High-efficiency nanoscale liquid chromatography coupled on-line with mass spectrometry using nanoelectrospray ionization for proteomics

We describe high-efficiency (peak capacities of approximately 10(3)) nanoscale (using column inner diameters down to 15 microm) liquid chromatography (nanoLC)/low flow rate electrospray (nanoESI) mass spectrometry (MS) for the sensitive analysis of complex global cellular protein enzymatic digests (i.e., proteomics). Using a liquid slurry packing method with carefully selected packing solvents, 87-cm-length capillaries having inner diameters of 14.9-74.5 microm were successfully packed with 3-microm C18-bonded porous (300-A pores) silica particles at a pressure of 18,000 psi. With a mobile-phase delivery pressure of 10,000 psi, these packed capillaries provided mobile-phase flow rates as low as approximately 20 nL/min at LC linear velocities of approximately 0.2 cm/s, which is near optimal for separation efficiency. To maintain chromatographic efficiency, unions with internal channel diameters as small as 10 microm were specially produced for connecting packed capillaries to replaceable nanoESI emitters having orifice diameters of 2-10 microm (depending on the packed capillary dimensions). Coupled on-line with a hybrid-quadrupole time-of-flight MS through the nanoESI interface, the nanoLC separations provided peak capacities of approximately 10(3) for proteome proteolytic polypeptide mixtures when a positive feedback switching valve was used for quantitatively introducing samples. Over a relatively large range of sample loadings (e.g., 5-100 ng, and 50-500 ng of cellular proteolytic peptides for 14.9- and 29.7-microm-i.d. packed capillaries, respectively), the nanoLC/nanoESI MS response for low-abundance components of the complex mixtures was found to increase linearly with sample loading. The nanoLC/nanoESI-MS sensitivity also increased linearly with decreasing flow rate (or approximately inversely proportional to the square of the capillary inner diameter) in the flow range of 20-400 nL/min. Thus, except at the lower loadings, decreasing the separation capillary inner diameter has an effect equivalent to increasing sample loading, which is important for sample-limited proteomic applications. No significant effects on recovery of eluting polypeptides were observed using porous C18 particles with surface pores of 300-A versus nonporous particles. Tandem MS analyses were also demonstrated using the high-efficiency nanoLC separations. Chromatographic elution time, MS response intensity, and mass measurement accuracy was examined between runs with a single column (with a single nanoESI emitter), between different columns (same and different inner diameters with different nanoESI emitters), and for different samples (various concentrations of cellular proteolytic peptides) and demonstrated robust and reproducible sensitive analyses for complex proteomic samples.     

Optimization and evaluation of a sheathless capillary electrophoresis-electrospray ionization mass spectrometry platform for peptide analysis: comparison to liquid chromatography-electrospray ionization mass spectrometry

In this study we have evaluated the suitability of a sheathless capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS) interface with a porous tip as the nanospray emitter for use in peptide analysis. A positively charged capillary coating and 0.1% formic acid as background electrolyte were used for separation upstream from mass spectrometry characterization. The influence of the distance between emitter tip and MS inlet, ESI voltage applied, and of the electroosmotic flow (EOF) on electrospray performance and efficiency of the system was investigated in detail. Under optimized conditions, less than 30 amol of a model peptide (angiotensin I) was required for a detection in the base peak electropherogram and positive identification via tandem MS. Three different cationic capillary coatings were investigated for stability, resolution, and EOF and were found to enable reproducible separations by CE-ESI-MS. After optimizing MS settings, the effectiveness of the CE-ESI-MS method developed was compared with a state-of-the-art nano-liquid chromatography (LC)-ESI-MS method by analyzing Arg-C-digested rat testis linker histones with both systems. With comparable amounts of sample applied, the number of identified peptides increased by more than 60% when using CE-ESI-MS. We found that low molecular mass peptides (below 1400 Da) were preferentially identified by CE-ESI-MS, since this group of peptides poorly interacted with the reversed-phase material in the nano-LC system. Finally, total analysis time in LC-ESI-MS for three runs including equilibration was nearly 4 times longer than that of CE-ESI-MS: 246 versus 66 min.     

