Rice1 project protocol

Using hair proteomics to distinguish 11 strains of mice   (2012)

Rice RH, Sundberg JP
With: Bradshaw KM, Durbin-Johnson BP, Rocke DM, Eigenheer RA, Phinney BS




Rice1 Protocol

Project protocol - Contents

Workflow and sampling

Step
Procedure
 Equipment
Data collected
1
 Hair collected and digested  
-
2
 Samples analyzed Mass spectrometer
-
3 Proteins identified Software Spectral counts

Equipment and supplies

  • Aspire RP30 C18 desalting tips (Thermo)
  • Agilent ZORBAX 300SB C18 reverse-phase trap cartridge
  • Michrom Magic C18 AQ column (200 µm x 150 mm nano-LC)
  • Michrom Advance Plug and Play nanospray source
  • CTC Pal autosampler
  • Thermo-Finnigan LTQ ion trap mass spectrometer
  • X! Tandem, version TORNADO (thegpm.org)

Reagents and solutions

  • SDS
  • NaHPO4
  • dithioerythritol
  • iodoacetamide
  • ethanol
  • ammonium bicarbonate
  • methylated bovine trypsin
  • acetonitrile
  • formic acid
  • trifluoroacetic acid

Procedure: Hair collection and digestion

    1. Samples (5 mg) are rinsed twice in 2% SDS.
    2. Samples are incubated overnight at 37°C in 0.4 mL of 2% SDS, 0.1M NaHPO4, 25 mM dithioerythritol.
    3. Samples are alkylated for 1h with iodoacetamide at room temperature with magnetic stirring.
    4. Proteins are precipitated with 1 mL of ethanol.
    5. Each fraction is rinsed twice with 67% ethanol, once with 0.1 M ammonium bicarbonate.
    6. Samples are digested at room temperature with reductively methylated bovine trypsin (1% by weight) in fresh 0.1 M ammonium bicarbonate-10% acetonitrile; trypsin is added daily for 3 days.
    7. Samples are clarified and submitted for mass spectrometry (see below).

Procedure: Mass spectrometry

    1. Salts and polypeptides resistant to elution from C18 reversed phase material are depleted from samples by solid phase extraction with Aspire RP30 C18 desalting tips; tips are rinsed exhaustively with 60% acetonitrile and then 0.1% trifluoroacetic acid.
    2. Samples are adjusted to approximately equal peptide amounts by A280.
    3. Samples are directly loaded onto an Agilent ZORBAX 300 SB C18 reverse-phase trap cartridge, which is switched in-line with a Michrom Magic C18 AQ nano-LC column connected to a Thermo-Finnigan LTQ ion trap mass spectrometer through a Michrom Advance Plug and Play nanospray source and CTC Pal autosampler.
    4. The nano-LC column is used with a binary solvent gradient (buffer A: 0.1% formic acid; buffer B: 100% acetonitrile); the 120-min gradient consisted of the following steps: 2-35% buffer B in 85 min, 35-80% buffer B in 23 min, hold for 1 min, 80-100% buffer B in 1 min, then hold for 10 min; flow rate of 2 µL/min.
    5. An MS survey scan is obtained for the m/z range 375-1400, and the MS/MS spectra are acquired from the 10 most intense ions in the MS scan by subjecting them to automated low energy CID.
    6. An isolation mass window of 2 Da is used for the precursor ion selection, and normalized collision energy of 35% is used for the fragmentation; a two min duration is used for the dynamic exclusion.

Procedure: Protein identification

    1. Tandem mass spectra are extracted with Xcalibur version 2.0.7.
    2. All MS/MS samples are analyzed using X! Tandem, version TORNADO (thegpm.org). See Rice et al., for details.
    3. Numbers of assigned spectra are tabulated; assigned spectra are adjusted for shared peptides using a locally developed script.

Data collected by investigator

  • Spectral counts

Investigator Notes: Data provided to MPD are corrected for shared peptides, permitting MPD users to do their own analyses. Results using MPD analysis tools may differ somewhat from those reported in Rice et al. (which combine males and females) due to different statistical procedures.