High-speed, high-resolution monolithic capillary LC-MALDI MS using an off-line continuous deposition interface for proteomic analysis

High-speed, high-resolution LC separations, using a poly(styrene-divinylbenzene) monolithic column, have been coupled to MALDI MS and MS/MS through an off-line continuous deposition interface. The LC eluent was mixed with alpha-cyano-4-hydroxycinnamic acid matrix solution and deposited on a MALDI plate that had been precoated with nitrocellulose. Deposition at subatmospheric pressure (80 Torr) formed a 250-microm-wide serpentine trace with uniform width and microcrystalline morphology. The deposited trace was then analyzed in the MS mode using a MALDI-TOF/TOF MS instrument. Continuous deposition allowed interrogation of the separation with a high data sampling rate in the chromatographic dimensions, thus preserving the high resolution of narrow peaks (3-5-s peak width at half-height) of the fast monolithic LC. No extracolumn band broadening due to the deposition process was observed. Over 2000 components were resolved in a 10-min linear gradient separation of the model sample, and 386 unique peptides were identified in the subsequent MS/MS analysis. The continuous deposition interface allows the coupling of high-resolution separations to MALDI MS without degradation in separation efficiency, thus enabling high-throughput proteome analysis.     

Automated iterative MS/MS acquisition: a tool for improving efficiency of protein identification using a LC-MALDI MS workflow

We have developed an information-dependent, iterative MS/MS acquisition (IMMA) tool for improving MS/MS efficiency, increasing proteome coverage, and shortening analysis time for high-throughput proteomics applications based on the LC-MALDI MS/MS platform. The underlying principle of IMMA is to limit MS/MS analyses to a subset of molecular ions that are likely to identify a maximum number of proteins. IMMA reduces redundancy of MS/MS analyses by excluding from the precursor ion peak lists proteotypic peptides derived from the already identified proteins and uses a retention time prediction algorithm to limit the degree of false exclusions. It also increases the utilization rate of MS/MS spectra by removing "low value" unidentifiable targets like nonpeptides and peptides carrying large loads of modifications, which are flagged by their "nonpeptide" excess-to-nominal mass ratios. For some samples, IMMA increases the number of identified proteins by ～20-40% when compared to the data dependent methods. IMMA terminates an MS/MS run at the operator-defined point when "costs" (e.g., time of analysis) start to overrun "benefits" (e.g., number of identified proteins), without prior knowledge of sample contents and complexity. To facilitate analysis of closely related samples, IMMA's inclusion list functionality is currently under development.     

Enhanced characterization of complex proteomic samples using LC-MALDI MS/MS: exclusion of redundant peptides from MS/MS analysis in replicate runs

Due to the complexity of proteome samples, only a portion of peptides and thus proteins can be identified in a single LC-MS/MS analysis in current shotgun proteomics methodologies. It has been shown that replicate runs can be used to improve the comprehensiveness of the proteome analysis; however, high-intensity peptides tend to be analyzed repeatedly in different runs, thus reducing the chance of identifying low-intensity peptides. In contrast to commonly used online ESI-MS, offline MALDI decouples the separation from MS acquisition, thus allowing in-depth selection for specific precursor ions. Accordingly, we extended a strategy for offline LC-MALDI MS/MS analysis using a precursor ion exclusion list consisting of all identified peptides in preceding runs. The exclusion list eliminated redundant MS/MS acquisitions in subsequent runs, thus reducing MALDI sample depletion and allowing identification of a larger number of peptide identifications in the cumulative dataset. In the analysis of the digest of an Escherichia coli lysate, the exclusion list strategy resulted in a 25% increase in the number of unique peptide identifications in the second run, in contrast to simply pooling MS/MS data from two replicate runs. To reduce the increased LC analysis time for repeat runs, a four-column multiplexed LC system was developed to carry out separation simultaneously. The multiplexed LC-MALDI MS provides a high-throughput platform to utilize the exclusion list strategy in proteome analysis.     

