Protocols
TCGA Breast Cancer CompRef Samples

Dataset: 03CompRef_P5-25_P5-19_P6-85_P6-58_Phosphoproteome_BI_20130719

Analytical Sample Protocol

  1. Starting Amount 4 mg
  2. Proteolysis LysC/Trypsin
  3. Alkylation Iodoacetamide
  4. Enrichment Phosphopeptide enrichment with immobilized metal affinity chromatography (NiNTA beads stripped with EDTA and loaded with FeCl3; Qiagen)
  5. Labelling 4-plex iTRAQ
  6. Fractionation Peptide basic pH Reversed Phase LC (pH 10)
  7. Fractions 13


Chromatography Protocol

  1. Column Type New Objective; PicoFrit SELF/P
  2. Column Length 20 cm
  3. Inside Diameter 75 µm
  4. Particle Type Dr Maisch GmbH; ReproSil-Pur 120 C18-AQ; 1.9 um
  5. Particle Size 1.9 µm
  6. Injected 1 µg Proteome / 4 of 9 µl IMAC sample Phosphoproteome
  7. Gradient Length 110 min


Mass Spectrometry Protocol

  1. Type Orbitrap Mass Spectrometry Protocol
  2. Instrument Q Exactive
  3. Dissociation high-energy collision-induced dissociation (HCD)
  4. MS 1 Resolution 70,000
  5. MS 2 Resolution 17,500
  6. Collision Energy 28%
  7. Precursors Top 12