High Sensitivity Mass Spectrometry of Proteins and Peptides

The specific aim is to produce a small volume, high sensitivity protein identification system capable of examining the collected prteins from cell lines and tissues. To improve sensitivity in MALDI mass spectrometry we will refine liquid localization, crystallization processes and substrate patterning to increase ifficiency in the MALDI deposition and ionization processes. Surfaces patterned with silicon nanowires and vole surface chemistry will constrain the evaporating droplet of protein/matrix solution to a small sample area. The sample area will be patterned to cause preferential and controllable crystallization by graphoepitaxy to maximize the efficiency of desorption and ionization. Current approaches only detect approximately 1 in 100,000 to 10,000,000 proteins present on the MALDI target, and this disparity represents an attractie target for sensitivity improvements. Our objective is to be able to determine the mass of a protein or peptide, either within a mixture or purified, for as few as 10^4 molecules, ie few attomole detection limits. this basic approach will be combined with a small volume high resolution sample preparation and separation scheme to allow the examination of complex mixtures and weak interactions between proteins in complexes. We will develop superhydrophobic nanowire surfaces with hydrophilic spots and emostrate that these efficiently constrain the protein/matric cyrstals from an evaporating droplet and that there is a cooresponding increase in sensitivity/detection limits. We will develop the ability to fabricate theses in arrays and integrate these with both fraction collection from separations from sequence capture strategies. We will also demostrate significant qantitative improvements in detection limits by patterning the substate of MALDI targest. Combining these approaches we will pattern the superhydrophobic contrained wells to control cyrstallization within these wells.