Coupling of hydrodynamically closed large bore capillary isotachophoresis with electrospray mass spectrometry

Capillary isotachophoresis with coupled columns provides efficient means for rapid electrophoretic analysis of sample volumes of up to 10 μl or more. Commercially available instruments are commonly equipped with conductivity and UV absorbance detectors; however, their on-line coupling with electrospray mass spectrometry is highly desirable. In this work we have modified the commercial coupled column isotachophoresis system for direct connection to an ion trap mass spectrometer. The design included attachment of an elution block with a short capillary transfer line directing the separated zones towards the mass spectrometer. The modification further included separation of the injection and electrode blocks from the separation columns by semipermeable membranes eliminating unwanted fluid movements in the wide bore fluoropolymer separation capillaries. Fused silica capillaries with varying internal diameter were connected as a transfer line between the elution block and mass spectrometer. The transfer line served also as the ESI tip of the sheathless electrospray interface. During the analysis the first, wide bore preseparation capillary with 0.8 mm internal diameter served for removal of the bulk sample components and preseparation of the potentially interfering analytes. After the electronic column switching the separation was finished in a 0.3 mm internal diameter capillary and the separated ITP zones were transferred in-line by a spray liquid towards the mass spectrometer. The instrumentation was tested for determination of vitamins in whole blood analysis and separation of tryptic peptides.     

Capillary liquid chromatography/atmospheric-pressure matrix-assisted laser desorption/ionisation ion trap mass spectrometry: a comparison with liquid chromatography/matrix-assisted laser desorption/ionisation time-of-flight and liquid chromatography/electrospray ionisation quadrupole time-of-flight for the identification of tryptic peptides

The atmospheric-pressure matrix-assisted laser desorption/ionisation quadrupole ion trap (AP-MALDI-QIT) analysis of tryptic peptides is reported following capillary liquid chromatographic (LC) separation and direct analysis of a protein digest. Peptide fragments were identified by peptide mass fingerprinting from mass spectrometric data and sequence analysis obtained by tandem mass spectrometry of the principal mass spectral peaks using a data-dependent scanning protocol. These data were compared with those from mass spectrometric analysis using capillary LC/MALDI-time-of-flight (TOF) and capillary LC/electrospray ionisation (ESI)-quadrupole TOF. For all three configurations the resulting data were searched against the MSDB database, using MASCOT and the sequence coverage compared for each technique. Complementary data were obtained using the three techniques.     

Capillary high-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic columns and carbon fiber electrospray ionization emitters

Monolithic columns having long hydrocarbon chains were prepared by in-situ polymerization in capillary fused silica tubing. The capillary columns were coupled with a newly developed carbon fiber electrospray ionization (ESI) emitter for proteomic analysis using sheathless capillary HPLC-ESI mass spectrometry (MS). The sample loading capacity and chromatographic performance of the styrene-based monolithic column, which was prepared by photo-polymerization of octylstyrene (OS) and divinylbenzene (DVB) were compared with that of the methacrylate-based monolithic column composed of lauryl methacrylate (LMA) and ethylene dimethacrylate (EDMA). The sample loading ability of tryptic digested protein in poly-OS (POS)-DVB column was higher than that of poly-LMA (PLMA)-EDMA column, possibly due to the irregular and rugluous surface offering a greater surface area of POS-DVB stationary phase. The POS-DVB column also provided better separation efficiency in the separation of high concentration (10 microg) of tryptic digested albumin bovine serum (BSA). Due to the successful interface of a highly efficient monolithic column and a stable, durable carbon fiber emitter, low femtomole levels of peptides were successfully separated and identified in the presence of large amounts of tryptic digested protein.     

Proteomic identification of technologically modified proteins in malt by combination of protein fractionation using convective interaction media and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A method for the fast separation of proteins and identification of their modifications based on the use of monolithic chromatographic media and mass spectrometric techniques is described. This method has been developed and applied to the analysis of malt proteins and its posttranslational modifications (glycation). Glycation, one of the most common forms of posttranslational modifications (PTM), can be detected in both biological and industrial samples. Our attention was focused on the investigations of possible chemical modifications of water-soluble barley proteins during malting process by combination of anion-exchange chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The malt extract was directly fractioned by anion-exchange chromatography using short monolithic columns and a linear gradient from 0 to 700 mM NaCl. Sufficient fractionation was obtained for malt sample, which demonstrates the potential of anion-exchange chromatography on this type of column. Proteins in separated fractions were identified by MALDI-TOF/TOF MS. Our proteomic analysis provided the identification of the major proteins present in the malt that were found to be heterogeneously glycated after malting. One of these proteins: nonspecific lipid transfer protein 1 (LTP1) can be used as a marker for characterization of glycation during malting. This protein and its modifications can be easily determined by the developed method.     

