Nanoliter chemistry combined with microspot MALDI TOF MS for sensitive protein and peptide characterization.

摘要

A combination of subnanoliter volume sample handling employing small-diameter capillaries and direct sample deposition onto very thin preformed matrix-layers makes matrix-assisted laser desorption/ionization mass spectrometry (MALDI TOF MS) one of the most sensitive analytical methods currently available for protein and peptide detection. This research is focused on the technical development of this technique and demonstrates its applications in protein and peptide analysis. Employing microspot MALDI mass spectrometric analysis of peptides with a total sample loading of several tens of thousands of molecules (e.g. 25,000 molecules or 42 zeptomole of Substance P) is demonstrated. Several unique technical and fundamental issues in dealing with such a low level of analysis are discussed.; Extension of this technique's capabilities toward multiple chemical and/or enzymatic reactions within a nanoliter chemistry station allows for more detailed structural characterization of proteins. Required sample amounts of this technique are in the subfemtomole range, enabling, as an example, the distinction of hemoglobin variants from single red blood cells. Combined HPLC-fractionation and nanoliter chemistry is used to increase the number of identifiable proteins from bacteria extracts, thus contributing to the establishment of high quality databases for bacteria identification.; Application of this sensitive approach to high molecular weight proteins enables analysis of contaminated and dilute protein solutions in the low nanomolar range, however decreased resolution and formation of multiply-charged species have to be taken into account. The formation of multiply-charged species can be beneficial in reduction studies of proteins consisting of disulfide-linked subunits for obtaining high-order structural information.; Employing a modified setup of the nanoliter chemistry station for solvent extraction with a nanoliter droplet of organic phase in microliter volumes of aqueous sample solutions enables the simultaneous detection of hydrophobic and hydrophilic analytes by MALDI TOF MS. Extension of this newly developed technique towards investigation of metal-peptide complexes is demonstrated.