Two-dimensional capillary liquid chromatography: pH gradient ion exchange and reversed phase chromatography for rapid separation of proteins

In the present work, an orthogonal two-dimensional (2D) capillary liquid chromatography (LC) method for fractionation and separation of proteins using wide range pH gradient ion exchange chromatography (IEC) in the first dimension and reversed phase (RP) in the second dimension, is demonstrated. In the first dimension a strong anion exchange (SAX) column subjected to a wide range (10.5-3.5) descending pH gradient was employed, while in the second dimension, a large pore (4,000 A) polystyrene-divinylbenzene (PS-DVB) RP analytical column was used for separation of the protein pH-fractions from the first dimension. The separation power of the off-line 2D method was demonstrated by fractionation and separation of human plasma proteins. Seventeen pH-fractions were manually collected and immediately separated in the second dimension using a column switching capillary RP-LC system. Totally, more than 200 protein peaks were observed in the RP chromatograms of the pH-fractions. On-line 2D analysis was performed for fractionation and separation of ten standard proteins. Two pH-fractions (basic and acidic) from the first dimension were trapped on PS-DVB RP trap columns prior to back-flushed elution onto the analytical RP column for fast separation of the proteins with UV/MS detection.     

Two-dimensional strong cation exchange/porous layer open tubular/mass spectrometry for ultratrace proteomic analysis using a 10 microm id poly(styrene- divinylbenzene) porous layer open tubular column with an on-line triphasic trapping column

This study expands the capabilities for ultratrace proteomic analysis of our previous work by incorporating on-line sample desalting using a triphasic (RP/strong cation exchange (SCX)/micro-SPE) trapping column connected to a 3.2 m x 10 microm id poly(styrene-divinylbenzene) (PS-DVB) porous layer open tubular (PLOT) column. To minimize extra sample handling steps, C18 RP packing was incorporated in the capillary tubing upstream of the SCX column for the on-line desalting. For the micro-SPE column, a 50 microm id PS-DVB monolithic column was positioned downstream of the SCX column. High-performance separation was achieved on the PLOT column at a mobile phase flow rate of 20 nL/min. The sensitivity and high resolution capability of the new multidimensional platform was evaluated using an in-gel tryptic digested sample of a cervical cancer (SiHa) cell line. For the injected amount of 1200 cells ( approximately 500 ng), over 2700 peptides covering greater than 850 unique proteins were identified from the triphasic SCX/PLOT/MS analysis of a single SDS gel section (>40 kDa). The 2-D LC/MS platform demonstrated good separation performance, such that more than 85% of the identified peptides were detected from only one salt fraction. In a triplicate analysis of the above >40 kDa gel section, 4497 peptides and 1209 unique proteins were identified when applying stringent filtering criteria, with a false-positive rate of 2.4%. When all three SDS-PAGE gel sections of the lysed SiHa cells were analyzed, 5047 peptides and 1857 unique proteins (false-positive rate 1.8%), including cancer-related proteins such as MAP kinases, were identified.     

Tandem LC columns for the simultaneous retention of polar and nonpolar molecules in comprehensive metabolomics analysis

The tandem use of hydrophilic interaction LC columns with RP columns in series configuration has resulted in the retention of both polar and nonpolar components in complex biological samples (mouse serum) in a single analysis. This approach successfully coupled various columns with orthogonal separation characteristics, employed a single solvent gradient program compatible with the two columns and used ESI coupled to a TOF mass spectrometer for detection. Ion suppression, a common problem in ESI, was virtually eliminated for components eluting with apparent capacity factors >0.7. Retention time reproducibility with the tandem columns performed over three days with over 100 injections was comparable to that observed for single columns alone. This method was applied to the analysis of a pooled mouse serum sample and afforded highly reproducible data for up to 3000 mass spectral features. This approach was implemented with a conventional LC–MS system and should find broad applicability in the comprehensive analysis of complex mixtures containing a wide range of compound polarities.     

On-Line multitasking analytical proteomics: how to separate, reduce, alkylate and digest whole proteins in an on-Line multidimensional chromatography system coupled to MS

An on-Line multidimensional system has been developed, consisting of pH gradient strong anion exchange chromatography of native proteins in the first dimension with subsequent trapping and on-column reduction/alkylation on C4 trap columns and RP separation of the alkylated proteins in the second dimension followed by on-column tryptic digestion and electrospray MS detection. The system was evaluated using model proteins and a human urine sample. Compared to the commonly used in-solution alkylation method, the developed on-column method provides an equivalent efficiency. The recovery from the C4 trap columns of the alkylated proteins relative to the native state was from 94 to 102%. On-column tryptic digestion was satisfactory for many, but not for all proteins. The whole analytical procedure was performed on-Line with packed capillary columns for a total time of 320 min for the first ion exchange fraction, with additional 60 min for each subsequent fraction.     