Array of chemically etched fused-silica emitters for improving the sensitivity and quantitation of electrospray ionization mass spectrometry

An array of emitters has been developed for increasing the sensitivity of electrospray ionization mass spectrometry (ESI-MS). The linear array consists of 19 chemically etched fused-silica capillaries arranged with 500 microm (center-to-center) spacing. The multiemitter device has a low dead volume to facilitate coupling to capillary liquid chromatography (LC) separations. The high aspect ratio of the emitters enables operation at flow rates as low as 20 nL/min/emitter, effectively extending the benefits of nanoelectrospray to higher flow rate analyses. To accommodate the larger ion current produced by the emitter array, a multicapillary inlet to the mass spectrometer was also constructed. The inlet, which matched the dimensions of the emitter array, preserved ion transmission efficiency. Standard reserpine solutions of varying concentration were electrosprayed at 1 microL/min using the multiemitter/multi-inlet combination, and the results were compared to those from a standard, single-emitter configuration. A 9-fold sensitivity enhancement was observed for the multiemitter relative to the single emitter. A bovine serum albumin tryptic digest was also analyzed, and a sensitivity increase ranging from 2.4- to 12.3-fold for the detected tryptic peptides resulted; the varying response was attributed to reduced ion suppression under the nanoESI conditions afforded by the emitter array. An equimolar mixture of leucine enkephalin and maltopentaose was studied to verify that ion suppression is indeed reduced for the multiplexed ESI (multi-ESI) array relative to a single emitter over a range of flow rates.     

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.     

High-efficiency on-line solid-phase extraction coupling to 15-150-microm-i.d. column liquid chromatography for proteomic analysis

The ability to manipulate and effectively utilize small proteomic samples is important for analyses using liquid chromatography (LC) in combination with mass spectrometry (MS) and becomes more challenging for very low flow rates due to extra column volume effects on separation quality. Here we report on the use of commercial switching valves (150-microm channels) for implementing the on-line coupling of capillary LC columns operated at 10,000 psi with relatively large solid-phase extraction (SPE) columns. With the use of optimized column connections, switching modes, and SPE column dimensions, high-efficiency on-line SPE-capillary and nanoscale LC separations were obtained demonstrating peak capacities of approximately 1000 for capillaries having inner diameters between 15 and 150 microm. The on-line coupled SPE columns increased the sample processing capacity by approximately 400-fold for sample solution volume and approximately 10-fold for sample mass. The proteomic applications of this on-line SPE-capillary LC system were evaluated for analysis of both soluble and membrane protein tryptic digests. Using an ion trap tandem MS it was typically feasible to identify 1100-1500 unique peptides in a 5-h analysis. Peptides extracted from the SPE column and then eluted from the LC column covered a hydrophilicity/hydrophobicity range that included an estimated approximately 98% of all tryptic peptides. The SPE-capillary LC implementation also facilitates automation and enables use of both disposable SPE columns and electrospray emitters, providing a robust basis for automated proteomic analyses.     

Impulse-driven heated-droplet deposition interface for capillary and microbore LC-MALDI MS and MS/MS

