Application of micropellicular poly-styrene/divinylbenzene stationary phases for high-performance reversed-phase liquid chromatography electrospray-mass spectrometry of proteins and peptides

Abstract

Key WordsColumn liquid chromatography Electrospray ionization mass spectrometry Micropellicular poly-styrene/divinylbenzene Peptides and proteins SummaryShort columns packed with highly crosslinked 2.3 ~tm poly-styrene/divinylbenzene (PS/DVB) particles were used for rapid and efficient separation of proteins and peptides by reversed-phase high-performance liquid chromatography at elevated temperatures. Enhancement of the diffusivities of the sample components at elevated temperatures together with the short diffusion pathlength with the micropellicular polymeric stationary phases were responsible for high efficiency, high speed of analysis, and short column regeneration times. Underivatized PS/DVB beads as well as PS/DVB microspheres which have been modified with polyvinylalcohol or octadecyl chains on the surface were synthesized, employed, and compared to HY-TACH-C18, a commercially available micropellicular octadecyl-silica stationary phase, for the separation of proteins, octapeptides and tryptic protein digests. Highest performance was obtained with the silica-and PS/DVB-based octadecyl stationary phases, which exhibited similar column efficiencies but different selectivities for proteins and peptides. The minimum detectability at 214 nm and the maximum loading capacity for ribonuclease A using analytical 30 x 4.6 mm I.D. columns were 10 ng (0.6 pmol) and I pg, respectively. Finally, reversed-phase HPLC with a 60 • 2 mm I.D. narrow-bore column packed with micropellicular octadecyl PS/DVB was coupled successfully to electrospray mass spectrometry at a flow-rate of 0.15 mL min -1 and on-line full-scan mass spectra for molecular mass determination and identification of proteins in the lower picomol range were obtained.

1 mm ID poly(styrene‐co‐divinylbenzene) monolithic columns for high‐peak capacity one‐ and two‐dimensional liquid chromatographic separations of intact proteins

“…The in-situ preparation of poly(styrene-co-divinylbenzene) monoliths in 200 lm i.d. capillary column formats was further refined by Huber et al [23]. The excellent separation performance of these columns was demonstrated with gradient-elution analysis of complex mixtures of tryptic peptides, oligonucleotides and proteins, respectively [24,25].…”

Covalently coating dextran on macroporous polyglycidyl methacrylate microsphere enabled rapid protein chromatographic separation

“…Microspheres based on polymer matrix, as chromatographic separation media, have been popular in separation field [1][2][3][4]. Especially, the interest in and demand for high throughput separation of biomolecules accelerated the exploitation of effective polymer bioseparation media in chromatography.…”

Separation of intact proteins on γ‐ray‐induced polymethacrylate monolithic columns: A highly permeable stationary phase with high peak capacity for capillary high‐performance liquid chromatography with high‐resolution mass spectrometry

“…Furthermore, substantial improvements in terms of high permeability, low backpressure, rapid mass transfer and kinetic performances have been finally achieved by the use of organic monolithic columns, whose potential for the separation of intact proteins has been previously demonstrated . Besides the simplicity of their in situ preparation , another appealing aspect of monoliths is the possibility to easily create extra‐long miniaturized column formats that can be used in high‐resolution experiments, registering backpressure values compatible with currently available instrumentation. Porous polymeric stationary phases can be synthesized at any temperature and in almost any container using high energy radiation such as γ‐rays without the addition of initiators .…”