A Microscale Electrospray Interface Incorporating a Monolithic, Poly(styrene−divinylbenzene) Support for On-Line Liquid Chromatography/Tandem Mass Spectrometry Analysis of Peptides and Proteins

Abstract

A methodology is described for creating a monolithic chromatography support within a pulled fused-silica electrospray needle. The monolith was formed from a mixture of styrene, divinylbenzene, 1-dodecanol, and toluene using 2,2'-azobis(isobutyronitrile) as the catalyst. The mixture was loaded into 150-micron-i.d. fused-silica capillary tubing with a pulled 5-10-micron needle tip at one end. Polymerization at 65 degrees C followed by removal of the porogen material yielded a stable, porous, monolithic support which had excellent properties for the separation and on-line, electrospray, mass spectrometry analysis of peptides and proteins. The performance of the monolith-filled electrospray needles was compared with similar needles filled with commercial C18 silica and polymeric particulate supports. Separation efficiencies for both protein and peptide mixtures were generally equal to or better than the particulate supports at comparable pressures and flow rates. The ion chromatograms derived from the on-line MS analysis were remarkably free from chemical background signals that often complicate the LC/MS analysis of femtomole amounts of sample. Good sequence coverage was obtained by LC/MS/MS analysis of the peptide mixture obtained from a protein isolated by silver-stained gel electrophoresis. The capability of the monolith to do peak parking experiments was demonstrated by the characterization of an immunoreactive HPLC fraction. The simple fabrication method, chromatographic performance, and robust nature of these microscale integrated column electrospray sources make them ideally suited for high-sensitivity tandem LC/MS analyses.

Advances in capillary electrochromatography and micro‐high performance liquid chromatography monolithic columns for separation science

“…In 1998, a group at the Beckman research institute incorporated this monolith (with bimodal toluene/dodecanol porogen) to the needle of an electrospray interface [56].…”

Rigid Macroporous Polymer Monoliths

“…[62,91±93] Similarly, we have developed monolithic columns that change their chromatographic selectivity in response to external temperature, enabling the first reported isocratic hydrophobic interaction separation of proteins. [63] In addition, the ability to prepare monoliths within a mold of any shape was used by Lee et al [94] to prepare monolithic ST-DVB microbeds within pulled fused silica needles for the reversed-phase separation and on-line electrospray mass spectrometry detection of proteins and peptides. As illustrated by Figure 6, these monolithic microcolumns exhibited efficiencies far better than capillaries packed with commercial C18 silica or polymeric beads.…”

High‐efficiency peptide analysis on monolithic multimode capillary columns: Pressure‐assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization‐mass spectrometry

“…Several recent reports about useful microscale preparation of porous polymer monoliths that combine well-controlled porosity with appropriate surface chemistry using thermal or UV-initiated polymerization have been published [9][10][11]. Protein and peptide gradient capillary LC separations on monolithic reversed-phase chromatographic supports coupled to on-line ESI-MS detection have been described by several groups [21,22]. However, little has been reported on the separation of peptide mixtures by pCEC on polymeric monoliths [7,[23][24][25] and on the coupling of such separations to MS.…”