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Development and Characterization of a Novel Plug and Play Liquid Chromatography-Mass Spectrometry (LC-MS) Source That Automates Connections between the Capillary Trap, Column, and Emitter*

Open AccessPublished:February 19, 2013DOI:https://doi.org/10.1074/mcp.O112.024893
      We report the development and characterization of a novel, vendor-neutral ultra-high pressure-compatible (∼10,000 p.s.i.) LC-MS source. This device is the first to make automated connections with user-packed capillary traps, columns, and capillary emitters. The source uses plastic rectangular inserts (referred to here as cartridges) where individual components (i.e. trap, column, or emitter) can be exchanged independent of one another in a plug and play manner. Automated robotic connections are made between the three cartridges using linear translation powered by stepper motors to axially compress each cartridge by applying a well controlled constant compression force to each commercial LC fitting. The user has the versatility to tailor the separation (e.g. the length of the column, type of stationary phase, and mode of separation) to the experimental design of interest in a cost-effective manner. The source is described in detail, and several experiments are performed to evaluate the robustness of both the system and the exchange of the individual trap and emitter cartridges. The standard deviation in the retention time of four targeted peptides from a standard digest interlaced with a soluble Caenorhabditis elegans lysate ranged between 3.1 and 5.3 s over 3 days of analyses. Exchange of the emitter cartridge was found to have an insignificant effect on the abundance of various peptides. In addition, the trap cartridge can be replaced with minimal effects on retention time (<20 s).
      The tremendous progress in the field of proteomics over the last decade can largely be attributed to significant advancements in mass spectrometry instrumentation (
      • Syka J.E.
      • Marto J.A.
      • Bai D.L.
      • Horning S.
      • Senko M.W.
      • Schwartz J.C.
      • Ueberheide B.
      • Garcia B.
      • Busby S.
      • Muratore T.
      • Shabanowitz J.
      • Hunt D.F.
      Novel linear quadrupole ion trap/FT mass spectrometer: Performance characterization and use in the comparative analysis of histone H3 post-translational modifications.
      ,
      • Andrews G.L.
      • Simons B.L.
      • Young J.B.
      • Hawkridge A.M.
      • Muddiman D.C.
      Performance characteristics of a new hybrid quadrupole time-of-flight tandem mass spectrometer (TripleTOF 5600).
      ,
      • Hu Q.
      • Noll R.J.
      • Li H.
      • Makarov A.
      • Hardman M.
      • Graham Cooks R.
      The Orbitrap: a new mass spectrometer.
      ,
      • Olsen J.V.
      • Schwartz J.C.
      • Griep-Raming J.
      • Nielsen M.L.
      • Damoc E.
      • Denisov E.
      • Lange O.
      • Remes P.
      • Taylor D.
      • Splendore M.
      • Wouters E.R.
      • Senko M.
      • Makarov A.
      • Mann M.
      • Horning S.
      A dual pressure linear ion trap orbitrap instrument with very high sequencing speed.
      ,
      • Second T.P.
      • Blethrow J.D.
      • Schwartz J.C.
      • Merrihew G.E.
      • MacCoss M.J.
      • Swaney D.L.
      • Russell J.D.
      • Coon J.J.
      • Zabrouskov V.
      Dual-pressure linear ion trap mass spectrometer improving the analysis of complex protein mixtures.
      ). Mass spectrometers have improved in every imaginable analytical figure of merit, including the following: duty cycle, resolving power, mass measurement accuracy, sensitivity, and compatibility to various dissociation techniques (
      • Bereman M.S.
      • Canterbury J.D.
      • Egertson J.D.
      • Horner J.
      • Remes P.M.
      • Schwartz J.
      • Zabrouskov V.
      • MacCoss M.J.
      Evaluation of front-end higher energy collision-induced dissociation on a benchtop dual-pressure linear ion trap mass spectrometer for shotgun proteomics.
      ,
      • Williams Jr., D.K.
      • McAlister G.C.
      • Good D.M.
      • Coon J.J.
      • Muddiman D.C.
      Dual electrospray ion source for electron-transfer dissociation on a hybrid linear ion trap-orbitrap mass spectrometer.
      ,
      • Baba T.
      • Hashimoto Y.
      • Hasegawa H.
      • Hirabayashi A.
      • Waki I.
      Electron capture dissociation in a radio frequency ion trap.
      ). An area of advancement that is not always acknowledged is the usability of current commercial instruments. No longer is one required to be an expert in instrumentation, an electrical engineer, or an analytical chemist to operate, maintain, and acquire scientifically sound data from a mass spectrometer. This fact combined with the power of mass spectrometry to achieve absolute molecular specificity has expanded the breadth of users to experts outside the field of traditional chemistry (e.g. biology). However, in a typical proteomics experiment mass spectrometric analysis is preceded by liquid chromatography (i.e. LC-MS), which is significantly less robust.
      Chromatography is a major limitation in proteomics (e.g. throughput and robustness) but an absolute necessity. Separation of analytes as a function of time accomplishes two main feats: 1) it reduces charge competition inside an ESI droplet that mitigates ion suppression; 2) it allows the mass spectrometer time to interrogate more ions. Both of these are critical to maximizing proteome coverage and identifying potential physiologically relevant species that are present at low abundance in biological samples. However, with these advantages, the robustness of chromatography suffers due to its extreme sensitivity to sample preparation, detergents, particulate (i.e. clogging), fitting fatigue, and dead volume. Several of these problems are compounded by the necessity in proteomics to operate in the nanoliter flow-rate regime. The sensitivity of electrospray ionization is inversely proportional to flow rate (
      • Wilm M.
      • Mann M.
      Analytical properties of the nanoelectrospray ion source.
      ,
      • Luo Q.
      • Tang K.
      • Yang F.
      • Elias A.
      • Shen Y.
      • Moore R.J.
      • Zhao R.
      • Hixson K.K.
      • Rossie S.S.
      • Smith R.D.
      More sensitive and quantitative proteomic measurements using very low flow rate porous silica monolithic LC columns with electrospray ionization-mass spectrometry.
      ,
      • Schmidt A.
      • Karas M.
      • Dülcks T.
      Effect of different solution flow rates on analyte ion signals in nano-ESI MS, or: when does ESI turn into nano-ESI?.
      ), and low flow rates concentrate the analyte inside the ESI droplet as species elute in a smaller peak volume. Combined, these effects of low flow rates significantly increase the sensitivity of proteomic experiments via LC-MS. Nano-flow rates require the miniaturization of all components of an LC instrument. Although this is advantageous in that it requires lower amounts of material to achieve the desired signal, it increases the susceptibility to various problems such as clogging. Other factors become increasingly important when operating at nano-flow rates, including the ability to make low dead volume connections.
      As the field of proteomics continues to attract new researchers and as the seasoned proteomic laboratories move into clinical validation of putative biomarkers where thousands of analyses will be necessary, the robustness of chromatography needs improvement. Some of these factors are experimental/user-specific (e.g. good sample preparation) and are unlikely to be obviated by improved hardware design. However, there is a strong need for systems to be developed that automate connections between components, removing the variation inherently associated with manual connections. In addition, there is a need for systems where individual components can be rapidly and reproducibly exchanged independently of one another. Agilent Technologies has introduced a partial solution to this problem, referred to as HPLC-Chip Cube, in which the trapping, separation, and ESI is performed on a microfluidic chip (
      • Yin H.
      • Killeen K.
      • Brennen R.
      • Sobek D.
      • Werlich M.
      • van de Goor T.
      Microfluidic chip for peptide analysis with an integrated HPLC column, sample enrichment column, and nanoelectrospray tip.
      ,
      • Fortier M.-H.
      • Bonneil E.
      • Goodley P.
      • Thibault P.
      Integrated microfluidic device for mass spectrometry-based proteomics and its application to biomarker discovery programs.
      ). The chip contains all of the components and thus has eliminated the need to make conventional finger tight fittings. In addition, because these chips can be fabricated to extremely low tolerances, the inter-chip reproducibility is exceptional. However, the device is limited in that if a single component fails (e.g. ESI tip), it often requires replacement of the whole chip, which is expensive. Eksigent Technologies developed the modular NanoFlex where trapping and separation of mixtures are performed on separate microfluidic chips, both which are separated from the ESI emitter. However, it still suffers from the high cost in purchasing replacement or additional chips. Possibly, the most noteworthy drawback of both devices is the limited versatility. Consumers are limited to a finite selection of vendor stationary phases and short set column lengths (<15 cm). These factors significantly limit both the type and performance (i.e. peak capacity) of any individual proteomic experiment. Furthermore, neither device is currently ultra-high pressure compatible (>4000 p.s.i.). Recently, Thermo Fisher Scientific introduced an integrated column and emitter device that requires the user to make a single manual connection (
      • Kiyonami R.
      • Ravnsborg C.
      • Madsen O.
      • Zabrouskov V.
      Easy-to-Use, Plug-and-Spray Ion Source for Robust and Reproducible Ultra High Pressure Nanoflow LC/MS.
      ). Although this device is ultra-HPLC-compatible and undoubtedly simplifies the setup, it neither allows for the exchange of individual components nor does it accept user packed capillary columns.
      Herein, we describe the development and characterization of a vendor-neutral device that fills an empty “niche” in LC-MS instrumentation. The hybrid LC-MS device incorporates the advantages of the traditional setup (i.e. versatility and low cost) combined with the ability to automate connections between the individual components. The device accepts user or commercially packed capillary columns and is completely plug and play. It allows the user the ability to exchange individual components independently of one another. The device is described in detail, and several experiments are described to systematically evaluate the reproducibility of the system and the exchange of the individual components.