Comparison of comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry and gas chromatography-mass spectrometry for the analysis of tobacco essential oils

A sample of tobacco essential oil was analyzed using gas chromatography-mass spectrometry (GC/MS) and comprehensive two-dimensional gas chromatography coupled to a time-of-flight mass spectrometry (GC × GC/TOFMS), respectively. In the GC/MS analysis, serially coupled columns were used. By comparing the GC/MS results with GC × GC/TOFMS results, many more components in the essential oil could be found within the two-dimensional separation space of GC × GC. The quantitative determination of components in the essential oil was performed by GC × GC with flame ionization detection (FID), using a method of multiple internal standards calibration.     

Separation, detection, and identification of peptides by ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry at high and low pH

Bioactive peptides and tryptic digests of various proteins were separated under acidic and alkaline conditions by ion-pair-reversed-phase high-performance liquid chromatography (RP-HPIPC) in 200 microm I.D. monolithic, poly(styrene-divinylbenzene)-based capillary columns using gradients of acetonitrile in 0.050% aqueous trifluoroacetic acid, pH 2.1, or 1.0% triethylamine-acetic acid, pH 10.6. Chromatographic performances with mobile phases of low and high-pH were practically equivalent and facilitated the separation of more than 50 tryptic peptides of bovine serum albumin within 15-20 min with peak widths at half height between 4 and 10 s. Neither a significant change in retentivity nor efficiency of the monolithic column was observed during 17-day operation at pH 10.6 and 50 degrees C. Upon separation by RP-HPIPC at high-pH, peptide detectabilities in full-scan negative-ion electrospray ionization mass spectrometry (negESI-MS) were about two to three times lower as compared to RP-HPIPC at low-pH with posESI-MS detection. Tandem mass spectra obtained by fragmentation of deprotonated peptide ions in negative ion mode yielded interpretable sequence information only in a few cases of relatively short peptides. However, in order to obtain sequence information for peptides separated with alkaline mobile phases, tandem mass spectrometry (MS/MS) could be performed in positive ion mode. The chromatographic selectivities were significantly different in separations performed with acidic and alkaline eluents, which facilitated the fractionation of a complex peptide mixture obtained by the tryptic digestion of 10 proteins utilizing off-line, two-dimensional RP-HPIPC at high pH x RP-HPIPC at low pH and subsequent on-line identification by posESI-MS/MS.     

Qualitative drug analysis of hair extracts by comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry

A technique using comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC/TOFMS) is applied to a qualitative analysis of three sample extracts from hair suspected of containing various drug compounds. The samples were also subjected to a quantitative target analysis for codeine, morphine, 6-monoacetylmorphine (6-MAM), amphetamine, methamphetamine, methylenedioxyamphetamine (MDA), methylenedioxymethylamphetamine (MDMA), methadone, and benzylpiperazine (BZP) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). GC × GC/TOFMS provided a non-specific procedure that identified various drugs, metabolites, and impurities not included in the target analysis. They included cocaine, diazepam, and methaqualone (quaalude). Comprehensive GC × GC separation was achieved using twin-stage cryo-modulation to focus eluant from a DB-5ms (5% phenyl) to a BPX50 (50% phenyl) GC column. The TOF mass spectrometer provided unit mass resolution in the mass range m/z 5–1000 and rapid spectral acquisition (≤500 spectra/s). Clean mass spectra of the individual components were obtained using mass spectral deconvolution software. The ‘unknown’ components were identified by comparison with mass spectra stored in a library database.     

Capillary isoelectric focusing of probiotic bacteria from cow's milk in tapered fused silica capillary with off-line matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification

In this study, combination of capillary isoelectric focusing (CIEF) in tapered fused silica (FS) capillary with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is presented as an efficient approach for unambiguous identification of probiotic bacteria in real sample. For this purpose, bacteria within genus Lactobacillus were selected as model bioanalytes and cow's milk was selected as a biological sample. CIEF analysis of both the cultivated bacteria and the bacteria in the milk was optimized and isoelectric points characterizing the examined bacteria were subsequently determined independently of the bacterial sample origin. The use of tapered FS capillary significantly enhanced the separation capacity and efficiency of the CIEF analyses performed. In addition, the cell number injected into the tapered FS capillary was quantified and an excellent linearity of the calibration curves was achieved which enabled quantitative analysis of the bacteria by CIEF with UV detection. The minimum detectable number of bacterial cells was 2 × 10⁶ mL⁻¹. Finally, cow's milk spiked with the selected bacterium was analyzed by CIEF in tapered FS capillary, the focused and detected bacterial cells were collected from the capillary, deposited onto the cultivation medium, and identified using MALDI-TOF MS afterward. Our results have revealed that the proposed procedure can be advantageously used for unambiguous identification of probiotic bacteria in a real sample.     

