Summary

Title

Proteomic signature of neuroblastoma cells UKF-NB-4 reveals key role of lysosomal sequestration and the proteasome complex in acquiring chemoresistance to cisplatin

Description

Cisplatin (CDDP) is a widely used agent in the treatment of neuroblastoma. Unfortunately, the development of acquired chemoresistance limits its clinical use. To gain a detailed understanding of the mechanisms underlying the development of such chemoresistance, we comparatively analysed established cisplatin-resistant neuroblastoma cell line (UKF-NB- 4CDDP) and its sensitive counterpart (UKF-NB-4). We confirmed the decreased sensitivity of tested cells to cisplatin and identified a cross-resistance to carboplatin and oxaliplatin. Among the proteins responsible for UKF-NB-4CDDP chemoresistance, ion channels transport family proteins, ABC superfamily proteins, SLC-mediated trans-membrane transporters, proteasome complex subunits and V-ATPases were identified. Moreover, we detected markedly higher proteasome activity in UKF-NB-4CDDP cells and a remarkable lysosomal enrichment that can be inhibited by bafilomycin A to sensitize UKF-NB-4CDDP to CDDP. Our results indicate that lysosomal sequestration and proteasome activity may be one of key mechanisms responsible for intrinsic chemoresistance of neuroblastoma to CDDP.

Sample Processing Protocol

After protein extraction using RIPA buffer, proteins were reduced, alkylated, and digeste with trypsin overnight at 37°C. Resulting peptides were desalted and separated on an Easy-nLC 1000 nano system (Thermo Scientific). Then, the samples were loaded into a precolumn Acclaim PepMap 100 (Thermo Scientific) and eluted in a RSLC PepMap C18, 25 cm long, 75 μm inner diameter and 2 μm particle size (Thermo Scientific). The mobile phase flow rate was 300 nL/min using 0.1% formic acid in water (v/v) and 0.1% formic acid (v/v) and 100% acetonitrile(v/v). MS analysis was performed using a Q-Exactive mass spectrometer (Thermo Scientific). For ionization, 2000 V of liquid junction voltage and 270°C capillary temperature was used. The full scan method employed a m/z 400–1500 mass selection, an Orbitrap resolution of 70.000 (at m/z 200), a target automatic gain control (AGC) value of 3e6, and maximum injection times of 100 ms. After the survey scan, the 15 most intense precursor ions were selected for MS/MS fragmentation. Fragmentation was performed with a normalized collision energy of 27 eV and MS/MS scans were acquired with a starting mass of m/z 100, AGC target was 2e5, resolution of 17,500 (at m/z 200), intensity threshold of 8e3, isolation window of 2 m/z units. Charge state screening was enabled to reject unassigned, singly charged, and equal or more than seven protonated ions. A dynamic exclusion time of 20 s was used to discriminate against previously selected ions. The protein identification was carried out by nLC-MS/MS.

Data Processing Protocol

MS data were analysed with Proteome Discoverer (v.1.4.1.14) (Thermo Fisher Scientific) using standardized workflows. The mass spectrum *.raw file was searched against the human SwissProt 57.15 database (20,266 sequences protein entries) using MASCOT search engine (version 2.3, Matrix Science). Precursor and fragment mass tolerance were set to 10 ppm and 0.02 Da, respectively, allowing 2 missed cleavages, carbamidomethylation of cysteines as a fixed modification, methionine oxidation, phosphorylation serine, threonine and tyrosine acetylation N-terminal and as a variable modification. Identified peptides were filtered using Percolator algorithm with a q-value threshold of 0.01.

Contact

Vivian de los Rios, CIB.CSIC
Vivian de los Rios, Proteomics and Genomics Facility ( lab head )

Submission Date

20/12/2018

Publication Date

02/01/2019

Disease

neuroblastoma

Instrument

Q Exactive

Software

Not available

Experiment Type

Shotgun proteomics

Publication

    Merlos Rodrigo MA, Buchtelova H, de Los Rios V, Casal JI, Eckschlager T, Hrabeta J, Belhajova M, Heger Z, Adam V. Proteomic signature of neuroblastoma cells UKF-NB-4 reveals key role of lysosomal sequestration and the proteasome complex in acquiring chemoresistance to cisplatin. J Proteome Res. 2018 PubMed: 30592607