An automated off-line liquid chromatography-matrix-assisted laser desorption ionization (LC-MALDI) interface capable of coupling both capillary and microbore LC separations with MALDI mass spectrometry (MS) and tandem mass spectrometry (MS/MS) has been developed. The interface is a combination of two concepts: analyte concentration from heated hanging droplets and impulse-driven droplet deposition of LC fractions onto a MALDI sample plate. At room temperature the interface allows the coupling of capillary LC separations (i.e., flow rate of <5 microL/min) with MALDI MS. With heating, it can be used to combine microbore LC operated at a relatively high flow rate of up to 50 microL/min with MALDI MS. The collected fractions can be analyzed by MALDI MS and MS/MS instruments, such as time-of-flight (TOF) and quadrupole-TOF MS. Performance of the interface was examined using several peptide and protein standards. It was shown that, using MALDI-TOF MS, [GLU1]-fibrinopeptide B could be detected with a total injection amount of 5 fmol to microbore LC. Chromatographic performance was also monitored. A peak width of 12 s at half-height for [GLU1]-fibrinopeptide B showed no evidence of band broadening due to the interface. The ability of the interface to mitigate ion suppression was studied using a mixture of 100 fmol of [GLU1]-fibrinopeptide B and 10 pmol of cytochrome c tryptic digest. Although fully suppressed under direct MALDI conditions, LC-MALDI analysis was able to detect the 100 fmol peptide with 10 s fraction collection. Finally, the ability to inject relatively large sample amounts to improve detectability of low-abundance peptides was illustrated in the analysis of phosphopeptides from alpha-casein tryptic digests. A digest loaded on column to 2.4 microg and analyzed by LC-MALDI MS/MS resulted in 82% sequence coverage and detection of all nine phosphoserine residues. It is concluded that, being able to handle both high- and low-flow LC separations, the impulse-driven heated-droplet interface provides the flexibility to carry out MALDI analysis of peptides and proteins depending on the information sought after, analysis speed, and sample size.     

Comparison of packed-column supercritical fluid chromatography--tandem mass spectrometry with liquid chromatography--tandem mass spectrometry for bioanalytical determination of (R)- and (S)-ketoprofen in human plasma following automated 96-well solid-phase extraction

The popularity of packed-column supercritical fluid, subcritical fluid, and enhanced fluidity liquid chromatographies (pcSFC) for enantiomeric separations has increased steadily over the past few years. The addition of a significant amount (typically 20-95%) of a viscosity lowering agent, such as carbon dioxide, to the mobile phase provides a number of advantages for chiral separations. For example, higher mobile-phase flow rates can often be attained without a concomitant loss in chromatographic efficiency since diffusion coefficients, and optimum velocities, are typically higher in pcSFC. Ultratrace enantioselective quantitation of drugs in biomatrixes is an ideal application for these chromatographic attributes. To demonstrate the utility of this approach, a pcSFC tandem mass spectrometry (pcSFC-MS/MS) method was compared to a LC-MS/MS method for quantitation of the (R)- and (S)-enantiomers of ketoprofen (kt), a potent nonsteroidal, anti-inflammatory drug, in human plasma. After preparation using automated solid-phase extraction in the 96-well format, kt enantiomers were separated on a Chirex 3005 analytical column using isocratic conditions. Validation data and study sample data from patients dosed with either orally or topically administered ketoprofen were generated using both pcSFC and LC as the chromatographic methods to compare and contrast these analytical approaches. Generally, most analytical attributes, including specificity, linearity, sensitivity, accuracy, precision, and ruggedness, for both of these methods were comparable with the exception that the pcSFC separation provided a roughly 3-fold reduction in analysis time. A 2.3-min pcSFC separation and a 6.5-min LC separation provided equivalent, near-baseline-resolved peaks, demonstrating a significant time savings for analysis of large batch pharmacokinetic samples using pcSFC.     

Fully automated four-column capillary LC-MS system for maximizing throughput in proteomic analyses

We describe a four-column, high-pressure capillary liquid chromatography (LC) system for robust, high-throughput liquid chromatography-mass spectrometry (LC-MS(/MS)) analyses. This system performs multiple LC separations in parallel, but staggers each of them such that the data-rich region of each separation is sampled sequentially. By allowing nearly continuous data acquisition, this design maximizes the use of the mass spectrometer. Each analytical column is connected to a corresponding ESI emitter in order to avoid the use of postcolumn switching and associated dead volume issues. Encoding translation stages are employed to sequentially position the emitters at the MS inlet. The high reproducibility of this system is demonstrated using consecutive analyses of global tryptic digest of the microbe Shewanella oneidensis.     

