RIME proteomics of estrogen and progesterone receptors in breast cancer

Abstract

Nuclear receptors play an important role in transcriptional regulation of diverse cellular processes and is also relevant in diseases such as cancer. In breast cancer, the nuclear receptors - estrogen receptor (ER) and progesterone receptor (PR) are classical markers of the disease and are used to classify breast cancer subtypes. Using a recently developed affinity purification MS technique (RIME) [1], we investigate the protein interactors of ER and PR in breast cancer cell lines upon stimulation by the ligands - estrogen and progesterone. The data is deposited at proteomeXchange (PXD002104) and is part of a publication [2] that explains the link between the two nuclear receptors and potential consequences of this in breast cancer. In this manuscript, we describe the methodology used and provide details on experimental procedures, analysis methods and analysis of raw data. The purpose of this article is to enable reproducibility of the data and provide technical recommendations on performing RIME in hormonal contexts.

Fig. 1

Example mass spectra (MS1) of peptide ions used for quantification in an ER RIME experiment with or without progesterone treatment where ER and PR association is seen only after progesterone treatment (PRIDE Accession PDX002104>52234/JP517). (A) MS1 spectrum of the 2+ peptide ions corresponding to the peptide sequence SIILLNSGVYTFLSSTLK from the human estrogen receptor (AC# P03372). Both the light (SIILLNSGVYTFLSSTLK [⁶C₁₂²N₁₄], monoisotopic peak at m/z 978.55695) and heavy (SIILLNSGVYTFLSSTLK [⁶C₁₃²N₁₅], monisotopic peak at m/z 982. 56415) versions of the peptide are indicated. These originate from the estrogen+progesterone treated cells and the estrogen alone cells respectively. Quantification of the peak areas resulted in a 1:1 (H:L) ratio. (B) MS1 spectrum of the 3+ peptide ions corresponding to the peptide sequence ALSVEFPEMMSEVIAAQLPK from the human progesterone receptor (AC# P06401). Both the light (ALSVEFPEMMSEVIAAQLPK [⁶C₁₂²N₁₄], monoisotopic peak at m/z 730.71130) and heavy (ALSVEFPEMMSEVIAAQLPK [⁶C₁₃²N₁₅], monoisotopic peak at m/z 732.95355) versions of the peptide are indicated. These originate from the estrogen+progesterone treated cells and the estrogen alone cells respectively. Quantification of the peak areas resulted in a 1:15 (H:L)ratio.