Dataset Information

GASP and FASP are complementary for LC-MS/MS proteomic analysis of drug-metabolising enzymes and transporters in pig liver

ABSTRACT: Sample preparation is a critical step in the proteomic workflow. Numerous different approaches are used, tailored to the type of sample, the aims of the experiment, analytical method, and to an extent, user preference. This has resulted in large variation in reported protein abundances. In this study we demonstrate the complementarity of two different sample preparation techniques for the study of absorption, distribution, metabolism and excretion (ADME) related proteins from pig liver tissue. Filter-aided sample preparation (FASP) is a well-established and widely used method, whilst gel-aided sample preparation (GASP) is a relatively new method optimised and simplified from previous gel-associated digestion techniques. To investigate each method the number of peptides and proteins characterised, reproducibility of results and their real-time application were examined. Whilst both methods have their merits and limitations, for example, FASP is the less technical of the two methods, whilst GASP is time efficient, ultimately the two methods show significant differences in the peptides identified and therefore the use of both methods should be considered when examining and quantifying ADME related proteins.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Sus Scrofa Domesticus (domestic Pig)

TISSUE(S): Liver

SUBMITTER: Martyn Howard

LAB HEAD: Jill Barber

PROVIDER: PXD011324 | Pride | 2018-11-12

REPOSITORIES: Pride

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Publications

GASP and FASP are Complementary for LC-MS/MS Proteomic Analysis of Drug-Metabolizing Enzymes and Transporters in Pig Liver.

Howard Martyn M Achour Brahim B Al-Majdoub Zubida Z Rostami-Hodjegan Amin A Barber Jill J

Proteomics 20181130 24

Sample preparation is a critical step in the proteomic workflow. Numerous different approaches are used, tailored to the type of sample, the aims of the experiment, analytical method, and to an extent, user preference. This has resulted in large variation in reported protein abundances. In this study, the complementarity of two different sample preparation techniques is demonstrated for the study of absorption, distribution, metabolism, and excretion (ADME) related proteins from pig liver tissue ...[more]

PMID: 30371990

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Project description:The genus Lactobacillus contains over 100 different species that were traditionally considered to be uniformly non-motile. However, at least twelve motile species are known to exist in the L. salivarius clade of this genus. Of these, Lactobacillus rumnis is the only motile species that is also autochthonous to the mammalian gastrointestinal tract. The genomes of two L. ruminis strains, ATCC25644 (human isolate, non-motile) and ATCC27782 (bovine isolate, motile) were sequenced and annotated to identify the genes responsible for flagellum biogenesis and chemotaxis in this species. Transcriptome analysis revealed that motility genes were transcribed at a significantly higher level in motile L. ruminis ATCC27782 than in non-motile ATCC25644 during the motile growth phase. In preparation for RNA isolation from L. ruminis ATCC27782 and ATCC25644 cultures in the motile phase, 20 ml aliquots of MRS broth were inoculated (0.25 % inoculum) with a turbid culture of the desired strain. The cultures were incubated anaerobically at 37 Â°C for 15 hr. In preparation for RNA isolation from L. ruminis ATCC27782 and ATCC25644 cultures in the non-motile growth phase, 20 ml aliquots of MRS broth were inoculated (1 % inoculum) with a turbid culture of the desired strain. The cultures were incubated anaerobically at 37 Â°C for 16-20 hrs. RNAprotect Bacteria reagent (Qiagen) was used to stabilize gene expression in the target cultures, and total RNA was isolated according to the protocol for enzymatic and mechanical disruption of bacteria as described in the RNAprotect Bacteria Reagent handbook. RNA isolation was completed with the RNeasy Mini kit, and contaminating DNA was removed with the Turbo DNA-free kit (Ambion). An Oligo aCGH/ChIP-on chip hybridization kit (Agilent) was used for hybridization of the labelled cDNA to the microarrays. Probe hybridization took place at 65 Â°C for 20 hrs with constant rotation (10 rpm). Microarrays were scanned using the Agilent Microarray Scanner System (G2505B) and the scanned files were converted to data files with Feature Extraction software (Agilent). Three microarrays, (three biological replicates) were used to examine gene transcription during the motile growth phase. One microarray was used to examine gene transcription during the non-motile growth phase.

Dataset Information

GASP and FASP are complementary for LC-MS/MS proteomic analysis of drug-metabolising enzymes and transporters in pig liver

Publications

GASP and FASP are Complementary for LC-MS/MS Proteomic Analysis of Drug-Metabolizing Enzymes and Transporters in Pig Liver.

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