      CONCLUSIONS

      Motivated by the difficulties in nano-flow chromatography (e.g. making reproducible low dead volume connections) and the limitations in current commercial technologies aimed at circumventing these problems (e.g. expense and versatility), we developed a vendor-neutral device that automates connections among various components and accepts user packed capillary traps, columns, and emitters in a plug and play manner. These studies have been directed at investigating the reproducibility in retention time and ion abundance of exchanging the various components within the device. The ESI emitter, a component prone to failure throughout a series of experiments, can be exchanged reproducibly in a high throughput manner without disturbing the trap and column cartridges and without significant effects on the ESI process. The trap cartridge, while showing statistically significant variation, could still be exchanged with limited effect on RT (0.26 ± 0.13 min). Because of its versatility and plug and play attributes, we feel this device will find significant use across clinical laboratories, core laboratory facilities, and even seasoned proteomic laboratories interested in high throughput. Although some practice is needed to become familiar with the device, the ease at which low dead volume connections are made is significant, and we are certain that the device decreases the technical expertise needed to perform robust nano-LC-MS measurements. Future work is ongoing to develop hardware and software to completely automate the emitter exchange system centered on real time feedback from the instrument based on heuristics calculated from peptide abundance and spray stability. The source described in this study is now marketed as the CorConneX source and is commercially available from CorSolutions (Ithaca, NY).

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