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

High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 microm inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n-propanol and formamide as porogens and azobisisobutyronitrile as initiator. N-Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300 000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method.     

Intact-protein trapping columns for proteomic analysis in capillary high-performance liquid chromatography

A new type of monolithic trapping columns with high mechanical strength was prepared by thin-layer sol–gel coating method and applied to trapping intact proteins for on-line capillary liquid chromatography. Monolithic trapping columns were fabricated by entrapping C8 reversed-phase particles into the capillary columns through a sol–gel network, which was formed by hydrolysis and polycondensation of methyltriethoxysilane. Hundreds times of trapping/untrapping for intact proteins were carried out. The trapping columns showed long-term stability up to 300 bar. Recovery, loading capacity and reproducibility of trapping columns were evaluated using four proteins. The recovery of four protein mixtures for the C8 monolithic trapping columns was 99.3% on average. The loading capacity of 5 mm × 320 μm i.d. C8 trapping columns for the protein mixtures was 30 μg. Day-to-day relative standard deviation (RSD) values for recoveries of protein mixtures on the same C8 trapping column ranged from 2.34 to 5.87%, column-to-column RSD values were from 3.01 to 6.81%. The C8 trapping columns were used to trap normal mouse liver intact proteins in a capillary liquid chromatography system. Results demonstrated high efficiency of the monolithic trapping columns for trapping intact proteins for proteomic analysis in on-line capillary liquid chromatography system.     

Development and optimization of a method for the separation of platycosides in Platycodi Radix by comprehensive two-dimensional liquid chromatography with mass spectrometric detection

Comprehensive two-dimensional chromatography (LC × LC) using combinations of two columns (C₁₈ × CN and C₁₈ × NH₂) was employed with electrospray (ESI) mass spectrometry to analyze platycosides from root extract. Based on the capability of the C₁₈, CN and NH₂ columns to separate the platycosides, the orthogonality in two-dimensional space according to each combination of columns was predicted from the correlation coefficients between the retention times of the 17 compounds separated by the independent CN and C₁₈ columns, and NH₂ and C₁₈ columns. The expected distribution of the peaks was also compared with the two-dimensional plots obtained by practical separation in an LC × LC system. The increased peak capacities using C₁₈ × NH₂ allowed three minor components and five isomers of the platycosides to be newly separated, which were not identified with 1D-LC using the individual C₁₈ column, whereas the combination of C₁₈ × CN did not result in any improvement of the separation performance.     

N-terminal H3/D3-acetylation for improved high-throughput peptide sequencing by matrix-assisted laser desorption/ionization mass spectrometry with a time-of-flight/time-of-flight analyzer

A novel method for peptide sequencing by matrix-assisted laser desorption/ionization mass spectrometry with a time-of-flight/time-of-flight analyzer (MALDI-TOF/TOF) is presented. A stable isotope label introduced in the peptide N-terminus by derivatization, using a 1:1 mixture of acetic anhydride and deuterated acetic anhydride, allows for easy and unambiguous identification of ions belonging either to the N- or the C-terminal ion series in the product ion spectrum, making sequence assignment significantly simplified. The good performance of this technique was shown by successful sequencing of the contents of several peptide maps. A similar approach was recently applied to nanoelectrospray ionization (nanoESI) and nano-liquid chromatography/tandem mass spectrometry (LC/MS/MS). The MALDI-TOF/TOF technique allows for fast, direct sequencing of modified peptides in proteomics samples, and is complementary to the nanoESI and nanoLC/MS/MS approaches.     

