoassay DEvelopment (MS-GUIDE)
Data License: CC BY 4.0 | ProteomeXchange: PXD016976
  • Organism: Homo sapiens
  • Instrument: Orbitrap Fusion ETD,Q Exactive Plus
  • SpikeIn: Yes
  • Keywords: prostate cancer, serum, glycocapture, biomarker, ELISA, PRM
  • Lab head: Bernd Wollscheid Submitter: Sandra Goetze
We applied targeted MS (parallel reaction monitoring, PRM) to pre-qualify candidates for clinical ELISA assay development. Literature harvested potential biomarker candidates, for which typically no antibodies for sensitive detection in complex samples exist, were evaluated in a medium-sized patient cohort using PRM. After biomarker validation, clinical grade ELISAs were developed and tested in a larger independent patient cohort.
Experiment Description
For the study we included prostate cancer patients with the following inclusion criteria: initial biopsy; an elevated total PSA (tPSA) concentration between 2 and 10 ng/mL; a negative digital rectal examination (DRE); an enlarged prostate with a volume ≥35 mL (determined by transrectal ultrasound (TRUS)) ; an available serum sample and provision of informed consent to using their sample for research purposes. All men with a cancer-positive biopsy outcome needed to have undergone subsequent in‐house radical prostatectomy, so that the Gleason score (GS) from both the biopsy and the prostatectomy specimen were available. 78 pre-prostatectomy serum samples from patients were measured using protein glycocapture and PRM. Samples were received from the Martini-Klinik, Prostata Cancer Center at the University Medical Center Hamburg-Eppendorf, Germany. For all samples, follow-up data were available.
Sample Description
Glycoproteins were enriched from sera using the protocol published protein glycoapture. In short, two bovine reference glycoproteins (Alpha-1-acid glycoprotein (Q3SZR3) and Fetuin-B (Q58D62)) as well as two human heavy labeled GST reference proteins (Cathepsin D (P07339) and Hypoxia up-regulated protein 1 (Q9Y4L1)) were spiked into 50 μl of patient serum at a concentration of 1 pmol/μL or 50 ng respectively. References were used to evaluate and normalize for intra-sample variation during glycopature. Glycan-moieties of serum proteins were oxidized using sodium periodate (Sigma-Aldrich). Oxidized proteins were purified using G-10 gel filtration cartridges (Nest Group) and subsequently conjugated overnight to Affi-gel Hydrazine-resin (Bio-Rad). After coupling, samples were washed extensively to remove unbound proteins. Proteins were then digested on-beads overnight using a mix of trypsin (Promega) and lysyl endopeptidaseR (Wako). Cleaved non-glycopeptides were washed off the beads and N-linked glycopeptides were released by the addition of PNGase F (BioConcept). Released peptides were purified for mass spectrometry measurements via C18 reverse phase resin (Nest Group). For mass spectrometry measurements, peptides were solubilized in 100 μL of 0.1% aqueous formic acid (FA) and 2% acetonitrile (ACN). N-glycopeptides corresponding to 2 μL of patient blood were used for each MS run.
Created on 1/6/20, 9:48 PM
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20190906_heavy_light_jpt_all.sky.zip2020-01-06 21:47:41541262501,1935
20190920_HH_results_modeling.sky.zip2020-01-06 21:47:4133626424378

This data is available under the CC BY 4.0 license.