Multidimensional separation of tryptic peptides from human serum proteins using reversed‐phase,strong cation exchange,weak anion exchange,and fused‐core fluorinated stationary phases

Proteome profiling of crude serum is a challenging task due to the wide dynamic range of protein concentrations and the presence of high‐abundance proteins, which cover >90% of the total protein mass in serum. Peptide fractionation on strong cation exchange, weak anion exchange in the electrostatic repulsion hydrophilic interaction chromatography (ERLIC) mode, RP C₁₈ at pH 2.5 (low pH), fused‐core fluorinated at pH 2.5, and RP C₁₈ at pH 9.7 (high pH) stationary phases resulted in two to three times more identified proteins and three to four times more identified peptides in comparison with 1D nanoChip‐LC–MS/MS quadrupole TOF analysis (45 proteins, 185 peptides). The largest number of peptides and proteins was identified after prefractionation in the ERLIC mode due to the more uniform distribution of peptides among the collected fractions and on the RP column at high pH due to the high efficiency of RP separations and the complementary selectivity of both techniques to low‐pH RP chromatography. A 3D separation scheme combining ERLIC, high‐pH RP, and low‐pH nanoChip‐LC–MS/MS for crude serum proteome profiling resulted in the identification of 208 proteins and 1088 peptides with the lowest reported concentration of 11 ng/mL for heat shock protein 74.     

Application of nanoscale packed capillary liquid chromatography (75 μm id) and capillary zone electrophoresis/electrospray ionization mass spectrometry to the analysis of macrolide antibiotics

Nanoscale separation techniques, nanoscale packed capillary columns (75 μm id), and capillary zone electrophoresis (CZE), on-line with electrospray mass spectrometry (ESI/MS), were applied to the separation of a series of ten macrolide antibiotics. Both techniques use sub-microliter-per-minute flow rates through the analytical column and therefore require an electrospray probe that incorporates coaxial sheath flow. Positive ion electrospray mass spectra of these compounds yielded mainly protonated molecules. Fragmentation to yield structurally significant fragment ions was achieved by collision-induced dissociation (CID) at increased skimmer voltages. Separations were achieved using both techniques, with CZE/ESI/MS showing improved peak shapes and detection limits combined with faster analysis times. Nanoscale packed capillary columns provided better chromatographic resolution and was less susceptible to peak broadening caused by overloading of the analytes.     

A fully automated 2-D LC-MS method utilizing online continuous pH and RP gradients for global proteome analysis

The conventional 2-D LC-MS/MS setup for global proteome analysis was based on online and offline salt gradients (step and continuous) using strong-cation-exchange chromatography in conjunction with RP chromatography and MS. The use of the online system with step salt elution had the possibility of resulting in peptide overlapping across fractions. The offline mode had the option to operate with continuous salt gradient to decrease peak overlap, but exhibited decreased robustness, lower reproducibility, and sample loss during the process. Due to the extensive washing requirement between the chromatography steps, online continuous gradient was not an option for salt elution. In this report, a fully automated, online, and continuous gradient (pH continuous online gradient, pCOG) 2-D LC-MS/MS system is introduced that provided excellent separation and identification power. The pH gradient-based elution provided more basic peptides than that of salt-based elution. Fraction overlap was significantly minimized by combining pH and continuous gradient elutions. This latter approach also increased sequence coverage and the concomitant confidence level in protein identification. The salt and pH elution-based 2-D LC-MS/MS approaches were compared by analyzing the mouse liver proteome.     

