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A Robust Method for Packing High Resolution C18 RP-nano-HPLC Columns

Published: May 14th, 2021



1Department of Biological Chemistry, David Geffen School of Medicine at UCLA
* These authors contributed equally

Here, we present and evaluate a protocol for making low cost reversed phase nano-flow liquid chromatography columns for peptide characterization using LC-MS/MS proteomic workflows.

The high complexity prevalent in biological samples requires chromatographic separations with high sensitivity and resolution to be effectively analyzed. Here we introduce a robust, reproducible and inexpensive protocol for preparation of a nano-flow reversed phase high performance liquid chromatography (RP-HPLC) columns for on-line separation of analytical peptides before introduction into and detection by a mass-spectrometer in traditional bottom-up proteomics workflows. Depending on the goal of the experiment and the chemical properties of the analytes being separated, optimal column parameters may differ in their internal or outer diameters, length, particle size, pore size, chemistry of stationary phase particles, and the presence or absence of an integrated electrospray emitter at the tip. An in-house column packing system not only enables the rapid fabrication of columns with the desired properties but also dramatically reduces the cost of the process. The optimized protocol for packing a C18 AQ (aqueous) fused silica column discussed here is compatible with a wide range of liquid chromatographic instruments for achieving effective separation of analytes.

HPLC columns have contributed immensely to productivity in the fields of pharmaceutical, medical and environmental research1,2,3,4. Having access to high-quality chromatography columns is a pivotal step in the fractionation of complex analytes. In shotgun proteomics, high analytical sensitivity is routinely accomplished by coupling electrospray ionization (ESI) mass spectrometry (MS) to nanoflow chromatography5,6,7,8

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1. Preparation of the capillary tip

  1. Using a ceramic cleaving stone, cut about 60-70 cm of a polyimide coated fused silica capillary with an internal diameter (ID) of 75 µm and an outer diameter (OD) of 360 µm.
  2. Hold the capillary with your hands at approximately the middle of its length, leaving a 4-5 cm gap between fingers and heat the area in the gap while rotating it over the flame of an alcohol lamp. Polish the burnt area clean using a methanol-soaked low lint tissue until the glass is .......

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To evaluate the performance of the columns, 750 ng of tryptic peptide digests prepared from whole cell lysates of HEK293 cells were fractionated online using a 25 cm long, 75 µm ID fused-silica capillary packed in-house with bulk ReproSil-Pur 120 C18-AQ particles as described in the protocol. Prior to sample loading, the column was washed using 6 µL of a mixture of acetonitrile, isopropanol and H2O in a ratio of 6:2:2 and pre-equilibrated with buffer A (Buffer A: water with 3% DMSO). The tryptic pept.......

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Modern proteomic strategies are reliant upon high quality chromatographic separations to effectively analyze complex biological systems. Hence, high-performing and cost-effective nanoflow LC columns are crucial components of a successful tandem mass-spectrometry regime aimed at characterizing thousands of proteins in a single workflow.

In this study we evaluated the performance and reliability of a range of LC columns for LC-MS/MS made using the protocol described above. The performance of the.......

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This work was supported by the National Institutes of Health grant GM089778 to J.A.W.


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Name Company Catalog Number Comments
99.99% Formamide acid Sigma-Aldrich for making frit
alcohol lamp Any brand For providing heat
Brechbuehler helium pressure cell BioSurplus for packing column
Ceramic column cutter Any brand for cutting silica capillary
Dimethyl sulfoxide (DMSO) ≥ 99% Sigma-Aldrich Stored in a flammable cabinet
Formamide  ≥99.5% Sigma-Aldrich for making frit
Hydrofluoric acid (HF) (50%) Fisher Scientific for opening the emitter after polymerization
KASIL (Potassium Silicate Solution) PQ Corporation for making frit
Orbitrap Fusion Lumos Thermo Fisher Scientific for MS data acquisition
P2000 Laser Puller Sutter for pulling capillary
PTFE 1/16" Ferrule 0.4 mm ID (long) for Tube Fitting Chromre 214104 For bomb setting
Reprosil-Pur 120 C18-AQ, 1.9 um, 1g Dr. Masch GmbH Batch 5910
Soldering Any brand For initiating polimerization
Stainless Steel Pipe Fitting, Hex Coupling, 1/4 in. Female NPT Swagelok SS-4-HCG for bomb setting
TSP075375 fused silica, 75 µm ID x 360 µOD MOLEX/Polymicro 1068150019 For column tubing
Ultimate 3000 UHPLC Dionex HPLC type

