Merlin Bruening (University of Notre Dame)


Location: 127 Nieuwland Science Hall

Porous membranes are attractive for separations and catalysis because convective mass transport rapidly brings analyte molecules to binding or catalytic sites.  The minimal thickness of commercial membranes (~100 mm) also leads to low pressure drops, and variation of flow rate affords fine control (milliseconds) over residence times.  To create functional membranes, we adsorb polyelectrolytes in membrane pores and subsequently covalently or electrostatically attach affinity molecules or enzymes to the polyelectrolytes.  Immobilization of Ni2+-nitrilotriacetate complexes in pores yields membranes that capture polyhistidine-tagged proteins, and spin membranes (now available commercially) facilitate high-capacity purification of tagged proteins in under 5 min.  Antibody mimotopes anchored in membranes capture specific antibodies from human serum, and we hope to use these membranes for antibody quantitation.  Immobilized enzymes such as trypsin and pepsin digest proteins in residence times as short as a few milliseconds, and the size of the proteolytic peptides increases with increasing flow rates.  In the case of antibody digestion, fast flow and subsequent infusion electrospray ionization (ESI) mass spectrometry give 100% peptide coverage and identification of post-translational modifications. For de novo sequencing, we are exploring the use of multiple flow rates through the membrane to provide small peptides whose masses sum to those of larger ones.  Combined with the overlap of a few peptides, the relationships between peptide masses enable their arrangement for sequencing.  Finally, very short digestion times allow cleavage only at the most accessible, labile digestion sites.  Changes in cleavage patterns may identify alterations in protein conformations.