Inlet ionization: a new highly sensitive approach for liquid chromatography/mass spectrometry of small and large molecules

Inlet ionization is a new approach for ionizing both small and large molecules in solids or liquid solvents with high sensitivity. The utility of solvent based inlet ionization mass spectrometry (MS) as a method for analysis of volatile and nonvolatile compounds eluting from a liquid chromatography (LC) column is demonstrated. This new LC/MS approach uses reverse phase solvent systems common to electrospray ionization MS. The first LC/MS analyses using this novel approach produced sharp chromatographic peaks and good quality full mass range mass spectra for over 25 peptides from injection of only 1 pmol of a tryptic digest of bovine serum albumin using an eluent flow rate of 55 μL min(-1). Similarly, full acquisition LC/MS/MS of the MH(+) ion of the drug clozapine, using the same solvent flow rate, produced a signal-to-noise ratio of 54 for the major fragment ion with injection of only 1 μL of a 2 ppb solution. LC/MS results were acquired on two different manufacturer's mass spectrometers using a Waters Corporation NanoAcquity liquid chromatograph.     

Fast, high peak capacity separations in gas chromatography-time-of-flight mass spectrometry

Peak capacity production (i.e., peak capacity per separation run time) is substantially improved for gas chromatography-time-of-flight mass spectrometry (GC-TOFMS) and applied to the fast separation of complex samples. The increase in peak capacity production is achieved by selecting appropriate experimental conditions based on theoretical modeling of on-column band broadening, and by reducing the injection pulse width. Modeling to estimate the on-column band broadening from experimental parameters provided insight for the potential of achieving GC separations in the absence of off-column band broadening, i.e., the additional band broadening not due to the on-column separation process. To optimize GC-TOFMS separations collected with a commercial instrumental platform, off-column band broadening from injection and detection needed to be significantly reduced. Specifically for injection, a commercially available thermal modulator is adapted and applied (referred to herein as thermal injection) to provide a narrow injection pulse, while the TOFMS provided a data collection rate of 500 Hz, initially averaged to 100 Hz for data storage. The use of long, relatively narrow open tubular capillary columns and a 30 °C/min programming rate were explored for GC-TOFMS, specifically a 20 m, 100 μm inner diameter (i.d.) capillary column with a 0.4 μm film thickness to benefit column capacity, operated slightly below the optimal average linear gas velocity (at ~2 mL/min, due to the flow rate constraint of the TOFMS). Standard autoinjection with a 1:100 split resulted in an average peak width of ~1.2 s, hence a peak capacity production of 50 peaks/min. Metabolites in the headspace of urine were sampled by solid-phase microextraction (SPME), followed by thermal injection and a ~7 min GC separation (with a ~6 min separation time window), producing ~660 ms peak widths on average, resulting in a total peak capacity of ~550 peaks (at unit resolution) and a peak capacity production of ~90 peaks/min (~2-fold improvement relative to standard autoinjection with the 1:100 split). This total peak capacity production achieved is equivalent to, or greater than, that currently utilized in metabolomics studies using GC/MS, but with much slower separations, on the order of 40 to 60 min, corresponding to a 5-fold or greater GC/MS analysis throughput rate.     

Nanoflow low pressure high peak capacity single dimension LC-MS/MS platform for high-throughput, in-depth analysis of mammalian proteomes

The use of narrow bore LC capillaries operated at ultralow flow rates coupled with mass spectrometry provides a desirable convergence of figures of merit to support high-performance LC-MS/MS analysis. This configuration provides a viable means to achieve in-depth protein sequence coverage while maintaining a high rate of data production. Here we explore potential performance improvements afforded by use of 25 μm × 100 cm columns fabricated with 5 μm diameter reversed phase particles and integrated electrospray emitter tips. These columns achieve a separation peak capacity of ≈750 in a 600-min gradient, with average chromatographic peak widths of less than 1 min. At room temperature, a pressure drop of only ≈1500 psi is sufficient to maintain an effluent flow rate of ≤10 nL/min. Using mouse embryonic stem cells as a model for complex mammalian proteomes, we reproducibly identify over 4000 proteins across duplicate 600 min LC-MS/MS analyses.     

