Functionalized Porous Membranes for Rapid Protein Capture and Digestion Prior to Mass Spectrometry Analysis

Functionalized porous membranes are powerful tools for rapid protein isolation or digestion prior to mass spectrometry (MS) analysis. Aiming to increase protein accessibility to affinity binding sites in polyelectrolyte multilayers, layer-by-layer (LbL) adsorption of star-shaped polyelectrolytes in microporous membranes create porous coatings. Such coatings yield membranes that bind up to 120 mg of lysozyme per mL of membrane, which is double the capacity of commercial ion-exchange beads. To create membranes that selectively capture tagged proteins, we develop a convenient method to synthesize nitrilotriacetate (NTA)-containing polyelectrolytes. Subsequent adsorption of these polymers in membrane pores gives porous nylon membranes that capture 46 mg of His-tagged ubiquitin per mL. Due to the high affinity of NTA for metal ions, Ni²⁺ leaching in binding and rinsing buffers is minimal. Adsorption of either star polyelectrolytes or NTA-containing polymers in porous membranes could create materials that serve as ion-exchange or metal-ion affinity disposable membranes.

Analysis of protein sequences and post-translational modifications (PTMs) often requires proteolysis followed by MS. Conventional in-solution proteolytic digestion employs long incubation time and generates numerous small peptides, which complicates analysis. We recently developed a protease-containing spin-membrane platform to achieve rapid protein digestion. Centrifugation of monoclonal antibodies (mAbs) through these membranes leads to digestion <1 minute. Proteolytic peptides analyzed with MS cover ~100% of the mAb sequences with PTMs identified. Compared to in-solution digestion, spin digestion yields higher sequence coverages due to larger peptides. This method is easy to expand for high-throughput sample preparation and analysis.

Rapid, convenient methods for mAb isolation are critical for determining its concentrations in human serum. Conventional techniques such as enzyme-linked immunosorbent assays are costly and time-consuming. We created membranes containing mimotope peptides which could specifically isolate Herceptin from human serum with a binding capacity up to 10 mg per mL of membrane. LC-MS demonstrates the high purity of eluted Herceptin, and LC-MS/MS identifies nonspecific binding species in small amounts. Capture and elution followed by native fluorescence analysis allows determination of Herceptin concentrations over the range expected in diluted serum. Future studies with spin membranes or membranes in 96-well plates may afford convenient mAb analyses in <20 min.