Comparative membrane proteomics analyses of breast cancer cell lines to understand the molecular mechanism of breast cancer brain metastasis

Abstract

Breast cancer is the leading type of cancer in women. Breast cancer brain metastasis is currently considered an issue of concern among breast cancer patients. Membrane proteins play important roles in breast cancer brain metastasis, involving cell adhesion and penetration of blood-brain barrier. To understand the mechanism of breast cancer brain metastasis, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed in conjunction with enrichment of membrane proteins to analyze the proteomes from five different breast cancer and a brain cancer cell lines. Quantitative proteomic data of all cell lines were compared with MDA-MB-231BR which is a brain seeking breast cancer cell line, thus representing brain metastasis characteristics. Label-free proteomics of the six cell lines facilitates the identification of 1238 proteins and the quantification of 899 proteins of which more than 70% were membrane proteins. Unsupervised principal component analysis (PCA) of the label-free proteomics data resulted in a distinct clustering of cell lines, suggesting quantitative differences in the expression of several proteins among the different cell lines. Unique protein expressions in 231BR were observed for 28 proteins. The up-regulation of STAU1, AT1B3, NPM1, hnRNP Q, and hnRNP K and the down-regulation of TUBB4B and TUBB5 were noted in 231BR relative to 231 (precursor cell lines from which 231BR is derived). These proteins might contribute to the breast cancer brain metastasis. Ingenuity pathway analysis (IPA) supported the great brain metastatic propensity of 231BR and suggested the importance of the up-regulation of integrin proteins and down-regulation of EPHA2 in brain metastasis.

Integrated Transcriptomics, Proteomics, and Glycomics Reveals the Association between Up-regulation of Sialylated N-glycans/Integrin and Breast Cancer Brain Metastasis

“…Then the denatured proteins were reduced by 5 mM DTT at 60 °C for 45 min, alkylated by 20 mM IAA at 60 °C in darkness for 45 min, followed by quenching the reaction by adding another 5 mM DTT and incubating at 60 °C for 30 min. Next, protease Trypsin-Lys C (Promega, MS gold grade) was added to the sample with a 1:25 enzyme to protein ratio and incubated at 37.5 °C for 18 h. After tryptic digestion, formic acid was added (final concentration is 0.5%) to remove the SDC 48 . After centrifugation at max speed for 10 min, supernatant which contained peptides was collected and dried.…”

A rapid method for relative quantification ofN-glycans from a therapeutic monoclonal antibody during trastuzumab biosimilar development

“…MWCO filters were found to selectively bind certain glycans, and a detergent wash step (sodium deoxycholate, SDC) was added to the method (MWCO-SDC-IPC), which has been shown to be an improvement in filter-aided sample preparation in protein and glycoprotein digestion. 39,40 In addition, we used the PA-IPC method, which reliably identifies, quantifies and recovers more glycans, to compare the glycan profiles of innovator Herceptin® antibody products (6 lots, HER1 to HER6; 4 European and 2 US) with internal developmental biosimilar drug substance (scale-up bioreactor runs).…”

Proteome profiling in the aorta and kidney of type 1 diabetic rats

“…In this study, we aimed at identifying the global protein changes in response to T1DM-induced hyperglycemia in the aorta and kidney, by employing Liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique to comparatively quantitate the expression of different proteins among the different conditions, and to check the intensities of three different post-translational modifications, namely acetylation, phosphorylation and oxidation. In addition, systems biology analysis (Ingenuity Pathway Analysis, IPA) was used to model the effects of diabetes on different pathways in the two organs, to identify biological processes that are modified by the exposure conditions [ 17 – 19 ]. This approach allows the identification of possible novel biomarkers and development of new mechanisms aimed at defining the interplay of multiple biological pathways involved in the etiology of renal and vascular disease in diabetes.…”