Effects of the operation parameters on Hydrophilic Interaction Liquid Chromatography separation of phenolic acids on zwitterionic monolithic capillary columns

Strongly polar phenolic acids are weakly retained and often poorly separated in reversed-phase (RP) liquid chromatography. We prepared zwitterionic polymethacrylate monolithic columns for micro-HPLC by in situ co-polymerization in fused-silica capillaries. The capillary monolithic columns prepared under optimized polymerization conditions show some similarities with the conventional particulate commercial ZIC-HILIC silica-based columns, however have higher retention and better separation selectivity under reversed-phase conditions, so that they can be employed for dual-mode HILIC-RP separations of phenolic acids on a single column. The capillary polymethacrylate monolithic sulfobetaine columns show excellent thermal stability and improved performance at temperatures 60–80 °C. The effects of the operation conditions on separation were investigated, including the type and the concentration of the organic solvent in the aqueous-organic mobile phase (acetonitrile and methanol), the ionic strength of the acetate buffer and temperature. While the retention in the RP mode decreases at higher temperatures in mobile phases with relatively low concentrations of acetonitrile, it is almost independent of temperature at HILIC conditions in highly organic mobile phases. The best separation efficiency can be achieved using relatively high acetate buffer ionic strength (20–30 mmol L⁻¹) and gradient elution with alternately increasing (HILIC mode) and decreasing (RP mode) concentration of aqueous buffer in aqueous acetonitrile. Applications of the monolithic sulfobetaine capillary columns in alternating HILIC-RP modes are demonstrated on the analysis of phenolic acids in a beer sample.     

Practical aspects of fast HPLC separations for pharmaceutical process development using monolithic columns

A large number of samples can be generated during pharmaceutical process development. Fast separation for these samples is usually challenging due to the complexity of sample matrix, which requires high efficiency as well as high speed. Monolithic columns (E. Merck, Germany) were investigated as a possible tool for reducing separation time in reversed-phase HPLC without significantly sacrificing efficiency or resolution. Both van Deemter plots and separations of alkyl benzenes and in-process samples showed that monolithic columns were suitable for fast separations without significantly compromising resolution. Practical parameters including the pressure drop, retention factor, selectivity, and tailing factor of monolithic columns (Chromolith type) were compared to those of conventional YMC 150 mm × 4.6 mm (3-μm particles) and 250 mm × 4.6 mm (5-μm particles) packed columns. The batch-to-batch reproducibility of the 100 mm × 4.6 mm Chromolith columns from five randomly ordered batches was also compared to the 250 mm × 4.6 mm YMC particle-packed columns. Fast and efficient separations of complicated process samples including crude drug substances, reaction mixtures, and crystallized mother liquors were demonstrated for both monolithic columns and conventional packed columns. The analysis times were decreased by three to seven times on the coupled monolithic columns, while maintaining the comparable resolution to typical 5-μm particle-packed 250 mm × 4.6 mm columns.     

Preparation of a butyl–silica hybrid monolithic column with a “one-pot” process for bioseparation by capillary liquid chromatography

A butyl–silica hybrid monolithic column for bioseparation by capillary liquid chromatography (cLC) was prepared with butyl methacrylate and alkoxysilanes through a “one-pot” process. The effects of polycondensation temperature, volume percentage of N,N′-dimethylformamide, and content of cetyltrimethylammonium bromide and butyl methacrylate on the morphologies of the hybrid monolithic columns prepared were investigated in detail. Baseline separations of proteins and small peptides on the hybrid monolithic column were achieved by cLC with gradient elution. In addition, the resulting hybrid column was also applied for analysis of tryptic digests of bovine serum albumin by cLC coupled with tandem mass spectrometry. The results demonstrate its potential application in separation of complex biological samples.     

Peptide mapping with mobile phases of intermediate pH value using capillary reversed-phase high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry

This investigation describes the separation of tryptic peptides by capillary reversed-phase high-performance liquid chromatography (RP-HPLC) with eluents in the intermediate pH range, followed by in-line electrospray ionisation tandem mass spectrometry (ESI-MS/MS) analysis. For these purposes, gradient elution procedures with an aqueous eluent containing 20 mM ammonium formate, and an increasing content of acetonitrile or methanol, were employed. Compared to the analysis of the same tryptic peptides under low-pH conditions with an ion-pairing reagent, the increase in the pH with the 20 mM ammonium formate mobile phase led to significant changes in both peptide retention to the reversed-phase column and the collision-induced dissociation at the MS/MS stage as a consequence of the changes in the physico-chemical properties of these peptides, such as their overall charge, polarity and relative hydrophobicity. Thus, improved selectivity for the peptide separation and favourable tandem mass spectrometry analysis could be obtained with eluents in this intermediate pH range. The number of tryptic peptides identified by the new approach for the proteins investigated were significantly higher than that obtained by the conventional low-pH methods. Moreover, analysis of protein digests at very low concentrations was also performed under both acidic and intermediate pH conditions and similar improvements in selectivity and MS/MS detection limits were observed, i.e. identification of more distinct peptides and higher sequence coverage of the protein was obtained when eluents of intermediate pH were employed. This study therefore highlights the potential of conducting peptide mapping in the intermediate pH range to achieve more reliable and sensitive protein identifications with capillary RP-HPLC–ESI-MS/MS.     

