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Implementing 1.5 mm internal diameter columns into microflow LC-MS bottom-up proteomics

ABSTRACT: In this study, we systematically evaluated the 1.5 mm i.d. column and compared its performance with 1.0 mm i.d. columns in analyzing protein samples of varying complexity using a conventional chromatographic system.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Francisella Tularensis Subsp. Holarctica (strain Lvs)

SUBMITTER: Juraj Lenco

LAB HEAD: Juraj Lenco

PROVIDER: PXD057525 | Pride | 2025-02-11

REPOSITORIES: Pride

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			Action	DRS
	DataDescription.xlsx	Xlsx
	JLe0056_1i0mm_15min_DN195_Jurkat_0i025uGuL_00i05uG_01.raw	Raw
	JLe0056_1i0mm_15min_DN195_Jurkat_0i025uGuL_00i05uG_02.raw	Raw
	JLe0056_1i0mm_15min_DN195_Jurkat_0i025uGuL_00i16uG_01.raw	Raw
	JLe0056_1i0mm_15min_DN195_Jurkat_0i025uGuL_00i16uG_02.raw	Raw

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Project description:Glands were frozen at -80 ºC immediately after being retrieved and stored under this condition until initiating the proteomics analysis protocol. Glands were washed with cold PBS and immersed in 120 l of homogenization buffer (50 mM Tris-HCl, pH 6.8, 10 mM DTT, 4% (w/v) SDS). Glands were boiled for 5 min, incubated overnight at 4 ºC and centrifuged at 4 ºC and 13000 rpm for 10 min. The whole gland proteome of each gland was concentrated as described elsewhere (Bonzon-Kulichenko, F. Garcia-Marques, M. Trevisan-Herraz and J. Vazquez, Journal of Proteome Research, 2015, 14, 700-710 (DOI:10.1021/pr5007284).Briefly, samples were loaded into an SDS-PAGE gel (0.5 mm-thick, 4% stacking, and 10% resolving) and the electrophoresis process was stopped when the front entered 2 mm into the resolving gel. The unseparated protein bands were then visualized by Coomassie staining, excised and digested at 37 ºC overnight with 500 l of 25 g/l chymotrypsin (Promega) in 100 mM Tris-HCl, pH 7.8 containing 10 mM CaCl2. The cleaved peptides were extracted by incubation for 2 h in 100 mM Tris-HCl, pH 7.8 on shaking, acidized with 1% (v/v) trifluoroacetic acid, desalted onto C18 cartridges (Oasis, Waters Corporation, Milford, MA, USA), and dried down.The analysis of the samples proceeded through liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Easy nLC 1000 nano-HPLC coupled to a Q-Exactive Orbitrap mass spectrometer (Themo Scientific). Peptides were injected onto a C18 reverse-phase precolumn (Acclaim PepMap100, 75-m i.d., 3-m particle size, 2-cm length, Thermo Scientific), and a reverse-phase analytical column (Acclaim PepMap 100, 75-m, i.d., 3-m particle size, 50-cm length, Thermo Scientific) in buffer A [0.1% formic acid (v/v)] and eluted with a 60 min linear gradient of buffer B [90% acetonitrile, 0.1% formic acid (v/v)], at 200 nL/min. Mass spectrometry (MS) runs consisted of 70000 FT-resolution spectra in the 390-1200 m/z wide range, followed by data-dependent MS/MS spectra of the 15 most intense parent ions acquired along the chromatographic run. High-energy collisional (HCD) fragmentation was performed at 27% of normalized collision energy and the isolation window for the parent ion mass was established at 2 Da.

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Implementing 1.5 mm internal diameter columns into microflow LC-MS bottom-up proteomics

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