Large-Scale Analysis of Breast Cancer-Related Conformational Changes in Proteins Using SILAC-SPROX

Abstract

Proteomic methods for disease state characterization and biomarker discovery have traditionally utilized quantitative mass spectrometry methods to identify proteins with altered expression levels in disease states. Here we report on the large-scale use of protein folding stability measurements to characterize different subtypes of breast cancer using the Stable Isotope Labeling with Amino Acids in Cell Culture and Stability of Proteins from Rates of Oxidation (SILAC-SPROX) technique. Protein folding stability differences were studied in a comparison of two luminal breast cancer subtypes, luminal-A and -B (i.e., MCF-7 and BT-474 cells, respectively), and in a comparison of a luminal-A and basal subtype of the disease (i.e., MCF-7 and MDA-MB-468 cells, respectively). The 242 and 445 protein hits identified with altered stabilities in these comparative analyses, included a large fraction with no significant expression level changes. This suggests thermodynamic stability measurements create a new avenue for protein biomarker discovery. A number of the identified protein hits are known from other biochemical studies to play a role in tumorigenesis and cancer progression. This not only substantiates the biological significance of the protein hits identified using the SILAC-SPROX approach, but it also helps elucidate the molecular basis for their disregulation and/or disfunction in cancer.

Network-based method for drug target discovery at the isoform level

“…Splice variant proteins were generated based on our published identification method 24 . The steps are as follows: firstly, mass spectrometric results data of different breast cancer subtypes was download from the PRIDE archive (PRIDE archive download; available at http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD006703) 49 . Secondly, the Uniprot IDs marked with “Majority protein IDs” were extracted from these files and retained for the further study.…”

Proteome-Wide Characterization of Phosphorylation-Induced Conformational Changes in Breast Cancer

“…In the SILAC-SPROX experiment, the data were analyzed as previously described. 34 Briefly, a Mathematica script, developed in house, was used to process the data and to determine final hits. The script imported the output files from MaxQuant, and normalized the H/L ratio of methionine containing peptide with the H/L ratio of non-methionine peptide from the same protein.…”

Protein profiling and pseudo-parallel reaction monitoring to monitor a fusion-associated conformational change in hemagglutinin

“…HDX has several limitations, however, including incompatibility with biologically complex matrices, the requirement for rapid, highresolution separation, and usually high nanogram to microgram levels of sample. To overcome these challenges, oxidative and electrophilic labeling approaches have been taken to footprint water accessibility on protein surfaces, both in buffer and in complex media [6][7][8][9][10]. Among oxidative approaches, SPROX employs peroxide to oxidize methionine residues [9,11], while FPOP generates oxidizing radicals through photolysis [12][13][14][15].…”

“…To overcome these challenges, oxidative and electrophilic labeling approaches have been taken to footprint water accessibility on protein surfaces, both in buffer and in complex media [6][7][8][9][10]. Among oxidative approaches, SPROX employs peroxide to oxidize methionine residues [9,11], while FPOP generates oxidizing radicals through photolysis [12][13][14][15]. Electrophilic approaches include targeting lysine with NHS esters [16,17], oxyimidates, and thioimidates [18][19][20], and targeting glutamate and aspartate with glycine ethyl ester [21][22][23].…”