Silica‐based monoliths for rapid peptide screening by capillary liquid chromatography hyphenated with electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Abstract

Capillary liquid chromatography based on particulate and monolithic stationary phases was used to screen complex peptide libraries by fast gradient elution coupled on-line to electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS). A slightly modified commercial electrospray interface consisting of a fused-silica transfer capillary and low dead volume stainless steel union at which the electrospray voltage was grounded enabled the effluent of all the capillary columns to be directly sprayed into the mass spectrometer. Stable electrospray conditions were generated over a wide range of mobile phase compositions, alleviating the need for a tapered end of the spray capillary, pneumatic assistance or preheated nebulizer gas. Since the identification of complex samples containing numerous isobaric substances is facilitated by chromatographic separation prior to mass spectrometry, stationary phase materials have been employed which offer a fast, efficient elution and, due to the complexity of samples, a high loading capacity. Silica-based monolithic capillary columns combine these three characteristics in a unique manner due to a tailored adjustment of both macro- and mesopore sizes in the highly porous silica structure. As we demonstrate by a comparative study of the silica-based monolithic and packed capillaries for LC/MS analysis of complex peptide libraries, silica monoliths show superior performance over packed beds of small-diameter particles with respect to analysis time and separation efficiency. Libraries with more than 1000 different peptides could be screened in less than 20 min.

Silica monolithic columns: Synthesis, characterisation and applications to the analysis of biological molecules

“…To analyse combinatorial, synthetic peptide libraries, a 100-lm monolithic column (500 mm long) was coupled to a Fourier Transform mass spectrometer [74]. The first synthetic peptide library was based on the sequence, VSXLY (X = one out of all 20 natural amino acids), whereas the second library had the following characteristics, CWXXXG (X = amino acids E, N, R, F, P, S, W, Y, L or H).…”

Controlling the retention in capillary LC with solvents, temperature, and electric fields

“…Typical analysis of a protein or peptide mixture is performed on a 0.075 -0.200 mm ID capillary column in reversed-phase mode, using gradients of acetonitrile in water with addition of 0.1 -0.2% trifluoroacetic acid (in connection with UV detection) or formic acid (in hyphenated ESI-LC/MS techniques) [58]. Capillary columns packed with large-pore spherical C18 materials [60,61], silica-based monolithic capillary columns [62], or polystyrene -divinylbenzene polymer monolithic capillary columns, either non-modified [63] or alkylated with C18 chains [64], can be employed for this purpose. High-resolution separations of oligonucleotides were accomplished on capillary monolithic columns based on cross-linked polynorbornene using gradients of acetonitrile in 0.1 M aqueous triethylammonium acetate [65].…”

Performance of octadecylsilylated monolithic silica capillary columns of 530 μm inner diameter in HPLC

“…Also those columns having small domain size were shown to produce high column efficiency in a short time. Nano-scale HPLC combined with ESI-MS using monolithic columns smaller than 200 lm inner diameter showed excellent properties for analysis of peptides and proteins [18][19][20][21][22]. Notably, a monolithic column within an electrospray needle represents a powerful analytical tool in nano-LC/ESI-MS.…”