A low-flow ce/electrospray ionization MS interface for capillary zone electrophoresis,large-volume sample stacking,and micellar electrokinetic chromatography

A simple and versatile low-flow interface has been developed for interfacing capillary electrophoresis (CE) with electrospray ionization (ESI) mass spectrometry. This low-flow interface showed better sensitivity than a conventional sheath liquid interface, primarily attributed to a low dilution factor and a reduction in the sprayer orifice size. The interface was also found to be more tolerant to the presence of nonvolatile salts. Because of tolerance to the surfactant SDS, this interface can be used to couple micellar electrokinetic chromatography (MEKC) with ESI-MS. The performance of the interface in an MEKC-MS application, as demonstrated in the analysis of triazines, was significantly better than that obtained with a conventional sheath liquid interface. Moreover, this interface can be easily used for large-volume sample-stacking (LVSS) applications. Using a series of phenols as a test case, an approximate 500-fold enrichment was achieved by LVSS in conjunction with the low-flow CE/MS interface described.     

Micro-SPE method for sample introduction in capillary HPLC/MS

A new solid-phase extraction on-line device for micro-HPLC is presented. This device optimizes the injection of very dilute samples into a packed capillary column. It consists of two capillary, reversed-phase, HPLC columns of different length that can be linked together as a single chromatographic column. The first segment, only 2 cm long is connected to the HPLC injector. When disconnected from the longer column, several milliliters of an aqueous sample can be passed through at a high flow rate for fast trapping. On the basis of the retention mechanism, all suitable compounds are focused on the short column head in a sharp band. As soon as the chromatographic column is recomposed, the trapped analytes are eluted and separated at the optimal flow rate and gradient conditions. Due to the high preconcentration factor, trace-level analysis can be performed successfully. Different classes of analytes of various polarities and molecular weights can be determined, depending on the stationary phase and on the detector used. Some pesticides belonging to different classes were chosen to evaluate the performance of the device using an electron ionization mass spectrometer as HPLC detector. A fungicide in an irrigation canal water was determined at a concentration level of 4.5 microg x L(-1).     

Genotoxicity screening using biocatalyst/DNA films and capillary LC-MS/MS

Detection of DNA adducts can serve as a basis for genotoxicty screening of new chemicals and drugs. We report here a simple, sensitive procedure for this purpose using films containing DNA and a biocatalyst to mimic the metabolic action of human liver cytochrome P450s. DNA adducts formed from an in-situ-generated toxic metabolite (styrene oxide) were detected at subpicomole levels after neutral thermal hydrolysis of the DNA films and analysis with capillary liquid chromatography with on-line column preconcentration and MS/MS detection. An on-line column switching system allowed for increased sample loading volume and analyte preconcentration. This approach provides an estimate of the relative rate of DNA damage.     

Methodology utilizing MS signal intensity and LC retention time for quantitative analysis and precursor ion selection in proteomic LC-MALDI analyses

This study describes a methodology for performing relative quantitation in large-scale proteomic sample comparisons using an LC-MALDI mass spectrometry analytical platform without the use of isotope tagging reagents. The method utilizes replicate analyses of a sample to create a profile of constituent components that are aligned based on LC elution time and mass. Once components from individual runs have been grouped as common "features", the Student's t test is used to determine which components are systematically different between samples. In this study, five HPLC runs of human plasma were compared to five HPLC runs of human serum. About 3889 components were detected in all 10 runs. Of these, 1831 corresponded to approximately 100 known serum proteins, based on MS/MS analysis of one run each from serum and plasma. As expected, fibrinogen alpha, beta, and gamma chains accounted for many of the most significant differences. Therefore, using MALDI, samples containing thousands of peptides can be compared in a minimal amount of time. Moreover, the results of the comparison can be used to guide further MS/MS mode sample interrogation in a result dependent manner.     

