The lipid metabolite assay was conducted using the AbsoluteIDQ® p400 HR Kit (BIOCRATES Life Sciences AG, Austria). Logarithmically-growing lung cancer cells were harvested by treating them with 0.05% trypsin and resuspending in 100 μl phosphate buffered saline (PBS). Cell lysates were processed by a manual protocol. Samples were analyzed by the Q-Exactive LC/MS system (Thermo Fisher Scientific Inc, Waltham, MA) and data were processed using MetIDQ software (BIOCRATES Life Sciences AG, Austria).
Q exactive lc ms system
Manufactured by Thermo Fisher Scientific
The Q-Exactive LC/MS system is a high-resolution mass spectrometer that combines liquid chromatography (LC) with quadrupole-Orbitrap mass spectrometry (MS). It is designed to provide accurate, high-resolution data for a wide range of analytical applications.
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3 protocols using q exactive lc ms system
1
Lipid Metabolite Profiling of Lung Cancer Cells
2
Proteomic Analysis of YAP1 Interactome
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Cell lysates were prepared by lysing cells in buffer containing 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 1 mM EDTA, and protease and phosphatase inhibitors (#87787, Thermo Scientific). Total protein was then incubated with anti-YAP1 or anti IgG antibodies (16 h, 4°C, rotation), and protein A/G PLUS Agarose beads were added (20 μl, 1 h, 4°C, rotation). The captured agarose beads Ab–Ag complexes were washed (five times, PBS) and detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-page), followed by dyeing the strips with coomassie brilliant blue, cutting the strips in the lanes, and mixing them for mass spectrometry analysis, which was performed by Thermo’s Q Exactive™ LC/MS system. Next, the YAP1-interacting proteins of control and YO groups selected Score Sequest HT ≥ 1.5, deleted contaminating proteins, and removed high molecular weight proteins and negative reference proteins of the IgG group.
3
Proteomic Analysis of Protein Bands
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The proteins were identified by two-stage mass spectrometry, using a Q Exactive LC/MS system (Thermo Scientific Corporation). The protein bands with a relative molecular mass of 86 x 10 3 , 75 x 10 3 , and 60 x 10 3 were cut from the polyacrylamide gel for decolorization, reduction, and trypsin digestion. The samples were then fully dissolved and loaded onto a high-performance liquid chromatography-mass spectrometry (HPLC-MS) chamber. HPLC was conducted using a Diane NCS3500 system, while MS was performed on a Q Exactive system. The generated data and the downloaded proteome database (human-refseq-20140303-71465s.fasta; downloaded from NCBI) was analyzed using the Proteome Discoverer software.
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