  1. Richards, A. L., et al. One-hour proteome analysis in yeast. Nature Protocols. 10 (5), 701-714 (2015).
  2. Shishkova, E., Hebert, A. S., Coon, J. J. Now, More Than Ever, Proteomics Needs Better Chromatography. Cell Systems. 3 (4), 321-324 (2016).
  3. D'Atri, V., Fekete, S., Clarke, A., Veuthey, J. L., Guillarme, D. Recent Advances in Chromatography for Pharmaceutical Analysis. Analytical. Chemistry. 91 (1), 210-239 (2019).
  4. Gama, M. R., Collins, C. H., Bottoli, C. B. G. Nano-Liquid Chromatography in Pharmaceutical and Biomedical Research. Journal of Chromatographic Science. 51 (7), 694-703 (2013).
  5. Wilson, S. R., Vehus, T., Berg, H. S., Lundanes, E. Nano-LC in proteomics: recent advances and approaches. Bioanalysis. 7 (14), 1799-1815 (2015).
  6. Wilson, S. R., Olsen, C., Lundanes, E. Nano liquid chromatography columns. Analyst. 144 (24), 7090-7104 (2019).
  7. Cutillas, P. R. Principles of Nanoflow Liquid Chromatography and Applications to Proteomics. Current Nanoscience. 1 (1), 65-71 (2005).
  8. Dams, M., Dores-Sousa, J. L., Lamers, R. J., Treumann, A., Eeltink, S. High-Resolution Nano-Liquid Chromatography with Tandem Mass Spectrometric Detection for the Bottom-Up Analysis of Complex Proteomic Samples. Chromatographia. 82 (1), 101-110 (2019).
  9. Stehling, O., et al. MMS19 Assembles Iron-Sulfur Proteins Required for DNA Metabolism and Genomic Integrity. Science. 337 (6091), 195-199 (2012).
  10. Mayank, A. K., et al. An Oxygen-Dependent Interaction between FBXL5 and the CIA-Targeting Complex Regulates Iron Homeostasis. Molecular cell. 75 (2), 282 (2019).
  11. Nie, M., Oravcová, M., Jami-Alahmadi, Y., Wohlschlegel, J. W., Lazzerini-Denchi, E., Boddy, M. N. FAM111A induces nuclear dysfunction in disease and viral restriction. European Molecular Biology Organization. 22 (2), 50803 (2021).
  12. Wahab, M. F., Patel, D. C., Wimalasinghe, R. M., Armstrong, D. W. Fundamental and Practical Insights on the Packing of Modern HighEfficiency Analytical and Capillary Columns. Analytical Chemistry. 89 (16), 8177-8191 (2017).
  13. Blue, L. E., Jorgenson, J. W. 1.1 µm Superficially porous particles for liquid chromatography: Part II: Column packing and chromatographic performance. Journal of Chromatography A. 1380, 71-80 (2015).
  14. Shishkova, E., Hebert, A. S., Westphall, M. S., Coon, J. J. Ultra-High Pressure (>30,000 psi) Packing of Capillary Columns Enhancing Depth of Shotgun Proteomic Analyses. Analytical Chemistry. 90 (19), 11503-11508 (2018).
  15. Liu, H., Finch, J. W., Lavallee, M. J., Collamati, R. A., Benevides, C. C., Gebler, J. C. Effects of Column Length, Particle Size, Gradient Length and Flow Rate on Peak Capacity of Nano-Scale Liquid Chromatography for Peptide Separations. Journal of Chromatography A. 1147 (1), 30-36 (2007).
  16. Kovalchuk, S. I., Jensen, O. N., Rogowska-Wrzesinska, A., , FlashPack. Fast and Simple Preparation of Ultrahigh-performance Capillary Columns for LC-MS. Molecular and cellular proteomics. 18 (2), 383-390 (2019).
  17. Capriotti, F., Leonardis, I., Cappiello, A., Famiglini, G., Palma, P. A Fast and Effective Method for Packing Nano-LC Columns with Solid-Core Nano Particles Based on the Synergic Effect of Temperature, Slurry Composition, Sonication and Pressure. Chromatographia. 76, 1079-1086 (2013).
  18. Godinho, J. M., Reising, A. E., Tallarek, U., Jorgenson, J. W. Implementation of High SlurryConcentration and Sonication to Pack High-Efficiency, Meter-Long Capillary Ultrahigh Pressure Liquid Chromatography Columns. Journal of Chromatography A. 1462, 165-169 (2016).
  19. Pesek, J. J., Matyska, M. T. Our favorite materials: Silica hydride stationary phases. Journal of Separation Science. 32 (23), 3999-4011 (2009).
  20. Borges, E. M., Volmer, D. A. Silica, Hybrid Silica, hydride Silica and Non-Silica Stationary Phases for Liquid Chromatography. Part II: Chemical and Thermal Stability. Journal of Chromatographic Science. 53 (7), 580-597 (2015).
  21. Vyňuchalová, K., Jandera, P. Comparison of a C30 Bonded Silica Column and Columns with Shorter Bonded Ligands in Reversed-Phase LC. Chromatographia. 78 (13-14), 861-871 (2015).
  22. Novotny, M. V. Development of capillary liquid chromatography: A personal perspective. Journal of Chromatography A. 1523, 3-16 (2017).

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