Linear ion trap quadrupole mass spectrometer

Manufactured by Thermo Fisher Scientific

The linear ion trap quadrupole mass spectrometer is an analytical instrument that combines a linear ion trap and a quadrupole mass analyzer to perform high-performance mass spectrometry. It is designed to provide sensitive and accurate detection and identification of a wide range of molecular compounds.

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Lab products found in correlation

Mmessage mmachine t7 transcription kit, by Thermo Fisher Scientific (2 mentions) Pierce rna 3 end desthiobiotinylation kit, by Thermo Fisher Scientific (1 mentions) Magnetic rna protein pull down kit, by Thermo Fisher Scientific (1 mentions) Control anti igg, by Merck Group (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) T7 rna polymerase, by Roche (1 mentions) Rnase free dnase 1, by Promega (1 mentions) Biotin rna labeling mix kit, by Roche (1 mentions) Smart race cdna ampli cation kit, by Takara Bio (1 mentions) Sb c18 column, by Agilent Technologies (1 mentions)

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6 protocols using linear ion trap quadrupole mass spectrometer

Characterization of FAM83H-AS1 RNA Interactome

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RNA immunoprecipitation was performed as described previously,¹¹ and magnetic beads were conjugated with anti‐HNRNPK (ABCAM) or control anti‐IgG (Millipore) antibody. In vitro translation assays were performed using mMESSAGE mMACHINE T7 Transcription Kit (Invitrogen) according to the manufacturer's instructions. Then, FAM83H‐AS1 RNAs were labeled with desthiobiotinylation using the Pierce RNA 3′End Desthiobiotinylation Kit (Thermo Fisher). RNA pull‐down assays were performed with Magnetic RNA‐Protein Pull‐Down Kit (Thermo Fisher) according to the manufacturer's instructions. After elution of RNA‐interacting proteins, they were subjected to mass spectrometric analysis. Liquid chromatography–mass spectrometry experiments were performed with a linear ion trap quadrupole mass spectrometer (Thermo Finnigan) equipped with a micro‐spray source.

Wang S., Han C., Liu T., Ma Z., Qiu M., Wang J., You Q., Zheng X., Xu W., Xia W., Xu Y., Hu J., Xu L, & Yin R. (2021). FAM83H‐AS1 is a noncoding oncogenic driver and therapeutic target of lung adenocarcinoma. Clinical and Translational Medicine, 11(2), e316.

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Identification and Characterization of P. acnes CAMP Factor

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P. acnes bacteria including ATCC 6919, wild-type P. acnes (266; 1-1a, ST18) strain (Bruggemann et al., 2004) , and a knock-out mutant of CAMP factor 2 (Dcamp2, [PPA0687]) (Nakatsuji et al., 2008b; Sorensen et al., 2010) were used.
Mass spectrometry, vaccination, in vitro neutralization, and ex vivo acne model All procedures for the identification of P. acnes CAMP factor by isotope-coded protein labeling (Schmidt et al., 2005) using light ( 12 C 6 ) and heavy ( 13 C 6 ) forms of N-nicotinoyloxy-succinimide for liquid chromatography linear ion trap quadrupole mass spectrometer (Thermo Fisher Scientific, Waltham, MA), the expression of recombinant CAMP factor, vaccination, quantification of antibody titers, injection of P. acnes into mouse ear, in vitro neutralization, ELISA, bacteria counts, and establishment of ex vivo acne model were performed according to the methods described in Nakatsuji et al. (2008a) and are described in detail in the Supplementary Materials online.

Wang Y., Hata T.R., Tong Y.L., Kao M.S., Zouboulis C.C., Gallo R.L, & Huang C.M. (2018). The Anti-Inflammatory Activities of Propionibacterium acnes CAMP Factor-Targeted Acne Vaccines. The Journal of investigative dermatology, 138(11).

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Identification of ANXA2-Interacting lncRNAs

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RNA immunoprecipitation was performed as described previously^{48 (link)}, and magnetic beads were conjugated with anti-ANXA2 or control anti-IgG antibody. Biotin RNA Labeling Mix Kit (Roche) and T7 RNA polymerase (Roche) were used to biotin-label and transcribe the full-length as well as truncated fragments of MIR99AHG RNA, using MIR99AHG overexpression plasmid and PCR products of the truncated fragments as a template, respectively. Then, the RNAs were treated with RNase-free DNase I (Promega) and isolated with RNeasy Mini Kit (Qiagen). RNA pull-down assays were performed with a Magnetic RNA-Protein Pull-Down Kit according to the manufacturer’s instructions. After elution of lncRNA-interacting proteins, they were subjected to mass spectrometric analysis. Liquid chromatography mass spectrometry (LC–MS) experiments were performed with a linear ion trap quadrupole mass spectrometer (Thermo Finnigan) equipped with a microspray source.