A deoxynucleotide derivatization methodology for improving LC-ESI-MS detection

We have developed a novel LC-UV-MS derivatization method for the analysis of deoxyguanosine monophosphate adducts that demonstrates enhanced signal intensities relative to underivatized analytes in positive ion mode electrospray ionization MS. Detection of DNA nucleotide adducts is normally conducted in negative ion mode, which requires basic mobile phases that make chromatographic separations difficult and reduce MS sensitivity. Utilizing coupling reagents typically employed in peptide synthesis, several different deoxyguanosine nucleotide phosphoramidates and phosphomonoesters were synthesized in high conversion yield and under mild reaction conditions. The derivatives were characterized by MS/MS and reaction conversion yields determined from the DAD-UV traces. The derivatives were evaluated for ionization efficiencies, fragmentation patterns, and reversed-phase chromatographic properties by LC/ESI-MS/MS. Overall, the hydrophobic derivatives showed increases in ionization efficiency and improved peak shape. Rank ordering of the derivatizing agents was initially established using the dGp-modified derivatives. The best derivatizing agent, hexamethyleneimine, showed a 3-4-fold signal enhancement compared to underivatized dGp and was selected for additional evaluation. A model system using the carcinogen, N-acetoxy-2-acetylaminofluorene (AAAF), was used to synthesize a N-acetyl-(2-aminofluorenyl)-guanosine 5'-monophosphate (dGpAAF) adduct, which was subsequently derivatized with hexamethyleneimine. Detection limits for dGphex and dGpAAFhex, purified by HPLC, were 10- and 3-fold higher (S/N) than their respective underivatized analogues. Practical applicability, with similar improvements in sensitivity, was established by derivatizing adducts isolated from calf thymus DNA exposed to AAAF. Our results demonstrate the utility of simple reactions for the enhanced detection of a mononucleotide in positive ion mode ESI MS and the application of this technique for the detection of dGp-DNA adducts at the low-femtomole level.     

Ultrasensitive proteomics using high-efficiency on-line micro-SPE-nanoLC-nanoESI MS and MS/MS

Ultrasensitive nanoscale proteomics approaches for characterizing proteins from complex proteomic samples of <50 ng of total mass are described. Protein identifications from 0.5 pg of whole proteome extracts were enabled by ultrahigh sensitivity (<75 zmol for individual proteins) achieved using high-efficiency (peak capacities of approximately 10(3)) 15-microm-i.d. capillary liquid chromatography separations (i.e., using nanoLC, approximately 20 nL/min mobile-phase flow rate at the optimal linear velocity of approximately 0.2 cm/s) coupled on-line with a micro-solid-phase sample extraction and a nanoscale electrospray ionization interface to a 11.4-T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (MS). Proteome measurement coverage improved as sample size was increased from as little as 0.5 pg of sample. It was found that a 2.5-ng sample provided 14% coverage of all annotated open reading frames for the microorganism Deinococcus radiodurans, consistent with previous results for a specific culture condition. The estimated detection dynamic range for detected proteins was 10(5)-10(6). An improved accurate mass and LC elution time two-dimensional data analysis methodology, used to both speed and increase the confidence of peptide/protein identifications, enabled identification of 872 proteins/run from a single 3-h nanoLC/FTICR MS analysis. The low-zeptomole-level sensitivity provides a basis for extending proteomics studies to smaller cell populations and potentially to a single mammalian cell. Application with ion trap MS/MS instrumentation allowed protein identification from 50 pg (total mass) of proteomic samples (i.e., approximately 100 times larger than FTICR MS), corresponding to a sensitivity of approximately 7 amol for individual proteins. Compared with single-stage FTICR measurements, ion trap MS/MS provided a much lower proteome measurement coverage and dynamic range for a given analysis time and sample quantity.     

