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Deep LC-MS/MS analysis of the proteome from six cell lines

ABSTRACT: Mass spectrometry is a rational orthogonal method for antibody-based assays, but implementation of MS in the validation pipeline of antibody manufacturers is hampered by the high cost and low throughput. Here we present a rapid method for antibody validation based on denaturing gel electrophoresis of biotinylated cell lysates (PAGE) followed by mass spectrometry (MS) and antibody array analysis (MAP). The first step, PAGE, produces 12 fractions containing proteins of increasing molecular weight. The fractions are analyzed in parallel by MS and MAP. Antibodies to be tested are immobilized on color coded polymer beads to create antibody arrays, which can comprise up to several thousand various antibodies. MS data provide definite protein identifications in each fraction, creating a reference for antibody reactivity patterns obtained via MAP. The method employs automated software to compare both datasets and provide validation data for each antibody tested. Due to the high-throughput nature of the assay we were able to screen several thousands of antibodies against six different cell lines. The differences in protein expression between the cell lines provide an additional control of antibody specificity. Using PAGE-MAP it is possible to screen and validate thousands of antibodies in a matter of weeks. Moreover, antibodies are tested under standardized conditions, which allows for direct comparison of their performance.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Suspension Culture, Cell Culture, Jurkat Cell, Hela Cell

SUBMITTER: Krzysztof Sikorski

LAB HEAD: Fridtjof Lund-Johansen

PROVIDER: PXD005945 | Pride | 2018-07-19

REPOSITORIES: Pride

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Project description:We report the genome-wide mapping of time-series ChIP-seq data sets of human ERα cell lines (MCF-7). The 4 time points cover 0, 1, 4 and 24 hours, respectively. MCF7 cells were maintained in a hormone-free medium (phenol red–free MEM with 2 mmol/L L-glutamine, 0.1 mmol/L nonessential amino acids, 50 units/mL penicillin, 50 Ag/mL streptomycin, 6 ng/mL insulin, and 10% charcoal-stripped FBS) for three days. MCF7 cells were treated with DMSO (as 0 hour time point) or E2 (108 mol/L) for 1, 4 and 24 hours. 5 x 107 cells were cross-linked with 1% formaldehyde for 10 min, at which point 0.125 M glycine was used to stop the crosslinking. In brief, after crosslinking, cells were treated by lysis buffers and sonicated to fragment the chromatin to a size range of 500bp-1kb. Chromatin fragments were then immunoprecipitated with 10ug of antibody/magnetic beads. The antibodies against ERα were purchased from Santa Cruz Biotechnology (Santa Cruz, sc-8005 X). After immunoprecipitation, washing, and elution, ChIP DNA was purified by phenol:chloroform:isoamyl alcohol and solubilized in 70 μl of water. The ChIP DNA sample was run in 12% PAGE and the 100-300bp DNA fraction was excised and eluted from the gel slice overnight at 4 °C in 300 μl of elution buffer (5:1, LoTE buffer (3 mM Tris-HCl, pH 7.5, 0.2 mM EDTA)-7.5 M ammonium acetate) and was purified using a QIAquick purification kit (Qiagen, Cat#28104). The library was constructed using Illumina genomic DNA prep kit by following its protocol (Illumina, cat# FC-102-1002), clusters were generated on the Illumina cluster station (Illumina, cat# FC-103-1002), DNA samples (20 nM per sample) quantified by an Agilent Bioanalyzer, were loaded onto Illumina Genome Analyzer IIx (GAIIx) for sequencing according to the manufacturer’s protocol. Reads generated from the Illumina GAIIx pipeline were aligned to the Human Genome Assembly (NCBI build 36.1/hg18) using ELAND algorithm. Experiments on human ERα breat cancer cell lines (MCF-7) in 4 time points.

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	BproteinGroups.zip	Other
	P1261.raw	Raw
	P1262.raw	Raw
	P1263.raw	Raw
	P1264.raw	Raw

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Deep LC-MS/MS analysis of the proteome from six cell lines

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