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

Abstract

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.