Identifying the target proteins of bioactive small molecules is a key step in understanding mode‐of‐action of the drug and addressing the underlying mechanisms responsible for a particular phenotype. ...Proteomics has been successfully used to elucidate the target protein profiles of unmodified and ligand‐modified bioactive small molecules. In the latter approach, compounds can be modified via click chemistry and combined with activity‐based protein profiling. Target proteins are then enriched by performing a pull‐down with the modified ligand. Methods that utilize unmodified bioactive small molecules include the cellular thermal shift assay, thermal proteome profiling, stability of proteins from rates of oxidation, and the drug affinity responsive target stability (DARTS) determination (or read‐out). This review highlights recent proteomic approaches utilizing data‐dependent analysis and data‐independent analysis to identify target proteins by DARTS. When combined with liquid chromatography/tandem mass spectrometry, DARTS enables the identification of proteins that bind to drug molecules that leads to a conformational change in the target protein(s). In addition, an effective strategy is proposed for selecting the target protein(s) from within the pool of analyzed candidates. With additional complementary methods, the biologically relevant target proteins that bind to the small bio‐active molecules can be further validated.
Extracellular vesicles (EVs) are submicron, membrane-enclosed particles that are released from cells in various pathophysiological states. The molecular cargo of these vesicles is considered to ...reflect the composition of the cell of origin, and the EV proteome is therefore a potential source of biomarkers for various diseases. Our aim was to determine whether EVs isolated from plasma provide additional diagnostic value or improved pathophysiological understanding compared to plasma alone in the context of myocardial infarction (MI). A panel of proximity extension assays (n = 92) was employed to analyze EV lysates and plasma from patients with MI (n = 60) and healthy controls (n = 22). After adjustment for multiple comparisons, a total of 11 dysregulated proteins were identified in EVs of MI patients compared to the controls (q < 0.01). Three of these proteins: chymotrypsin C (CTRC), proto-oncogene tyrosine-protein kinase SRC (SRC) and C-C motif chemokine ligand 17 (CCL17) were unaltered in the corresponding plasma samples. As biomarkers for MI, rudimentary to no evidence exists for these proteins. In a separate group of patients with varying degrees of coronary artery disease, the decrease in EV-associated (but not plasma-related) SRC levels was confirmed by ELISA. Confirmation of the presence of SRC on EVs of different sizes and cellular origins was performed with ELISA, flow cytometry and nanoparticle tracking analysis. In conclusion, the data revealed that despite a similarity in the EV and plasma proteomes, analysis of isolated EVs does indeed provide additional diagnostic information that cannot be obtained from plasma alone.
After the successful completion of the Human Genome Project, the Human Proteome Organization has recently officially launched a global Human Proteome Project (HPP), which is designed to map the ...entire human protein set. Given the lack of protein-level evidence for about 30% of the estimated 20,300 protein-coding genes, a systematic global effort will be necessary to achieve this goal with respect to protein abundance, distribution, subcellular localization, interaction with other biomolecules, and functions at specific time points. As a general experimental strategy, HPP research groups will use the three working pillars for HPP: mass spectrometry, antibody capture, and bioinformatics tools and knowledge bases. The HPP participants will take advantage of the output and cross-analyses from the ongoing Human Proteome Organization initiatives and a chromosome-centric protein mapping strategy, termed C-HPP, with which many national teams are currently engaged. In addition, numerous biologically driven and disease-oriented projects will be stimulated and facilitated by the HPP. Timely planning with proper governance of HPP will deliver a protein parts list, reagents, and tools for protein studies and analyses, and a stronger basis for personalized medicine. The Human Proteome Organization urges each national research funding agency and the scientific community at large to identify their preferred pathways to participate in aspects of this highly promising project in a HPP consortium of funders and investigators.
The objective of the international Chromosome-Centric Human Proteome Project (C-HPP) is to map and annotate all proteins encoded by the genes on each human chromosome. The C-HPP consortium was ...established to organize a collaborative network among the research teams responsible for protein mapping of individual chromosomes and to identify compelling biological and genetic mechanisms influencing colocated genes and their protein products. The C-HPP aims to foster the development of proteome analysis and integration of the findings from related molecular -omics technology platforms through collaborations among universities, industries, and private research groups. The C-HPP consortium leadership has elicited broad input for standard guidelines to manage these international efforts more efficiently by mobilizing existing resources and collaborative networks. The C-HPP guidelines set out the collaborative consensus of the C-HPP teams, introduce topics associated with experimental approaches, data production, quality control, treatment, and transparency of data, governance of the consortium, and collaborative benefits. A companion approach for the Biology and Disease-Driven HPP (B/D-HPP) component of the Human Proteome Project is currently being organized, building upon the Human Proteome Organization's organ-based and biofluid-based initiatives (www.hupo.org/research). The common application of these guidelines in the participating laboratories is expected to facilitate the goal of a comprehensive analysis of the human proteome.
Mass spectrometry imaging (MSI) has the potential to reveal the localization of thousands of biomolecules such as metabolites and lipids in tissue sections. The increase in both mass and spatial ...resolution of today’s instruments brings on considerable challenges in terms of data processing; accurately extracting meaningful signals from the large data sets generated by MSI without losing information that could be clinically relevant is one of the most fundamental tasks of analysis software. Ion images of the biomolecules are generated by visualizing their intensities in 2-D space using mass spectra collected across the tissue section. The intensities are often calculated by summing each compound’s signal between predefined sets of borders (bins) in the m/z dimension. This approach, however, can result in mixed signals from different compounds in the same bin or splitting the signal from one compound between two adjacent bins, leading to low quality ion images. To remedy this problem, we propose a novel data processing approach. Our approach consists of a sensitive peak detection method able to discover both faint and localized signals by utilizing clusterwise kernel density estimates (KDEs) of peak distributions. We show that our method can recall more ground-truth molecules, molecule fragments, and isotopes than existing methods based on binning. Furthermore, it automatically detects previously reported molecular ions of lipids, including those close in m/z, in an experimental data set.
No therapeutic targets have been identified for lung squamous cell cancer (SqCC) which is the second most prevalent lung cancer because its molecular profiles remain unclear. This study aimed to ...unveil disease-related protein networks by proteomic and bioinformatic assessment of laser-microdissected cancerous cells from seven SqCCs compared with eight representative lung adenocarcinomas. We identified three network modules significant to lung SqCC using weighted gene co-expression network analysis. One module was intrinsically annotated to keratinization and cell proliferation of SqCC, accompanied by hypoxia-induced aerobic glycolysis, in which key regulators were activated (HIF1A, ROCK2, EFNA1-5) and highly suppressed (KMT2D). The other two modules were significant for translational initiation, nonsense-mediated mRNA decay, inhibited cell death, and interestingly, eIF2 signaling, in which key regulators, MYC and MLXIPL, were highly activated. Another key regulator LARP1, the master regulator in cap-dependent translation, was highly suppressed although upregulations were observed for hub proteins including EIF3F and LARP1 targeted ribosomal proteins, among which PS25 is the key ribosomal protein in IRES-dependent translation. Our results suggest an underlying progression mechanism largely caused by switching to the cap-independent, IRES-dependent translation of mRNA subsets encoding oncogenic proteins. Our findings may help to develop therapeutic strategies to improve patient outcomes.
PDF
