| This dissertation describes a surface science/mass spectrometry effort to develop and characterize patterned surfaces that serve as matrix-assisted laser desorption/ionization (MALDI) sample platforms capable of concentrating and purifying proteins and DNA. The use of these patterned surfaces can also enhance the detectability of peptides, especially phosphopeptides, from protein proteolytic digest mixtures. Using micro-contact printing, small (200-mum diameter) hydrophilic spots of bare gold, chemically anchored poly(acrylic acid) (PAA), or immobilized polyethylenimine (PEI) are patterned at 5-mm intervals in a hydrophobic field consisting of a self-assembled monolayer of hexadecanethiol. Dilute or salt-contaminated protein, DNA, or protein proteolytic digest samples are applied onto the hydrophilic spots, dried, and then rinsed with water to remove water-soluble contaminants, simplify a digestion mixture, or separate non-phosphopeptides from phosphopeptides. One of the key features in this work is the combination of a functionalized surface with a small spot to afford both concentration of analyte via evaporation to a small spot size and purification by selective adsorption. The polymeric anchors bind the analytes during the water-rinsing step, and the subsequently added acidic matrix solution releases analytes for their incorporation into the matrix crystals.; Use of these patterned surfaces decreases the detection limit for the analysis of dilute protein samples by MALDI-MS. For example, 1--5 fmol of insulin chain A, insulin chain B, insulin, and ribonulcease A can be routinely observed with patterned surfaces, while conventional stainless steel probes allow only 50--100 fmol detection limits. The detection limits for salt-containing samples decrease by at least one order of magnitude for use of patterned surfaces compared with use of non-patterned on-probe decontamination methods.; The patterned surfaces also allow the detection of more tryptic peptides in protein digestion mixtures. For example, use of a patterned PAA surface revealed 22 peptides as compared to only 11 peptides observed with a SS plate. Thus, the PAA surface allows a much higher confidence level for protein identification during myoglobin peptide mapping. Patterned surfaces also allowed 13% higher sequence coverage for a larger protein, bovine serum albumin.; Modified probes containing small spots modified with polycations show great promise for selectively enriching phosphorylated peptides directly on the probe prior to MALDI-MS analysis. The positively charged anchors selectively bind the negatively charged phosphopeptides, while the water-rinse removes other signal suppressing contaminants or non-phosphopeptides. |