Characterization of a capillary zone electrophoresis/electrospray-mass spectrometry interface

A dependable and stable CZE/ESI-MS interface has been constructed. To avoid instabilities in both, the capillary electrophoretic separation and the electrospray, the second of the three concentric capillaries in the three-layered sprayer has been replaced by an aluminum-coated fused-silica capillary with an inner diameter only slightly greater than the outer diameter of the separation capillary. By this means, the otherwise often observed destruction of the separation capillary ("electrodrilling") can be avoided completely due to the suppression of electrochemical processes leading to gas bubble formation at the tip of the sprayer. With some examples taken from different biochemical areas and by separation of natural compounds, the capability and the reliability of the modified sprayer as the central part of the interface are demonstrated.     

A low-makeup beveled tip capillary electrophoresis /electrospray ionization mass spectrometry interface for micellar electrokinetic chromatography and nonvolatile buffer capillary electrophoresis

A robust interface has been developed for interfacing micellar electrokinetic chromatography (MEKC) and nonvolatile buffer capillary electrophoresis (CE) to electrospray ionization mass spectrometry (ESI-MS). The interface consists of two parallel capillaries for separation (50 microm i.d. x 155 microm o.d.) and makeup (50 microm i.d. x 155 microm o.d.) housed within a larger capillary (530 microm i.d. x 690 microm o.d.). The capillaries terminate in a single tapered tip having a beveled edge. The use of a tapered beveled edge results in a greater tip orifice diameter (75 microm) than in a previous design from our laboratory (25 microm) that used a flat tip. While maintaining a similar optimum flow rate and consequently similar sample dilution, a 75-microm beveled emitter is more rugged than a 25-microm flat tip. Furthermore, the incorporation of a sheath liquid capillary allows the compositions of the final spray solution to be controlled. The application of this novel CE/ESI-MS interface was demonstrated for MEKC using mixtures of triazines (positive ion mode) and phenols (negative ion mode). The ability to perform CE/ESI-MS using a nonvolatile buffer was demonstrated by the analysis of gangliosides with a buffer consisting of 40 mM borate and 20 mM alpha-cyclodextrin.     

Characterization of the microdialysis junction interface for capillary electrophoresis/microelectrospray ionization mass spectrometry

A capillary electrophoresis/electrospray ionization mass spectrometry (CE/ESI-MS) interface, based on an electric circuit across a microdialysis membrane surrounding a short capillary segment closely connected to the separation capillary terminus, is demonstrated to be sensitive, efficient, and rugged. A microspray type ionization emitter produces a stable electrospray at the low flow rates provided by CE and thus avoids both the need for a makeup liquid flow provided by liquid junction or sheath flow interfaces and the subsequent dilution and reduction in sensitivity. Reproducibility studies and comparisons with CE/UV and the CE/sheath flow interface with ESI-MS are presented. Additionally, postrun acidification via the microdialysis junction interface is demonstrated and shown to be capable of denaturing the holomyoglobin protein noncovalent complex while maintaining separation efficiency.     

A metal nebulizer capillary electrophoresis/Fourier transform infrared spectrometric interface

A capillary electrophoretic (CE) system has been successfully interfaced to a Fourier transform infrared spectrometer. The advantage of such an interface is that analytes may be detected and often unequivocally identified without analyte derivatization. The interface consists of a stainless steel tube in which the CE capillary is placed and the two are held in contact with the use of a metal tee. A solvent elimination approach is used with the interface, so that analytes are deposited onto an infrared transparent window, that is, CaF2, and measured with the use of an infrared microscope. A critical component of this design is to provide an electrical connection at the end of the CE column to permit stable separations that allow for efficient transport of the sample onto the window. The interface produces an aerosol that is directed at the surface of the infrared transparent window. The use of a volatile electrolyte, along with the flow of helium, allows for partial evaporation of the electrolyte in flight and complete evaporation of the solvent and electrolyte on the surface of the window to produce a "dry", or neat, analyte deposit.     

