High-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic capillary columns for proteomic studies.

The use of tetrahydrofuran/decanol as porogens for the fabrication of micropellicular poly(styrene/divinylbenzene) monoliths enabled the rapid and highly efficient separation of peptides and proteins by reversed-phase high-performance liquid chromatography (RP-HPLC). In contrast to conventional, granular, porous stationary phases, in which the loading capacity is a function of molecular mass, the loadability of the monoliths both for small peptides and large proteins was within the 0.40.9-pmol range for a 60- x 0.2-mm capillary column. Lower limits of detection obtained by measuring UV-absorbance at 214 nm with a 3-nl capillary detection cell were 500 amol for an octapeptide and 200 amol for ribonuclease A. Upon reduction of the concentration of trifluoroacetic acid in the eluent from the commonly used 0.1-0.2 to 0.05%, the separation system was successfully coupled to electrospray ionization mass spectrometry (ESI-MS) at the cost of only a small decrease in separation efficiency. Detection limits for proteins with ESI-MS were in the lower femtomole range. High-quality mass spectra were extracted from the reconstructed ion chromatograms, from which the masses of both peptides and proteins were deduced at a mass accuracy of 50-150 ppm. The applicability of monolithic column technology in proteomics was demonstrated by the mass fingerprinting of tryptic peptides of bovine catalase and human transferrin and by the analysis of membrane proteins related to the photosystem II antenna complex of higher plants.

Determination of nociceptin-orphanin FQ metabolites by capillary LC-MS.

Nociceptin-orphanin FQ (OFQ/N) is a newly discovered peptide involved in pain transmission. The method is described to identify metabolic pathway of this neuropeptide in the spinal cord of rats using capillary size-exclusion liquid chromatography coupled to electrospray ionization mass spectrometry. The applied technique is rapid and selective, and allows for simultaneous measurement and quantitation of several fragments in the incubation mixture.

Size-exclusion chromatography performed in capillaries. Studies by liquid chromatography-mass spectrometry.

Miniaturization of the chromatographic column led to increased sensitivity and shortened time of analysis. In our work we applied 300 microm I.D. capillaries packed with a novel stationary phase Superdex Peptide for the size-exclusion chromatography, capable of separating molecules within the mass range of 0.1-7 kDa. Here we proved that such capillary columns can operate effectively at high sensitivity. Several peptide mixtures were efficiently chromatographed and analyzed on line with electrospray ionization mass spectrometry as a detection technique. A CNBr peptide map, derived from human globin alpha subunit, was effectively separated using this method. These fragments are difficult to elute from the reversed-phase column at low pH, therefore, such approach can be considered as a complementary to other separation techniques, in particular for analyzing hydrophobic components and complex mixtures.

Automated on-line ionic detergent removal from minute protein/peptide samples prior to liquid chromatography-electrospray mass spectrometry.

An automated on-line ionic detergent removal pre-column system coupled to capillary liquid chromatography-electrospray mass spectrometry is described. The system involves two micro precolumns, composed of a specific ionic detergent trapping column and a preconcentration column, respectively, and a packed 300 microns I.D. analytical column. Sample loading to the micro precolumns and regeneration of the detergent trapping column were performed at a flow-rate of 50 microliters/min, while the flow-rate through the analytical column was set at 5.0 microliters/min. Ionic detergent-containing tryptic-digested protein samples were directly applied to the micro precolumns without sample pretreatment and were analysed by UV absorption detection and electrospray mass spectrometry. The presented system allows for the fully automated removal of SDS with virtually no loss in protein/peptides. Maximum SDS load and breakthrough have been determined. Excellent protein recovery and complete removal of SDS is found. The chromatographic separation after SDS removal was completely restored and equalled the reference chromatogram. Mass spectral data confirm these findings. Finally, this technique allows for SDS removal from minute protein samples without the need for any sample handling.

Sodium dodecyl sulphate removal from tryptic digest samples for on-line capillary liquid chromatography/electrospray mass spectrometry.

An on-line microcolumn switching method was developed for the removal of sodium dodecyl sulphate (SDS) from tryptic digest samples. The system includes two micro-precolumns: a specific ionic detergent trapping column and a preconcentration column. Characterization of the proteinaceous samples, after isolation from the SDS, was performed by capillary liquid chromatography (LC) with UV absorption detection and electrospay mass spectrometry (ESI-MS). Loading and clean-up of the samples and regeneration of the detergent trapping column were performed at 50 microl min(-1), resulting in sample clean-up times of only 30 s. SDS-containing tryptic digested protein samples were directly applied to the micro-precolumns without any previous sample pretreatment. The developed microcolumn switching method permits the on-line analysis of small tryptic digest samples by capillary LC/ESI-MS in the presence of SDS. The method is completely automated and can be performed unattended. The maximum amount of SDS, in terms of loadability and breakthrough, were determined. Also studied were the selection of the loading and clean-up solvents and the recovery of the peptides. Chromatographic separations and mass spectral data confirmed the removal of SDS.

Instrumental requirements for nanoscale liquid chromatography.

Nanoscale liquid chromatography (nano-LC), with packed columns of typically 75 µm i.d. × 15 cm length, packed with C18, 5 µm of stationary phase, and optimal flow rates of 180 nL/min, can be considered as a miniaturized version of conventional HPLC. Using the down-scaling factor, which corresponds to the ratio of the column diameter in square, (d(conv)/d(micro))(2), excellent agreement between the theoretically calculated values and the values obtained using the down-scaling factor (∼3800) has been observed. This factor was applied to all system components, including flow rate, injection and detection volumes, and connecting capillaries. Down-scaling of a conventional HPLC system to a nano-LC system is easy to realize in practice and involves using a microflow processor for nanoflow delivery (50-500 nL/min), a longitudinal nanoflow cell (≤3 nL), a microinjection valve (≤ 20 nL), low-dispersion connecting tubing, and zero dead volume connections. Excellent retention time reproducibility was measured with RSD values of ±0.1% for isocratic and ±0.2% for gradient elution. Plates counts of more than 100 000/m indicate the excellent performance of the entire nano-LC system. With minimal detectable amounts of proteins in the low femtomole and subfemtomole ranges (e.g., 520 amol for bovine serum albumin), high mass sensitivity was found, making nano-LC attractive for the microcharacterization of valuable and/or minute proteinaceous samples. Coupling nano-LC with concomitant mass spectrometry using nanoscale ion spray or electrospray interfaces looks very promising and is obviously the next step for future work.

Determination of sub-nanogram amounts of dihydroergotamine in plasma and urine using liquid chromatography and fluorimetric detection with off-line and on-line solid-phase drug enrichment.

A highly sensitive and selective high-performance liquid chromatographic method, involving sample pre-treatment, column switching and fluorimetric detection, is described for the determination of dihydroergotamine in plasma and urine samples. The pre-chromatographic sample treatment consists of extraction by means of an Extrelut column for plasma samples, and pre-separation with enrichment steps on a Sep-Pak column for urine samples. The samples are then injected onto a pre-separation column (Aquapore), and the fraction containing dihydroergotamine are automatically diverted onto an analytical column (ODS reversed phase). An acetonitrile-ammonium carbamate gradient is used as the mobile phase. High recovery of dihydroergotamine from both plasma (87%) and urine (100%) and a detection limit as low as 100 pg/ml were achieved, with a linear response up to 5 ng/ml. The assay demonstrated a high degree of selectivity with regard to the extensive metabolism of dihydroergotamine especially to the main metabolite 8'-hydroxydihydroergotamine. The assay was successfully applied for more than one year to the determination of plasma and urine concentrations of dihydroergotamine after parenteral administration.