Nanoliter solvent extraction combined with microspot MALDI TOF mass spectrometry for the analysis of hydrophobic biomolecules

A nanoliter solvent extraction technique combined with microspot matrix-ass isted laser desorption/ionization (MALDI) mass spectrometry is presented. T his method involves the use of a nanoliter droplet containing organic solve nts at the tip of a small capillary for extraction. The droplet is formed i nside a microliter aqueous sample containing the analyte of interest. After extraction, the droplet is deposited onto a MALDI target precoated with a thin matrix layer. Since the nanoliter droplet never touches the sample con tainer wall, any possible extraction of contaminants adsorbed on the plasti c or glassware is avoided. In addition, there is no need to concentrate the organic phase after the extraction, thus avoiding any possible loss during the concentration step. The nanoliter volume can be readily deposited onto a MALDI target, producing a high analyte concentration within a microspot, Combined with microspot MALDI, this technique allows for very sensitive an alysis of the extracted analyte, The performance of this technique is illus trated in several applications involving the detection of hydrophobic pepti des or phospholipids, It is shown that very hydrophobic analytes can be ext racted from small-volume samples containing a large amount of salts and/or more hydrophilic analytes, which tend to give dominant signals in conventio nal MALDI experiments. Nanoliter extraction of analyte from samples contain ing less than 100 nM hydrophobic analyte and over 1 muM easily ionized hydr ophilic species is demonstrated. Finally, using the analysis of the ionopho re valinomycin as an example, it is demonstrated that the technique is a mo re reliable tool for probing metal-peptide complexes than regular MALDI sam ple preparations.