Peptide electroextraction for direct coupling of in-gel digests with capillary LC-MS/MS for protein identification and sequencing

An electrophoretic method has been developed for the extraction of peptides following in-gel digests of SDS-PAGE separated proteins. During electroextraction, the peptides are trapped on a strong cation-exchange microcartridge, before analysis by capillary LC--ESI-tandem mass spectrometry. The spectra obtained by tandem mass spectrometry are searched directly against a protein database for identification of the protein from which the peptide originated. By minimizing surface exposure of the peptides during electroextraction, a reduction of the detection limits for protein identification is realized. The performance of the peptide electroextraction was compared directly with the standard extraction method for in-gel protein digests, using a standard dilution series of phosphorylase B and carbonic anhydrase, separated by SDS-PAGE. The lowest gel loading in which phosphorylase B was identified using the standard extraction method was 2.5 ng or 25 fmol, and the lowest gel loading in which phosphorylase B was identified using electroextraction was 1.25 ng or 12.5 fmol. The design of the microextraction cartridge allows for direct interfacing with capillary LC, which is crucial for maintaining low detection limits. Furthermore, this method can be used for high-throughput proteomics since it can be easily multiplexed and requires only voltage control and low pressures (approximately 15 psi) for operation. We believe that peptide electroextraction is a significant advance for identification of proteins separated by one-dimensional or two-dimensional gel electrophoresis, as it can be easily automated and requires less protein than conventional methods.     

Low-attomole electrospray ionization MS and MS/MS analysis of protein tryptic digests using 20-microm-i.d. polystyrene-divinylbenzene monolithic capillary columns

This work explores the use of 20-microm-i.d. polymeric polystyrene-divinylbenzene monolithic nanocapillary columns for the LC-ESI-MS analysis of tryptic digest peptide mixtures. In contrast to the packing of microparticles, capillary columns were prepared, without the need of high pressure, in fused-silica capillaries, by thermally induced in situ copolymerization of styrene and divinylbenzene. The polymerization conditions and mobile-phase composition were optimized for chromatographic performance leading to efficiencies over 100000 plates/m for peptide separations. High mass sensitivity (approximately 10 amol of peptides) in the MS and MS/MS modes using an ion trap MS was found, a factor of up to 20-fold improvement over 75-microm-i.d. nanocolumns. A wide linear dynamic range (approximately 4 orders of magnitude) was achieved, and good run-to-run and column-to-column reproducibility of isocratic and gradient elution separations were found. As samples, both model proteins and tissue extracts were employed. Gradient nano-LC-MS analysis of a proteolytic digest of a tissue extract, equivalent to a sample size of approximately 1000 cells injected, is presented.     

An automated noncontact deposition interface for liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry

A new multichannel deposition system was developed for off-line liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry (LC/MALDI-MS). This system employs a pulsed electric field to transfer the eluents from multiple parallel columns directly onto MALDI targets without the column outlets touching the target surface. The deposition device performs well with a wide variety of solvents that have different viscosities, vapor pressures, polarities, and ionic strengths. Surface-modified targets were used to facilitate concentration and precise positioning of samples, allowing for efficient automation of high-throughput MALDI analysis. The operational properties of this system allow the user to prepare samples using MALDI matrixes whose properties range from hydrophilic to hydrophobic. The latter, exemplified by alpha-cyano-4-hydroxycinnamic acid, were typically processed with a multistep deposition method consisting of precoating of individual spots on the target plate, sample deposition, and sample recrystallization steps. Using this method, 50 amol of angiotensin II was detected reproducibly with high signal-to-noise ratio after LC separation. Experimental results show that there is no significant decrease in chromatographic resolution using this device. To assess the behavior of the apparatus for complex mixtures, 5 microg of a tryptic digest of the cytosolic proteins of yeast was analyzed by LC/MALDI-MS and more than 13,500 unique analytes were detected in a single LC/MS analysis.     

