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

Published: May 14, 2021

doi:

Yasaman Jami-Alahmadi*¹, Vijaya Pandey*¹, Adarsh K. Mayank*¹, James A. Wohlschlegel

¹Department of Biological Chemistry,David Geffen School of Medicine at UCLA

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

1. Preparation of the capillary tip 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. 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 …

Representative Results

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…

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…

Divulgations

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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