Improving peak capacity in fast online comprehensive two-dimensional liquid chromatography with post-first-dimension flow splitting

The use of flow splitters between the two dimensions in online comprehensive two-dimensional (2D) liquid chromatography (LC × LC) has not received very much attention, in comparison with their use in 2D gas chromatography (GC × GC), where they are quite common. In principle, splitting the flow after the first dimension column and performing online LC × LC on this constant fraction of the first dimension effluent should allow the two dimensions to be optimized almost independently. When there is no flow splitting, any change in the first-dimension flow rate has an immediate impact on the second dimension. With a flow splitter, one could, for example, double the flow rate into the first dimension column and perform a 1:1 flow split without changing the sample loop size or the sampler's collection time. Of course, the sensitivity would be diminished, but this can be partially compensated through the use of a larger injection; this will likely only amount to a small price to pay for this increased resolving power and system flexibility. Among other benefits, we found a 2-fold increase in the corrected 2D peak capacity and the number of observed peaks for a 15-min analysis time, using a post-first-dimension flow splitter. At a fixed analysis time, this improvement results primarily from an increase in the gradient time, resulting from the reduced system re-equilibration time, and, to a smaller extent, it is due to the increased peak capacity achieved by full optimization of the first dimension.     

Anion and cation mixed-bed ion exchange for enhanced multidimensional separations of peptides and phosphopeptides

Shotgun proteomics typically uses multidimensional LC/MS/MS analysis of enzymatically digested proteins, where strong cation-exchange (SCX) and reversed-phase (RP) separations are coupled to increase the separation power and dynamic range of analysis. Here we report an on-line multidimensional LC method using an anion- and cation-exchange mixed bed for the first separation dimension. The mixed-bed ion-exchange resin improved peptide recovery over SCX resins alone and showed better orthogonality to RP separations in two-dimensional separations. The Donnan effect, which was enhanced by the introduction of fixed opposite charges in one column, is proposed as the mechanism responsible for improved peptide recovery by producing higher fluxes of salt cations and lower populations of salt anions proximal to the SCX phase. An increase in orthogonality was achieved by a combination of increased retention for acidic peptides and moderately reduced retention of neutral to basic peptides by the added anion-exchange resin. The combination of these effects led to approximately 100% increase in the number of identified peptides from an analysis of a tryptic digest of a yeast whole cell lysate. The application of the method to phosphopeptide-enriched samples increased by 94% phosphopeptide identifications over SCX alone. The lower pKa of phosphopeptides led to specific enrichment in a single salt step resolving acidic phosphopeptides from other phospho- and non-phosphopeptides. Unlike previous methods that use anion exchange to alter selectivity or enrich phosphopeptides, the proposed format is unique in that it works with typical acidic buffer systems used in electrospray ionization, making it feasible for online multidimensional LC/MS/MS applications.     

MicroSPE-nanoLC-ESI-MS/MS using 10-microm-i.d. silica-based monolithic columns for proteomics

Silica-based monolithic capillary columns (25 cm x 10 microm i.d.) with integrated nanoESI emitters have been developed to provide high-quality and robust microSPE-nanoLC-ESI-MS analyses. The integrated nanoESI emitter adds no dead volume to the LC separation, allowing stable electrospray operation at flow rates of approximately 10 nL/min. In an initial application with a linear ion trap MS, we identified 5510 unique peptides that covered 1443 distinct Shewanella oneidensis proteins from a 300-ng tryptic digest sample in a single 4-h LC-MS/MS analysis. The use of an integrated monolithic ESI emitter provided enhanced resistance to clogging and provided good run-to-run reproducibility.     

