A Robust Method for Packing High Resolution C18 RP-nano-HPLC Columns
Summary
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.
Abstract
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.
Introduction
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. The efficient separation of thousands of peptides is paramount in this application as it allows the mass spectrometer to identify and quantify analytes with high sensitivity and resolution.
The field of column packing for mass-spectrometric applications has witnessed tremendous growth in recent years with advances in the understanding of fundamental column packing principles related to stationary phase morphology, solvent-particle interactions and hardware design, making possible the detailed characterization of a wide range of biomolecules in complex biological settings9,10,11,12,13,14. Efforts highlighting practical considerations in packing analytical columns for LC-MS purposes have paved the way for proteomic laboratories to develop in-house packing systems to meet their specific interests with the promise of maximum performance15,16,17,18.
Nanospray columns with internal diameters in the range of 50-150 μm and tapered ends are well-suited for the purpose of electrospray ionization. In the field of shotgun proteomics, separations are typically carried out using a solvent gradient flowing through a packed non-polar stationary phase, most commonly hydrophobic carbon chain bonded silica (C8-C30) with particle sizes varying between 1.7 to 3.5 μm19,20,21,22. The eluting analytes are emitted through an ESI emitter integrated within the column, which ensures soft ionization of solution phase analytes to gaseous ions. Coupling LC columns with ESI-MS has significantly advanced the application of tandem mass spectrometry to proteomic strategies in biomedical sciences.
LC columns with narrow inner diameters result in narrower chromatographic peaks and higher sensitivity relative to higher bore, microflow columns and hence are particularly advantageous with proteomic workflows. Although commercially available pre-packed LC columns are attractive options due to their convenience and ease-of-use, they can be prohibitively expensive and less flexible than in-house options. The goal of this work is to describe a technically simple and low-cost slurry packing approach to prepare narrow inner diameter reversed phase HPLC columns using fused-silica capillaries and an in-house built pressure bomb system for proteomic applications.
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the National Institutes of Health grant GM089778 to J.A.W.
Materials
| 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 | r119.aq.0001 | 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 |
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