Multidimensional capillary array liquid chromatography and matrix-assisted laser desorption/ionization tandem mass spectrometry for high-throughput proteomic analysis

A two-dimensional capillary array liquid chromatography system coupled with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) was developed for high-throughput comprehensive proteomic analysis, in which one strong cation-exchange (SCX) capillary chromatographic column was used as the first separation dimension and 10 parallel reversed-phase liquid chromatographic (RPLC) capillary columns were used as the second separation dimension. A novel multi-channel interface was designed and fabricated for on-line coupling of the SCX to RPLC column array system. Besides the high resolution based on the combination of SCX and RPLC separation, the developed new system provided the most rapid two-dimensional liquid chromatography (2D-LC) separation. Ten three-way micro-splitter valves used as stop-and-flow switches in transferring SCX fractions onto RPLC columns. In addition, the three-way valves also acted as mixing chambers of RPLC effluent with matrix. The system enables on-line mixing of the LC array effluents with matrix solution during the elution and directly depositing the analyte/matrix mixtures on MALDI plates from the tenplexed channels in parallel through an array of capillary tips. With the novel system, thousands of peptides were well separated and deposited on MALDI plates only in 150min for a complex proteome sample. Compared with common 2D-LC system, the parallel 2D-LC system showed about 10-times faster analytical procedure. In combination with a high throughput tandem time of flight mass spectrometry, the system was proven to be very effective for proteome analysis by analyzing a complicated sample, soluble proteins extracted from a liver cancer tissue, in which over 1202 proteins were identified.     

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.     

Fabrication of a poly(styrene-octadecene-divinylbenzene) monolithic column and its comparison with a poly(styrene-divinylbenzene) monolithic column for the separation of proteins

In this paper, a poly(styrene-octadecene-divinylbenzene) (PS-OD-DVB) monolithic column was prepared in one step by introducing a C18 carbon chain as monomer. N,N-Dimethylformamide and decanol served as porogens to make a homogeneous polymerization mixture in a fused silica capillary (320 microm inner diameter). Its physical and chromatographic properties were compared with those of poly(styrene-divinylbenzene) (PS-DVB) monolithic column, which was also fabricated by in-situ polymerization in a fused silica capillary with the same inner diameter. Six standard proteins were used to evaluate the columns and their potential application for the separation of human hemoglobin was also discussed. It was shown that the PS-OD-DVB and PS-DVB monoliths appeared to have similar efficiency for rapid separation of six proteins within 3.5 min. The PS-OD-DVB monolith was found to have higher loading capacity and higher resolution for the separation of alpha and beta chains of hemoglobin because of the introduction of C18 carbon chains, and shows great potential for the separation of bio-macromolecules.     

Two-dimensional reverse phase-reverse phase chromatography: A simple and robust platform for sensitive quantitative analysis of peptides by LC/MS. Hardware design

We have revised current two-dimensional RP-RP approaches and developed a new robust 2-D RP-RP platform. This platform was implemented on an Agilent 1100 2-D liquid chromatography system and is based on high pressure switching between two high-resolution RP columns. An independent binary gradient was implemented for each dimension. The powerful combination of dual analytical columns with independent gradient elution achieves high analyte purity, effectively eliminates matrix effects, and maximizes MS sensitivity in Q1 SIM comparable to the sensitivity enhancements of MS/MS-based methods. Implementation of dual simultaneous gradient profiles (overlapped gradients) reduces 2-D method run-time to the scale of 1-D method run-times. This robust and sensitive approach is particularly suitable for hydrophobic peptides and small proteins and can be used as a routine standard technique for enhanced on-line peptide purification coupled with mass spectrometric detection.     

Better understanding of carbon nanoparticles via high‐performance liquid chromatography‐fluorescence detection and mass spectrometry

RP‐HPLC coupled with fluorescence detection for separation of carbon nanoparticles (CNP) synthesized with microwave‐assisted pyrolysis of citric acid and 1,2‐ethylenediamine is presented. The influence of methanol content and pH of mobile phase on the separation of CNP has been investigated. Under optimal mobile phase and elution gradient conditions, the effect of mole ratio of amine to carboxylic groups (NH₂/COOH) in the initial reagents on CNP product is studied. At NH₂/COOH = 0.67, the strongest fluorescence CNP sample is obtained. The separated CNP fractions are collected and further characterized by UV‐visible absorption and photoluminescence (PL) spectroscopy, CE, transmission electron microscopy (TEM), and MALDI‐TOF MS. The absorption and PL emission bands of the fractions are bathochromatically shifted with the elution order of CNP on RP‐HPLC. The TEM images prove that CNP are eluted from the smallest to the largest. The MS data show that CNP undergo fragmentations, closely relating to their surface‐attached carboxylic acid and amide/amine moieties. This work highlights the merit of RP‐HPLC coupled with fluorescence detection, TEM, and MS for isolation and characterization of individual CNP species present in a CNP sample.     

