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Use of a modified Multiplexed Inhibitor Bead assay to study kinome responses to cytostatic treatment in human cancer cell lines Triplicate Test: pilot study

ABSTRACT: In order to improve elution of proteins from the column, to reduce the cost as well as the hands on time of the analysis, we here present a modification of the Multiplexed Inhibitor Bead assay (MIB assay), where we elute the proteins from beads containing covalently bound inhibitors using trypsin digestion. Three human cancer cell lines treated with the same chemotherapeutics, cisplatin and docetaxel, were used to monitor and evaluate the performance of the assay. Using this method, we see a good coverage of the kinome as well as other important signaling proteins using only three kinase inhibitors. We demonstrate that the method is reproducible and that the column is re-usable for more than 10 times without loss in performance. The MIB assay revealed large differences in the cellular signaling responses between the cell lines.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: Animesh Sharma

LAB HEAD: Voin Petrovic

PROVIDER: PXD005402 | Pride | 2017-02-13

REPOSITORIES: Pride

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On-column trypsinization allows for re-use of matrix in modified multiplexed inhibitor beads assay.

Petrovic Voin V Olaisen Camilla C Sharma Animesh A Nepal Anala A Bugge Steffen S Sundby Eirik E Hoff Bård Helge BH Slupphaug Geir G Otterlei Marit M

Analytical biochemistry 20170203

The Multiplexed Inhibitor Bead (MIB) assay is a previously published quantitative proteomic MS-based approach to study cellular kinomes. A rather extensive procedure, need for multiple custom-made kinase inhibitors and an inability to re-use the MIB-columns, has limited its applicability. Here we present a modified MIB assay in which elution of bound proteins is facilitated by on-column trypsinization. We tested the modified MIB assay by analyzing extract from three human cancer cell lines treat ...[more]

PMID: 28167071

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Project description:Purpose: The goal of this study is to compare the transcriptomes expressed during submergence stress of two Brachypodium distachyon ecotypes with contrasting survival under this stress. Bd21 is a submergence sensitive ecotype with EC50 of 2.5 days and Bd2-3 is a tolerant ecotype with EC50 of 4 days. Methods (Stress): Brachypodium Bd21 and Bd2-3 plants (14-day-old, 6 leaves stage) were submerged in a water column of 30 cm inside opaque-wall plastic tanks. Light still reached the plants at 40 uE m-2 s-??1. Ecotypes were submerged side-by-side in a randomized manner; only plants submerged in the same tank were compared. Controls were grown in plastic tanks without a water column. Submergence stress started at ZT14 (2h before night, long-day regime 16h light, 8h dark). Above ground tissue was collected after 48 h submergence stress in liquid nitrogen and stored at -80C in an ultra freezer until further processing. Tissue was ground to powder with mortar, pestle and liquid nitrogen avoiding thawing. Control and submerged total RNA was extracted with TRIzol reagent (Invitrogen, 15596018), purified with Direct-zol RNA mini prep columns (Zymo Research, R2050) and digested in-column with DNAse I (ThermoScientific, EN0521). RNA integrity and concentration was verified in denaturing 1.0% agarose gels, Nanodrop 2000 (ThermoScientific) and in a Bioanalyzer 2100 (Agilent) with the integrated software 2100 Expert, samples had a RNA Integrity Number (RIN) between 6.4-7.2 characteristic of aerial plant tissue (Babu and Gassman, 2011). Total RNA extracted from control and submerged tissue from three independent experiments consisting each of four individuals were used to construct cDNA indexed libraries and sequenced in a HiSeq2500 (Illumina) at 1x50 format, making a total of 12 sequenced libraries (tolerant and intolerant ecotype, control and submerged, all experimental triplicates) in a 2-lane format. RNA integrity, library construction and sequencing was performed as a service at the Unidad Universitaria de Secuenciacion Masiva, Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico (IBT-UNAM). Differential Gene Expression (DGE) analysis was performed with edgeR using a generalized linear model and false discovery rate <0.05 (FDR). To group differentially expressed transcripts a logFC value of 1.5 (up-regulated) or -1.5 (down-regulated) and a FDR <0.05x10-5 were selected. GO analysis of differential transcripts was performed at phytozome.org Results: We identified commonly up-regulated genes (317) and exclusively up-regulated in Bd2-3 (466) or Bd21 (706). Regarding down-regulation, 330 transcripts were common, an exclusively 851 and 1026 for Bd2-3 and Bd21, respectively. GO analysis indicated that oxidative stress, pathogen responses and nitric oxide homeostasis were the most differential characteristics of tolerant ecotype Bd2-3. Conclusions: The use of triplicate RNAseq data of transcriptomes expressed in ecotypes with contrasting tolerance to submergence under long-day light regime, allowed us to identify common responsive routes such as SUSY, glycolysis, anaerobic routes (alanine, ethanol, lactate, GABA) and glyoxylate cycle. It also enabled us to discover integrated oxidative stress and NO homeostasis pathways that are differentially expressed in the tolerant ecotype. We expect that this information can be translated to agricultural relevant plants to increase our knowledge and biotechnological possibilities on plant submergence stress. Sequenced libraries (triplicates, HiSeq2500 Illumina, 1x50 format) of aerial tissue (control and 48h submergence stress) of Brachypodium distachyon Bd21 (sensitive) and Bd2-3 (tolerant).

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	20161012_Voin_Rep_Day1.raw	Raw
	20161101_Voin_Rep_Day2.raw	Raw
	20161104_Voin_Rep_Day3.raw	Raw
	txt.zip	Other