Microfluidic platform for liquid chromatography-tandem mass spectrometry analyses of complex peptide mixtures

A microfluidic chip that integrates all the fluidic components of a gradient liquid chromatography (LC) system is described. These chips were batch-fabricated on a silicon wafer using photolithographic processes and with Parylene as the main structural material. The fabricated chip includes three electrolysis-based electrochemical pumps, one for loading the sample and the other two for delivering the solvent gradient; platinum electrodes for delivering current to the pumps and establishing the electrospray potential; a low-volume static mixer; a column packed with silica-based reversed-phase support; integrated frits for bead capture; and an electrospray nozzle. The fabricated structures were able to withstand pressures in excess of 250 psi. The device was used to perform a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of a mixture of peptides from the trypsin digestion of bovine serum albumen (BSA). Gradient elution through the 1.2-cm column was performed at a flow rate of 80 nL/min. Compared to the analysis of the same sample using a commercial nanoflow LC system, the chromatographic resolution was nearly as good, and the total cycle time was significantly reduced because of the minimal volume between the pumps and the column. Results demonstrate the potential of mass-produced, low-cost microfluidic systems capable of performing LC separations for proteomics applications.     

Preparation of 20-microm-i.d. silica-based monolithic columns and their performance for proteomics analyses

We describe the preparation and performance of high-efficiency 70 cm x 20 microm i.d. silica-based monolithic capillary LC columns. The monolithic columns at a mobile-phase pressure of 5000 psi provide flow rates of approximately 40 nL/min at a linear velocity of approximately 0.24 cm/s. The columns provide a separation peak capacity of approximately 420 in conjunction with both on-line coupling with microsolid-phase extraction and nanoelectrospray ionization-mass spectrometry. Performance was evaluated using a Shewanella oneidensis tryptic digest, and approximately 15-amol detection limits for peptides were obtained using a conventional ion trap and MS/MS for peptide identification. The sensitivity and separation efficiency enabled the identification of 2367 different peptides covering 855 distinct S. oneidensis proteins from a 2.5-microg tryptic digest sample in a single 10-h analysis. The number of identified peptides and proteins approximately doubled when the effective separation time was extended from 200 to 600 min. The number of identified peptides increased from 32 to 390 as the injection amount was increased from 0.5 to 100 ng. Both the run-to-run and column-to-column reproducibility for proteomic analyses were also evaluated.     

Microfluidic chip for peptide analysis with an integrated HPLC column, sample enrichment column, and nanoelectrospray tip

Current nano-LC/MS systems require the use of an enrichment column, a separation column, a nanospray tip, and the fittings needed to connect these parts together. In this paper, we present a microfabricated approach to nano-LC, which integrates these components on a single LC chip, eliminating the need for conventional LC connections. The chip was fabricated by laminating polyimide films with laser-ablated channels, ports, and frit structures. The enrichment and separation columns were packed using conventional reversed-phase chromatography particles. A face-seal rotary valve provided a means for switching between sample loading and separation configurations with minimum dead and delay volumes while allowing high-pressure operation. The LC chip and valve assembly were mounted within a custom electrospray source on an ion-trap mass spectrometer. The overall system performance was demonstrated through reversed-phase gradient separations of tryptic protein digests at flow rates between 100 and 400 nL/min. Microfluidic integration of the nano-LC components enabled separations with subfemtomole detection sensitivity, minimal carryover, and robust and stable electrospray throughout the LC solvent gradient.     

Capillary LC coupled with high-mass measurement accuracy mass spectrometry for metabolic profiling

We have developed an efficient and robust high-pressure capillary LC-MS method for the identification of large numbers of metabolites in biological samples using both positive and negative ESI modes. Initial efforts focused on optimizing the separation conditions for metabolite extracts using various LC stationary phases in conjunction with multiple mobile-phase systems, as applied to the separation of 45 metabolite standards. The optimal mobile and stationary phases of those tested were determined experimentally (in terms of peak shapes, theoretical plates, retention of small, polar compounds, etc.), and both linear and exponential gradients were applied in the study of metabolite extracts from the cyanobacterium Cyanothece sp. ATCC 51142. Finally, an automated dual-capillary LC system was constructed and evaluated for the effectiveness and reproducibility of the chromatographic separations using the above samples. When coupled with a commercial LTQ-orbitrap MS, approximately 900 features were reproducibly detected from Cyanothece sp. ATCC 51142 metabolite extracts. In addition, 12 compounds were tentatively identified, based on accurate mass, isotopic distribution, and MS/MS information.