FlashPack: Fast and simple preparation of ultra-high performance capillary columns for LC-MS

Abstract

Capillary ultra-high-pressure liquid chromatography (cUHPLC) is essential for in-depth characterization of complex biomolecule mixtures by LC-MS. We developed a simple and fast method called FlashPack for custom packing of capillary columns of 50-100 cm length with sub-2-m sorbent particles. FlashPack uses high sorbent concentrations of 500-1000 mg/ml for packing at relatively low pressure of 100 bar. Column blocking by sorbent aggregation is avoided during the packing of sorbent particles by gentle mechanical tapping of the capillary proximal end by a slowly rotating magnet bar. Utilizing a standard 100 bar pressure bomb, Flashpack allows for production of 15-25 cm cUHPLC columns within a few minutes and of 50 cm cUHPLC columns in less than an hour. Columns exhibit excellent reproducibility of back-pressure, retention time and resolution (CV 8,7 %). FlashPack cUHPLC columns are inexpensive, robust and deliver performance comparable to commercially available cUHPLC columns. The FlashPack method is versatile and enables production of cUHPLC columns using a variety of sorbent materials.

Complex-dependent histone acetyltransferase activity of KAT8 determines its role in transcription and cellular homeostasis

“…The samples were analyzed using DIA strategy with high resolution orbitrap MS1 and low resolution ion trap MS2 scans (Sidoli et al, 2015) on Orbitrap Velos coupled to nanoAcquity UPLC system (Waters). Peptides (600 ng per injection) were directly loaded on 30 cm long column with 150 uM inner diameter packed with intertsil 2 um sorbent using flashPack method (Kovalchuk et al, 2019). The flow rate was kept at 1 ul/min and column temperature was 30 C during the analysis.…”

Epinephrine Affects Ribosomes, Cell Division, and Catabolic Processes in Micrococcus luteus Skin Strain C01: Revelation of the Conditionally Extensive Hormone Effect Using Orbitrap Mass Spectrometry and Proteomic Analysis

“…For the MS experiments, samples were loaded to a homemade trap column 20 mm × 0.1 mm, packed with Inertsil ODS3 3 µm sorbent (GL Sciences, Tokyo, Japan), in the loading buffer (2% acetonitrile, 98% H 2 O, 0.1% trifluoroacetic acid) at 10 µL/min flow and separated at RT in a home-packed [26] fused-silica column 300 mm × 0.1 mm packed with Reprosil PUR C18AQ 1.9 (Dr. Maisch, Ammerbuch-Entringen, Germany) into an emitter prepared with P2000 Laser Puller (Sutter, Atlanta, GA, USA). Reverse-phase chromatography was performed with an Ultimate 3000 Nano LC System (ThermoFisher, Waltham, MA, USA), which was coupled to the Q Exactive Plus Orbitrap mass spectrometer (ThermoFisher, USA) via a nanoelectrospray source (ThermoFisher, USA).…”

Mapping Proteome and Lipidome Changes in Early-Onset Non-Alcoholic Fatty Liver Disease Using Hepatic 3D Spheroids

“…One μg of the peptide mixture of each sample was utilized for proteomics analysis. Peptides were captured on a pre-column (Acclaim PepMap 100 C18, Thermo Fisher Scientific) and separated using an in-house packed column (120 Å pore size, C18) using the FlashPack method [ 36 ]. The column was heated to 50 °C, and the gradient was 8–35% solution B (99.99% ACN, 0.01% formic acid) over 160 min at 550 nL/min using a Dionex UltiMate 3000 nanoLC system (Thermo Fisher Scientific).…”