Assembly of live micro-organisms on microstructured PDMS stamps by convective/capillary deposition for AFM bio-experiments

Immobilization of live micro-organisms on solid substrates is an important prerequisite for atomic force microscopy (AFM) bio-experiments. The method employed must immobilize the cells firmly enough to enable them to withstand the lateral friction forces exerted by the tip during scanning but without denaturing the cell interface. In this work, a generic method for the assembly of living cells on specific areas of substrates is proposed. It consists in assembling the living cells within the patterns of microstructured, functionalized poly-dimethylsiloxane (PDMS) stamps using convective/capillary deposition. This versatile approach is validated by applying it to two systems of foremost importance in biotechnology and medicine: Saccharomyces cerevisiae yeasts and Aspergillus fumigatus fungal spores. We show that this method allows multiplexing AFM nanomechanical measurements by force spectroscopy on S. cerevisiae yeasts and high-resolution AFM imaging of germinated Aspergillus conidia in buffer medium. These two examples clearly demonstrate the immense potential of micro-organism assembly on functionalized, microstructured PDMS stamps by convective/capillary deposition for performing rigorous AFM bio-experiments on living cells.     

On-line hyphenation of capillary isoelectric focusing and capillary gel electrophoresis by a dialysis interface

An on-line two-dimensional (2D) capillary electrophoresis (CE) system consisting of capillary isoelectric focusing (CIEF) and capillary gel electrophoresis (CGE) was introduced. To validate this 2D system, a dialysis interface was developed by mounting a hollow fiber on a methacrylate resin plate to hyphenate the two CE modes. The two dimensions of capillary shared a cathode fixated into a reservoir in the methacrylate plate; thus, with three electrodes and only one high-voltage source, a 2D CE framework was successfully established. A practical 2D CIEF-CGE experiment was carried out to deal with a target protein, hemoglobin (Hb). After the Hb variants with different isoelectric points (pIs) were focused in various bands in the first-dimension capillary, they were chemically mobilized one after another and fed to the second-dimension capillary for further separation in polyacrylamide gel. During this procedure, a single CIEF band was separated into several peaks due to different molecular weights. The resulting electrophoregram is quite different from that of either CIEF or CGE; therefore, more information about the studied Hb sample can be obtained.     

An etched porous interface for on-line capillary electrophoresis-based two-dimensional separation system

The construction and evaluation of an on-column etched fused-silica porous junction for on-line coupling of capillary isoelectric focusing (CIEF) with capillary zone electrophoresis (CZE) are described. Where two separation columns were integrated on a single piece of fused-silica capillary through the etched approximately 4 to 5-mm length porous junction along the capillary. The junction is easily prepared by etching a short section of the capillary wall with HF after removing the polyimide coating. The etched section becomes a porous glass membrane that allows only small ions related to the background electrolyte to pass through when high voltage is applied across the separation capillary. The primary advantages of this novel porous junction interface over previous designs (in which the interface is usually formed by fracturing the capillary followed by connecting the two capillaries with a section of microdialysis hollow fiber membrane) are no dead volume, simplicity, and ruggedness, which is particularly well suited for an on-line coupling capillary electrophoresis-based multiple dimensional separation system. The performance of the 2D CIEF-CZE system constructed by such an etched porous junction was evaluated by the analyses of protein mixtures.     

A sheathless nanoflow electrospray interface for on-line capillary electrophoresis mass spectrometry

A novel, rugged capillary electrophoresis-electrospray ionization (CE-ESI) interface where the separation column, an electrical porous junction, and the spray tip are integrated on a single piece of a fused-silica capillary is described. ESI is accomplished by applying an electrical potential through an easily prepared porous junction across a 3-4-mm length of fused silica. A stable electrospray is produced at nanoflow rates generated in the capillary by electrophoretic and electroosmotic forces. The interface is particularly well suited for the detection of low-femtomole levels of proteins and peptides. The ruggedness of this interface was evident by the continuous operation of the same column for over a 2-week period with no detectable deterioration in separation or electrospray performance. The new interface was used for the LC-ESI-MS separation and analysis of peptides and proteins. Injection of 25 fmol of [Glu1]-fibrinopeptide B using the new device produced a CE-ESI-MS electropherogram with a signal-to-noise ratio of over 100 for this peptide.