Separation and characterisation of sphingoceramides by high-performance liquid chromatography-electrospray ionisation mass spectrometry

We developed a simple and reliable analytical method for the quantification and the characterization of ceramides extracted from biological samples by high-performance liquid chromatography (HPLC) coupled to electrospray ionisation tandem mass spectrometry (ESI/MS/MS). The chromatographic separation of analytes was carried out in a RP8 column, eluting with a methanol-water mixture in gradient elution mode. The separated lipids were detected by total ion monitoring and characterised by MS/MS spectra; quantitative analysis was performed by integrating the extracted ion peaks obtained in the negative ion mode. Good repeatability was obtained for retention time (0.3-2%), peak area ratio (A(S)/A(IS), 2-8%), as well as limit of detection (LOD, 5-26 pg) and quantification (LOQ, 13-53 pg). The method was validated for the analysis of N-palmitoyl-D-erythro-sphingosine (Cer16), N-stearoyl-D-erythro-sphingosine (Cer18), N-tetracosanoyl-D-erythro-sphingosine (N24:0, lignoceric ceramide, Cer24:0), and N-tetracos-15'-enoyl-D-erythro-sphingosine (N24:1, nervonic ceramide, Cer24:1), giving good results. Lipid mixtures, extracted from skin and epidermal cells, were analysed for their content of the studied ceramides.     

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.     

Hollow-fiber flow field-flow fractionation: a gentle separation method for mass spectrometry of native proteins

Low-impact ionization sources like electrospray ionization (ESI) and matrix-assisted, laser desorption/ionization (MALDI) equipped with time-of-flight (TOF) mass analyzers provide intact protein analysis over a very wide molar mass range. ESI/TOFMS provides also indications on the higher-order structure of intact proteins and non-covalent protein complexes. However, direct analysis of intact proteins mixtures in real samples shows limited success, mainly because spectra become very complex to interpret. This is also due to sample contaminants, and to the mechanism of competitive ionization in ESI or MALDI. Rapid and efficient sample clean-up and separation methods can significantly enhance the power of TOFMS for intact protein analysis. However, if protein native conditions want to be maintained, the methods should affect neither the three-dimensional structure nor the non-covalent chemistry of the proteins. Reversed-phase (RP) HPLC, size-exclusion chromatography (SEC), and capillary zone electrophoresis (CZE) are on-line or off-line coupled to ESI/TOFMS or MALDI/TOFMS. In fact, these separation methods often show limitations when applied to the analysis of native proteins. Organic modifiers and saline buffers are required in the case of RP HPLC or CZE. They can induce protein degradation or affect ionization when MS is performed after separation. High voltages used in CZE can contribute to alter proteins from their native form. In the case of high molar mass proteins, SEC is scarcely selective, and barely able to detect protein aggregates. Sample entanglement/adsorption on the stationary phase can also occur.     

Short monolithic columns for purification and fractionation of peptide samples for matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry analysis in proteomics

This study records a novel application of methacrylate-based monolithic columns for MALDI-TOF/TOF MS analyses in proteomics for pre-concentration and separation of peptides derived from protein digestion. Reversed-phase monolithic capillary columns (30 mm × 0.32 mm i.d.) were created inside the fused silica capillary via thermal-initiated free-radical polymerization of ethylene glycol dimethacrylate and lauryl methacrylate monomers in the presence of 1-propanol and 1,4-butandiol as a porogen system. The elution of peptides was achieved using a linear gradient of acetonitrile from 0 to 60% in water with 0.1% trifluoroacetic acid formed in a microsyringe. Individual fractions of separated peptides were collected on the MALDI target spots covered with alpha-cyano-4-hydroxycinnamic acid used as a matrix and then they were analyzed using MALDI-TOF/TOF mass spectrometry. The developed method was tested with a mixture of tryptic peptides from bovine serum albumin and its applicability was also tested for tryptic in-gel digests from barley grain extracts of water soluble proteins separated using SDS gel electrophoresis. The number of detected peptides was approximately three to four times higher compared to the analysis without previous separation. These results show an improved quality of sample information with the higher amount of identified peptides which increased protein sequence coverage and improved sensitivity of mass spectrometry measurements.     

