Applications of Monolithic Silica Capillary Columns in Proteomics

Abstract

The use and applicability of silica based capillary monolithic reversed-phase columns in proteomic analysis has been evaluated by liquid chromatography−mass spectrometry (LC−MS). Chromatographic performance of the monolithic capillaries was evaluated with a tryptic digest of cytochrome C showing very good resolution and reproducibility in addition to the known advantages of a low pressure drop over a time period of 6 months. Monoliths were subsequently tested for their suitability to separate proteins and peptides from samples typically encountered in proteomic research such as in-gel digested tryptic peptide mixtures or fractions of proteolytically digested human serum. The monolithic capillaries also proved useful in the analysis of phospholipid species in bronchoalveolar lavage fluid. Compared to particle-filled conventional capillary columns, rapid and highly efficient separation of peptides and proteins was achieved using these bimodal pore size distribution columns, and good quality collision induced dissociation (CID) mass spectra were obtained on an ion trap mass spectrometer. These novel monolithic separation media are thus a promising addition to the methodological toolbox of proteomics research. Keywords: proteomics • monoliths • Bronchoalveolar Lavage Fluid • serum • cancer • mass spectrometry • HPLC

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

“…The high resolving power and low operating pressure of the silica monolith added to the high selectivity of the Fourier Transform mass spectrometer allowed to separate all peptides from these libraries in 30 min, while sensitivity remained adequate due to the capillary format of the monolith. A similar monolith (15060.1 mm) was coupled to an IT mass spectrometer for the analysis of different proteomics samples using a commercially available microESI source [70]. Samples of high complexity and of very different nature were used to demonstrate the capacity of RP silica monoliths for the analysis of these biomedical samples.…”

“…The monolith with wider throughpores could be used with flow rates twice as high as the monolith with the smaller throughpores, though at the cost of decreased capacity and selectivity due to the loss of surface area. Monolithic columns (10064.6 mm [36]; 15060.1 mm and 50060.1 mm [70] and 56060.05 mm, unpublished results) were used to evaluate their efficacy for high-speed gradient elution RPLC of peptides using tryptic digests of cytochrome C from bovine heart [63] and horse heart [70]. Resolution and retention volume were found to vary very little when the linear flow was increased from 1.1 to 11 mm/s (10 mL/ min) for use with the 4.6-mm ID monolith.…”

“…Due to their very high porosity, capillary monoliths are often used with higher flow rates. Barroso [70] used flows up to 4.5 lL/min with a 100-lm ID RP silica monolith, coupled to an ion trap (IT) mass spectrometer via a commercially available pneumatically assisted microESI source. In an attempt to eliminate postcolumn dead volumes altogether, a 100-lm ID silica monolith was also directly coupled to the mass spectrometer [71].…”

An Automated and Multiplexed Method for High Throughput Peptide Immunoaffinity Enrichment and Multiple Reaction Monitoring Mass Spectrometry-based Quantification of Protein Biomarkers

“…Therefore, we were able to run a relatively short 10-min LC gradient analysis with trap column loading for shortened LC-MS analysis time (total run times about 45 min) and improved throughput (about 25 samples/day). New techniques in the area of nano-LC separations, such as chip-based separations (22) and fast monolithic columns (23,24), are expected to make the technique even more suitable for high throughput. Alternatively, adapting the SISCAPA technique to quantitative MALDI mass spectrometry (25) has the potential for greatly increasing the sample throughput.…”

Comparison of monolithic silica and polymethacrylate capillary columns for LC

“…Problems connected with fixing the monolithic beds inside fused-silica capillaries have been successfully solved and silica-based monolithic capillary columns are now available commercially and are employed for separation of various samples, including proteins and peptides in proteomic research [24]. In addition to sol-gel binding of silica particles to the walls of fused silica capillaries, capillary columns can be first packed conventionally with 5 lm octadecyl silica gel particles and then sintered to produce a continuous bed [25].…”