April 2021 Issue
Feasibility of Phosphoproteomics on Leftover Samples After RNA Extraction With Guanidinium Thiocyanate
In Brief We performed phosphoproteomics after acid guanidinium thiocyanate–phenol–chloroform (AGPC) isolation for cells and tissue and compared it with a classical urea lysis protocol. For both, we show high similarity of data in steady state and after DNA damage induction through irradiation of U2OS cells. Since AGPC extraction can also yield DNA and RNA for molecular characterization, our results could be of potential use in situations when sample material is limited such as in clinical biopsy workflows.Mapping Isoform Abundance and Interactome of the Endogenous TMPRSS2-ERG Fusion Protein by Orthogonal Immunoprecipitation–Mass Spectrometry Assays
In Brief Orthogonal immunoprecipitation-mass spectrometry assays quantified TMPRSS2-ERG fusion protein (∼27,000 copies/cell) and its four distinct isoforms, and revealed that T1E4-ERG isoform accounted for 52 ± 3% of the total ERG in VCaP cells and 50 ± 11% in FFPE prostate cancer tissues. Methionine-truncated and N-acetylated peptide TASSSSDYGQTSK unique for T1/E4 TMPRSS2-ERG fusion was identified. Unlike the N-terminal antibodies, C-terminal antibodies identified 29 ERG-interacting proteins, including mutually exclusive BRG1- and BRM-associated canonical SWI/SNF chromatin remodeling complexes. Clinical perspectives of assays were discussed.Quantitative Proteomics Reveals that the OGT Interactome Is Remodeled in Response to Oxidative Stress
In Brief The goal of these studies was to provide insight into the regulation of the O-GlcNAc transferase (OGT) basally and in response to oxidative stress, as well as the role that O-GlcNAc plays in promoting cytoprotection. Using quantitative proteomics, the basal and injury-induced interactome of OGT has been defined and validated. Protein interactors are anticipated to regulate either the activity or substrate targeting of OGT, or to be substrates of OGT, thus affecting cytoprotection.Quantifying Positional Isomers (QPI) by Top-Down Mass Spectrometry
In Brief Here we determine the quantities of the same protein modified differentially by PTMs, creating so-called positional isomers (i.e., double phosphorylation, one isomer with positions Y64 and T98, and one isomer with positions Y64 and Y120). Knowledge of the relative abundances of these separate positional isomers is highly relevant, as researchers uncovered many thousands of phosphorylation sites many of which have no clear biological function. Uncovering their relative abundance will assist in determining the relative importance of each modification site.Data Management of Sensitive Human Proteomics Data: Current Practices, Recommendations, and Perspectives for the Future
In Brief Availability of proteomics data in the public domain has become the norm, as it has been the case in genomics and transcriptomics for many years. Analogously to sequencing data, there are increasing ethical issues and legal requirements related to sensitive human clinical proteomics data. We review the current state of the art and make concrete recommendations to address these issues in the proteomics field, which are summarized in four different areas.Quantitative Proteomics and Phosphoproteomics Support a Role for Mut9-Like Kinases in Multiple Metabolic and Signaling Pathways in Arabidopsis
In Brief The MUT9-like kinases are a family of plant-specific nuclear-localized kinases with roles in diverse signaling pathways, including light sensing, phytohormone perception, and the circadian clock. The proteome and phosphoproteome of compound mlk mutant seedlings have been determined under light and dark conditions. These experiments identify new roles for these kinases regulating secondary plant metabolism and stress responses, tested through metabolite analysis and assaying seedling sensitivity to DNA damaging agents.Reflections on the HUPO Human Proteome Project, the Flagship Project of the Human Proteome Organization, at 10 Years
In Brief Starting from several organ-oriented projects, HUPO in 2010 launched the Human Proteome Project to identify and characterize the protein parts list and integrate proteomics into multiomics research. Key steps were partnerships with neXtProt, PRIDE, PeptideAtlas, Human Protein Atlas, and instrument makers; global engagement of researchers; creation of ProteomeXchange; adoption of HPP Guidelines for Interpretation of MS Data and SRMAtlas for proteotypic peptides; annual metrics of finding “missing proteins” and functionally annotating proteins; and initiatives for early career scientists.Proximity-dependent Mapping of the Androgen Receptor Identifies Kruppel-like Factor 4 as a Functional Partner
In Brief A proximity interaction network for the androgen receptor (AR) was obtained from androgen-responsive prostate cancer cells. A total of 267 candidates were identified, most associating following ligand stimulation, including Krüppel-like factor 4 (KLF4). KLF4 and AR were found to colocalize genome-wide on 4097 genes including PSA (KLK3), for which KLF4 acts as a repressor, without regulating the expression of AR. These results are instrumental to further dissect the molecular mechanisms underlying androgen signaling in prostate cells.Stable Isotope Labeling of Amino Acids in Flies (SILAF) Reveals Differential Phosphorylation of Mitochondrial Proteins Upon Loss of OXPHOS Subunits
In Brief We advanced a fully composed food source for Drosophila melanogaster into a highly efficient, versatile, and cheap labeling method, termed SILAF. The larval proteome incorporates more than 99% heavy lysine-6 label within 6 days, while the adult fly metabolism allows protein turnover studies. We obtained a phosphoproteome with site-specific occupancy in a fly model of mitochondrial metabolic disease, highlighting the regulation of two novel conserved phosphosites on subunits of the electron transport chain.
