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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biochemistry

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

Published: May 14th, 2021

DOI:

10.3791/62380

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 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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