On-line amino acid-based capillary isoelectric focusing-ESI-MS/MS for protein digests analysis

Six amino acids with pIs that ranged from 3.2 to 9.7 were used as ampholytes to establish a pH gradient in capillary isoelectric focusing. This amino acid-based capillary isoelectric focusing (cIEF) was coupled with ESI-MS/MS using an electrokinetically pumped sheath-flow interface for peptide analysis. Amino acid-based isoelectric focusing generates a two-order of magnitude lower background signal than commercial ampholytes in the important m/z range of 300–1800. Good focusing was achieved for insulin receptor, which produced ∼10 s peak width. For 0.1 mg mL⁻¹ bovine serum albumin (BSA) digests, 24 ± 1 peptides (sequence coverage 47 ± 4%) were identified in triplicate analysis. As expected, the BSA peptides were separated according to their pI. The concentration detection limit for the BSA digests is 7 nM and the mass detection limit is 7 fmole. A solution of six bovine protein tryptic digests spanning 5 orders of magnitude in concentration was analyzed by amino acid based cIEF-ESI-MS/MS. Five proteins with a concentration range spanning 4 orders of magnitude were identified in triplicate runs. Using amino acid based cIEF-ESI-MS/MS, 112 protein groups and 303 unique peptides were identified in triplicate runs of a RAW 264.7 cell homogenate protein digest. In comparison with ampholyte based cIEF-ESI-MS/MS, amino acid based cIEF-ESI-MS/MS produces higher resolution of five acidic peptides, much cleaner mass spectra, and higher protein spectral counts.     

De novo sequencing of novel neuropeptides directly from Ascaris suum tissue using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight

Direct analysis of tissue by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) allows for the rapid profiling of biological molecules with minimal loss of sample or degradation and reduced likelihood of chemical modification. However, there are still considerable challenges to overcome due to the complexity of tissue and the low quantity of endogenous peptide in a single cell. These problems are exacerbated in the nematode Ascaris suum because of the small size of individual neurons and the paucity of peptide per cell. In an effort to address these difficulties, the recently developed matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) technology was used in combination with an on-target derivatization in order to sequence novel neuropeptides directly from Ascaris nervous tissue. Direct MALDI-TOF/TOF analysis of Ascaris tissue provided the complete amino acid sequences for a previously characterized neuropeptide as well as for three novel peptides with homologues found in other nematodes. These results demonstrate a method for the rapid characterization of sub-femtomolar amounts of peptide directly from tissue using MALDI-TOF/TOF.     

Potential of poly(styrene-<Emphasis Type="Italic">co</Emphasis>-divinylbenzene) monolithic columns for the LC-MS analysis of protein digests

Two polystyrene-based capillary monolithic columns of different length (50 and 250 mm) were used to evaluate the effects of column length on gradient separation of protein digests. A tryptic digest of a 9-protein mixture was used as a test sample. Peak capacities were determined from selected extracted ion chromatograms, and tandem mass spectrometry data were used for database matching using the MASCOT search engine. Peak capacities and protein identification scores were higher for the long column with all gradients. Peak capacities appear to approach a plateau for longer gradient times; maximum peak capacity was estimated to be 294 for the short column and 370 for the long column. Analyses with similar gradient slope produced a ratio of the peak capacities of 3.36 for the long and the short column, which is slightly higher than the expected value of the square root of the column length ratio. The use of a longer monolith improves peptide separation, as reflected by higher peak capacity, and also increases protein identification, as observed from higher identification scores and a larger number of identified peptides. Attention has also been paid to the peak production rate (PPR, peak capacity per unit time). For short analysis times, the short column produces a higher PPR, while for analysis times longer than 40 min, the PPR of the 250-mm column is higher.     

Automation of nanoflow liquid chromatography-tandem mass spectrometry for proteome and peptide profiling analysis by using a monolithic analytical capillary column

An automated nano-LC-MS/MS platform without trap column was established, which only used a 20 cm lauryl methacrylate-ethylene dimethacrylate (LMA-EDMA) monolithic capillary column to allow preconcentration and separation of peptides. The monolithic column had the advantages of good permeability and low backpressure resulting in higher flow rates for capillary columns. Tryptic digests of bovine albumin and yeast protein extract were tested using the monolithic column system. High proteomic coverage using this approach were demonstrated in this study. Furthermore, peptide samples extracted from mouse liver were separated by using the monolithic column system combined with size-exclusion chromatography prefractionation. This monolithic column system might be a promising alternative for the automated system previously using a trap column for routine proteome and peptide profiling analysis.