Intact Protein LS-ESI-TOF Protocol (P0710)

Samples are analyzed by reverse phase liquid chromatography (LC) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS).
  1. Deglycosylated antibody samples are denatured using 10 mM DTT for 30 minutes at room temperature, and diluted to 0.1% formic acid with water.
  2. A custom reverse-phase chip, containing an integrated trapping column (40 nl capacity), separation column and nano-ESI emitter (75 μm x 150 mm both packed with PLRP-S, 5 μm particles, 1000 A pore size) are used for the separation of proteins (1).
  3. The chip trap column is loaded at 2 μl/min and the separation column is developed at a flow rate of 500 nl/min using an Agilent 1200 series nano-LC connected directly to an Agilent 6210 series ESI-TOF MS.
  4. The column is equilibrated with 0.1% formic acid in water containing 5% acetonitrile.
  5. The sample (1 μl) of deglycosylated antibody is injected onto the column and developed after two minutes with a fifteen-minute linear gradient from 5% to 95% acetonitrile and then held at 95% acetonitrile for five minutes.
    Protein is found to elute at approximately 10 minutes after injection. The mass spectra is acquired from 150 to 3200 m/z, one cycle/sec and 10,000 transients per scan using an ionization energy of 1800 V, fragmentor of 215 V and drying gas of 325° C at 4.0 l/min. The acquired spectra is extracted and the protein spectra deconvoluted. 

(1) Vollmer, M and van de Goor (2009) Methods in Molecular Biology, 544, 3-15.