Application of trilobal capillary-channeled polymer (C-CP) fibers for reversed phase liquid chromatography and ESI-MS for the determination of proteins in different biological matrices

Abstract

Electrospray ionization mass spectrometry (ESI-MS) analysis provides a great deal of analytical information as a detection technique for biological species determinations. However, because of the common high ionic strength matrices and processing buffers, including salt and small molecules, additional sample preparation/desalting processes are necessary prior to introduction into the ESI source. The most typical form of processing is reversed phase liquid chromatography (RPLC). Limitations to low flow rates (<0.5 mL min⁻¹) for optimal spectral clarity and greatest sensitivity for conventional ESI generally imply use of low linear velocities and slow RPLC separations. Previous efforts in this laboratory have demonstrated the use of capillary-channeled polymer (C-CP) fibers for high throughput protein separations. More recently, a trilobal version of the fiber has proven to yield superior separations due to better packing quality. To further investigate the potential application of the trilobal-shaped C-CP fiber in LC-MS, trilobal polypropylene (PP) fiber columns were applied for RP-LC-ESI-MS protein determinations in highly-diverse biological matrices, including urine, saliva and HL5 cell culture media. Several parameters, including fiber packing density, mobile phase flow rate, and gradient rate were evaluated using a four-protein mixture (ribonuclease A, cytochrome c, lysozyme, and myoglobin). In addition, differences in ion-pairing performance between trifluoroacetic acid, formic acid, and acetic acid were evaluated. The effectiveness of the matrix removal is reflected in the near-identical qualitative and quantitative ESI-MS responses in all cases for a five-protein mixture. The results of this study demonstration the utility in the application of trilobal, polypropylene C-CP fibers in LC-MS analysis of biomacromolecules.