Han C., Li H., Ma Z., Dong G., Wang Q., Wang S., Fang P., Li X., Chen H., Liu T., Xu L., Wang J., Wang J, & Yin R. (2021). MIR99AHG is a noncoding tumor suppressor gene in lung adenocarcinoma. Cell Death & Disease, 12(5), 424.

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Characterization of FAM83H-AS1 lncRNA

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5′-RACE, 3′-RACE, and full-length ampli cation of FAM83H-AS1 were performed using a SMART RACE cDNA Ampli cation Kit (Clontech) according to the manufacturer's instructions. The gene-speci c primers used for RACE analysis are presented in Additional le 1: Table S1.
RNA immunoprecipitation and pull-down assays RNA immunoprecipitation was performed as described previously [16] , and magnetic beads were conjugated with anti-HNRNPK or control anti-IgG antibody. In vitro translation assays were performed using mMESSAGE mMACHINE T7 Transcription Kit (Invitrogen) according to the manufacturer's instructions. Then, FAM83H-AS1 RNAs were labeled with desthiobiotinylation using the Pierce RNA 3′ End Desthiobiotinylation Kit (Magnetic RNA-Protein Pull-Down Kit, Components; Thermo Fisher). RNA pulldown assays were performed with Magnetic RNA-Protein Pull-Down Kit according to the manufacturer's instructions. After elution of lncRNA-interacting proteins, they were subjected to mass spectrometric analysis. Liquid chromatography mass spectrometry (LC-MS) experiments were performed with a linear ion trap quadrupole mass spectrometer (Thermo Finnigan) equipped with a micro-spray source.

Wang S., Han C., Liu T., Ma Z., Qiu M., Wang J., Zheng X., Xu W., Xia W., Xu Y., Hu J., Lin X., & Yin R. (2020). LncRNA FAM83H-AS1 Amplification is Associated With a Poor Prognosis in Lung Adenocarcinoma and Can Serve as A Therapeutic Target.

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Glucuronide Derivatization by LQIT-MS

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The experiments were carried out in a Thermo Scientific linear quadrupole ion trap (LQIT) mass spectrometer modified with an external reagent mixing manifold.^{18 (link)} The studied glucuronides were ionized by negative mode electrospray ionization (ESI). The deprotonated glucuronides were isolated in the ion trap with an isolation width of 2 mass units and allowed to react with HSiCl₃ for 30– 100 ms. SiHCl₃ was introduced into the ion trap via the external reagent mixing manifold at a flow rate 3 μL/hour. No harmful effects to the instrumentation have been observed, likely because of the very small amount of the reagent introduced. A detailed description of the instrumentation used here for performing ion/molecule reactions can be found in the literature.^{15 (link)}

Kong J.Y., Yu Z., Easton M.W., Niyonsaba E., Ma X., Yerabolu R., Sheng H., Jarrell T.M., Zhang Z., Ghosh A.K, & Kenttämaa H.I. (2018). Differentiating Isomeric Deprotonated Glucuronide Drug Metabolites via Ion/Molecule Reactions in Tandem Mass Spectrometry. Analytical chemistry, 90(15), 9426-9433.

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HPLC-MS Analysis of Deprotonated Analytes

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HPLC-MS analysis was performed using a Surveyor Plus HPLC system with a quaternary pump, an auto sampler, and a photodiode array (PDA) detector. The HPLC was coupled to a Thermo Scientific linear quadrupole ion trap (LQIT) mass spectrometer equipped with an electrospray ionization (ESI) source. Separation was performed using a Zorbax SB-C18 column. A non-linear gradient of water (A) and acetonitrile (B) was used as follows: 0.00 minutes, 95% A and 5% B; 10.00 minutes, 95% A and 5% B; 30.00 minutes, 40% A and 60% B; 35.00 minutes, 5% A and 95% B; 38.00 minutes, 5% A and 95% B; 38.50 minutes, 95% A and 5% B, 45.00 minutes, 95% A and 5% B. The mobile phase flow rate was kept at 500 μL/min throughout the gradient. HPLC eluents were mixed via a T-connector with 1% sodium hydroxide solution at a flow rate of 0.1 μL/min to facilitate deprotonation of the analytes. ESI source conditions were set as: 3.5 kV spray voltage; 50 (arbitrary unit) sheath gas (N₂) flow and 20 (arbitrary unit) auxiliary gas (N₂) flow. Ion detection was performed under negative ion mode. The PDA detector was set at a wavelength of 254 nm.

Klose T., Herbst D.A., Zhu H., Max J.P., Kenttämaa H.I, & Rossmann M.G. (2015). A Mimivirus enzyme that participates in viral entry. Structure (London, England : 1993), 23(6), 1058-1065.

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