Immobilized Metal Affinity Beads for Recombinant Protein Analysis by Desorption Electrospray Ionization Mass Spectrometry.

We address the limitations of the low binding capacity of smooth metal affinity surfaces (below 1.661 femtomoles per mm2) and the well-documented low sensitivity of DESI-MS toward larger proteins by increasing the specific surface area of the sampling substrate, thereby providing a greater binding capacity per unit area of the desorption footprint. This was achieved using Ni-NTA-functionalized magnetic beads adhered to a flat surface. Close packing of the spherical beads provides a greater surface area, yielding an increased protein binding capacity as high as 4.028 nmol per mm2 of sampled surface. The magnetic properties of these beads allow for simplified workflows and efficient protein recovery directly from cell lysate prior to mass spectrometric analysis. The approach is demonstrated by purifying known concentrations of His-labeled S-adenosylhomocysteine nucleosidase and lactate oxidase. The known enzymatic reactions for each protein are shown directly from the captured proteins adsorbed on the beads by DESI-MS. Acidifying the desorption solvent disrupts interactions between chelated nickel and histidine, allowing for the release of the protein and its analysis by DESI-MS.