凝胶过滤/离子交换全二维液相色谱系统的构建与评价

基于蛋白质的尺寸及带电性质,将凝胶过滤色谱(GFC)与离子交换色谱(IEC)两种分离模式结合,采用双捕集柱接口构建了GFC/2×IEC二维液相色谱(2-D LC)分离系统,同时考虑离子交换色谱分离蛋白质对等电点范围的限制,进一步结合中心切割平行柱的方法实现对蛋白质的全二维分离。为与后续蛋白质在线酶解、多肽分离及质谱鉴定匹配,系统中采用常规柱以保证蛋白质质谱鉴定对样品量的要求,3种常规分离柱分别选用凝胶过滤色谱柱TSK-GEL G3000SW_(XL)(300 mm×7.8 mm,5μm)、强阴离子交换色谱柱Hypersil SAX(100 mm×4.6 mm,10μm)和强阳离子交换色谱柱Hypersil SCX(100 mm×4.6 mm,10μm)。最终以酵母细胞蛋白质提取液为样品,对构建的二维系统加以评价,在总蛋白质浓度13.5 mg/mL、上样体积100μL的条件下,将第一维分离等时间切割17次,并将切割馏分全部导入第二维继续分离,二维系统在148 min内获得的总峰容量达到884。说明所构建的系统可以用于蛋白质的在线全二维分离。     

Evaluation and application of a mixed‐mode chromatographic stationary phase in two‐dimensional liquid chromatography for the separation of traditional Chinese medicine

In this study, two mixed‐mode chromatography stationary phases (C8SAX and C8SCX) were evaluated and used to establish a two‐dimensional liquid chromatography system for the separation of traditional Chinese medicine. The chromatographic properties of the mixed‐mode columns were systematically evaluated by comparing with other three columns of C8, strong anion exchanger, and strong cation exchanger. The result showed that C8SAX and C8SCX had a mixed‐mode retention mechanism including electrostatic interaction and hydrophobic interaction. Especially, they were suitable for separating acidic and/or basic compounds and their separation selectivities could be easily adjusted by changing pH value. Then, several off‐line 2D‐LC systems based on the C8SAX in the first dimension and C8SAX, C8SCX, or C8 columns in the second dimension were developed to analyze a traditional Chinese medicine—Uncaria rhynchophylla. The two‐dimensional liquid chromatography system of C8SAX (pH 3.0) × C8SAX (pH 6.0) exhibited the most effective peak distribution. Finally, fractions of U. rhynchophylla prepared from the first dimension were successfully separated on the C8SAX column with a gradient pH. Thus, the mixed‐mode stationary phase could provide a platform to separate the traditional Chinese medicine in practical applications.     

全二维微柱液相色谱-质谱鉴定人胃癌组织中的差异蛋白质

构建了以阳离子交换色谱-反相色谱(SCX-RPLC)为分离模式的新型全二维微柱液相色谱-质谱分离平台.采用了醋酸铵缓冲液梯度洗脱,实现了第一维肽段的分步洗脱,洗脱的肽段经富集除盐后通过接口进入反相色谱微柱,通过线性梯度实现第二维进一步分离,最后进入质谱进行检测.采用此平台分析了人胃癌组织与正常组织提取蛋白质信息,其中正常胃组织鉴定蛋白质数为537个,而癌症组织鉴定蛋白质数目为506个.对胃癌和正常组织两种提取蛋白质酶解产物的蛋白质检索结果进行比较分析,将鉴定的蛋白质按照物理性质进行分布,找出正常组织与癌症组织间蛋白质差异,筛选出一种可能发生变异的癌症特有蛋白.     

Miniaturized on-line proteolysis-capillary liquid chromatography-mass spectrometry for peptide mapping of lactate dehydrogenase

In this study, methodology was developed for on-line and miniaturized enzymatic digestion with liquid chromatographic (LC) separation and mass spectrometric (MS) detection. A packed capillary LC-MS system was combined with on-line trypsin cleavage of a model protein, lactate dehydrogenase, to provide an efficient system for peptide mapping. The protein was injected onto an enzymatic capillary reactor and the resulting peptides were efficiently trapped on a capillary trapping column. Different trapping columns were evaluated to achieve a high binding capacity for the peptides generated in the enzyme reactor. The peptides were further eluted from the pre-column and separated on an analytical capillary column by a buffer more suitable for the following an electrospray ionisation (ESI) MS process. An important aspect of the on-line approach was the desalting of peptides performed in the trapping column to avoid detrimental signal suppression in the ESI process. The developed on-line system was finally compared to a classical digestion in solution, with reference to peptide sequence coverage and sensitivity. It was shown that the on-line system gave more than 100% higher peptide sequence coverage than traditional digestion methods.