A parallel method for desalting and concentrating microliter volume samples for mass spectrometry.
A method is described for constructing spin columns for reverse-phase centrifugal desalting of proteolytic digests. The technique employs small, self-packed, reusable cartridges and required less than 30 minutes to process six samples, making the procedure useful as a parallel technique. Up to 15 microL of sample could be loaded and eluted with 2 to 7 microL of a solvent compatible with electrospray ionization. The method was not limited to large-diameter resins or short column heights; a relative centrifugal driving force as low as 30 (500 rpm) applied for 1 to 3 minutes usually was sufficient for sample loading. Subsequently, data were obtained with 1 5-mm columns of 3- 5-, and 10-microm silica resins. The efficiently of recovery in the range of 0.5 to 250 pmol of peptides was measured as 60% to 90%, depending on resin type and sample load. Successful nanospray data were obtained with peptides that had been adulterated with 2 M urea and processed with a spin column. Matrix-assisted laser desorption and ionization/time-of-flight mass spectrometry data greatly improved after desalting of an in-gel digest of a 280-kd protein. Data are presented on the preparation of columns, optimization of procedures, the use of various types of C18 resins, and the efficiency of peptide recovery. The effect of rotor speed and the rate of sample processing are discussed.
Rusnak
F
F
California Institute of Technology, Beckman Institute, Pasadena, CA 91125, USA.
Zhou
J
J
Hathaway
G M
GM