Published: May 14th, 2021
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
1. Preparation of the capillary tip
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.......
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.......
|99.99% Formamide acid
|for making frit
|For providing heat
|Brechbuehler helium pressure cell
|for packing column
|Ceramic column cutter
|for cutting silica capillary
|Dimethyl sulfoxide (DMSO) ≥ 99%
|Stored in a flammable cabinet
|for making frit
|Hydrofluoric acid (HF) (50%)
|for opening the emitter after polymerization
|KASIL (Potassium Silicate Solution)
|for making frit
|Orbitrap Fusion Lumos
|Thermo Fisher Scientific
|for MS data acquisition
|P2000 Laser Puller
|for pulling capillary
|PTFE 1/16" Ferrule 0.4 mm ID (long) for Tube Fitting
|For bomb setting
|Reprosil-Pur 120 C18-AQ, 1.9 um, 1g
|Dr. Masch GmbH
|For initiating polimerization
|Stainless Steel Pipe Fitting, Hex Coupling, 1/4 in. Female NPT
|for bomb setting
|TSP075375 fused silica, 75 µm ID x 360 µOD
|For column tubing
|Ultimate 3000 UHPLC
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