Optimization of capillary-channeled polymer (C-CP) fiber stationary phase extractions of proteins from MALDI-MS suppressing media

Solid phase extraction (SPE) is an essential component in many proteomic analyses involving matrix-assisted laser desorption/ionization mass spectrometry detection (MALDI-MS). Parametric variables affecting the efficiency of protein extractions from buffer (Tris-HCl) and urine media using a capillary-channeled polymer (C-CP) fiber stationary phase in a tip-based format are evaluated. Proteins are immobilized on to polypropylene (PP) C-CP fibers and eluted in organic solvents conducive to high analytical performance using a simple benchtop centrifuge to drive the fluids. Experimental variables, including elution solvent strength, aqueous wash volume, the interactive aspects of fiber tip length (2.5-10 mm), and loading and elution volumes, were evaluated to gain insights into fundamental processes and to optimize protein recoveries/ analytical signals. Based on the MALDI-MS responses of the test proteins cytochrome c, lysozyme, and myoglobin, the optimized elution solvent was determined to be a 60-40 ACN-H₂O mixture with 0.07% trifluoroacetic acid (TFA). It was determined that different aqueous wash volumes were required to flush test solution remnants, depending on the buffer concentration, concluding that a 100 μL wash was effective at concentrations of up to 1 M. Fiber tip length was explored to determine the limits of loading volume/protein mass for each bed size while efficiently extracting from the buffer environment. Interestingly, the shortest C-CP fiber tips provided the best efficiency, isolating nanogram levels of protein from 1 μL aliquots of sample. After optimization, a proof of practice experiment was performed to extract the three-protein suite (<5 μM of each) from a synthetic urine matrix. The previously undetected proteins could be readily distinguished with high spectral clarity due to the SPE procedure utilizing C-CP fiber packed micropipette tips.