Proteomic and molecular dynamic investigations of PTM-induced structural fluctuations in breast and ovarian cancer

Post-translational processing leads to conformational changes in protein structure that modulate molecular functions and change the signature of metabolic transformations and immune responses. Some post-translational modifications (PTMs), such as phosphorylation and acetylation, are strongly related to oncogenic processes and malignancy. This study investigated a PTM pattern in patients with gender-specific ovarian or breast cancer. Proteomic profiling and analysis of cancer-specific PTM patterns were performed using high-resolution UPLC-MS/MS. Structural analysis, topology, and stability of PTMs associated with sex-specific cancers were analyzed using molecular dynamics modeling. We identified highly specific PTMs, of which 12 modified peptides from eight distinct proteins derived from patients with ovarian cancer and 6 peptides of three proteins favored patients from the group with breast cancer. We found that all defined PTMs were localized in the compact and stable structural motifs exposed outside the solvent environment. PTMs increase the solvent-accessible surface area of the modified moiety and its active environment. The observed conformational fluctuations are still inadequate to activate the structural degradation and enhance protein elimination/clearance; however, it is sufficient for the significant modulation of protein activity.


CANCER-SPECIFIC PROTEOME
The defined proteome specified for group of patients with ovarian cancer or patients with breast cancer Comparative symmetry analysis showed that the mutual proteome size (between all studied groups) was n = 147 protein identifications. The common proteome size of two cancer-specific groups was n = 50 protein identifications, whereas the group-specific proteomes were n = 74 and n = 25 protein identifications amongst patients with ovarian and breast cancer, correspondingly. Proteins, attributed for the pathology groups and featured by a statistically significant between-groups quantitative difference, are given below in Supplementary  Table S1.
Supplementary Table S1: Plasma-based differentially expressed proteins (DEP) selected upon results of semiquantitative and symmetry analysis at statistical significance cut-off level of p<0.05. We focused in on three known types of PTMs to identify them amongst patients under consideration ( Figure  S1). The discovered PTMs were ranged and plotted against each study group to reveal their input. It has been found, that ubiquitination is the least prevalent type of PTM in cancer phenotypes, but can be readily extracted from healthy donors. At the same time, phosphorylation was found to be most frequent type of PTM in ovarian cancer patients with the local input of 55%.

OVARIAN CANCER
Supplementary Figure S1: Distribution of identified PTMs types within studied groups. The defined PTMs of interest are distributed unequally amigo different cancer groups and the control group. Particularly, ubiquitination of the most prevalent type of PTM among healthy donors in contrast to cancer phenotype patients, where this PTM made only 32% and 7% in ovarian and breast cancer, correspondingly. Likewise, acetylation is more general PTM for cancer phenotype and constitutes only 8% on the control group of healthy donors. Altogether, it may partially conform the known phenomenon of actively ongoing chromatin remodeling and significantly suppressed apoptosis processes in tumor cells. Bottom panel (C) shows the fragmentation spectrum of the modified peptide with amino acid sequence MDAKMYLGYEYVTAIR derived from TRFE and found in both ovarian and breast cancer groups of study. The peptide is acetylated at lysine and was detected as an ion with m/z=983.4725 (mass error is -0.91 pm) at 34.47 minutes.

MOLECULAR GEOMETRY FEATURES OF THE MODIFIED ALBUMIN (ALBU)
There were five different PTMs identified specifically in serum samples of patients with breast cancer or ovarian cancer only and were not detected amongst patients of the control group. Each of selected PTMs were further categorized by the spatial geometry feature assisted by the molecular dynamic experiments.
The following characteristics were calculated along the molecular dynamic itemizing: ▪ LocPTM -type and localization of the modified amino acid residue in the chain-A; ▪ rA, rB -is a root mean square deviations (RMSD) for helices; ▪ d -mean value of inter-planar distance; ▪ r -minimal distance; ▪ α -angle between helices axis; ▪ θ -torsion angle; ▪ S -area of intercept between helices; ▪ P -polygonal perimeter between helices; ▪ sd, sr, sα, sθ, sS, sP -root mean square deviation of the above-mentioned parameters; ▪ stat -qualification pattern of the original (initial) structure; ▪ nmd -qualification of the unmodified (intact) structure; ▪ mod -qualification of the modified structure; Supplementary  Figure S3: Types and rules for qualification of secondary structures comprised of two distinct α-helices and unstructured