Membrane contact sites (MCSs) are morphologically defined intracellular structures where cellular membranes are closely apposed. Recent progress has significantly advanced our understanding of MCSs with the use of new tools and techniques. Visualization of MCSs in living cells by split fluorescence proteins or FRET-based techniques tells us the dynamic property of MCSs. Manipulation of MCSs by chemically-induced dimerization (CID) or light-induced dimerization (LID) greatly contributes to our understanding of their functional aspects including inter-organelle lipid transport mediated by lipid transfer proteins (LTPs). Here we highlight recent advances in these tools and techniques as applied to MCSs, and we discuss their advantages and limitations.

Platelets are small anucleate cell fragments (2-4 μm in diameter) in the blood, which play an essential role in thrombosis and hemostasis. Genetic or acquired platelet dysfunctions are linked to bleeding, increased risk of thromboembolic events and cardiovascular diseases. Advanced proteomic approaches may pave the way to a better understanding of the roles of platelets in hemostasis, and pathophysiological processes such as inflammation, metastatic spread and thrombosis. Further insights into the molecular biology of platelets are crucial to aid drug development and identify diagnostic markers of platelet activation. Platelet activation is known to be an extremely rapid process and involves multiple post-translational mechanisms at sub second time scale, including proteolysis and phosphorylation. Multi-omics technologies and biochemical approaches can be exploited to precisely probe and define these posttranslational pathways. Notably, the absence of a nucleus in platelets significantly reduces the number of present proteins, simplifying mass spectrometry-based proteomics and metabolomics approaches.

Purine nucleotides, generated by de novo synthesis and salvage pathways, are essential for metabolism and act as building blocks of genetic material. To avoid an imbalance in the nucleotide pool, nature has devised several strategies to regulate/tune the catalytic performance of key purine metabolic enzymes. Here, we discuss some recent examples, such as stress-regulating alarmones that bind to select pathway enzymes, huge ensembles like dynamic metabolons and self-assembled filaments that highlight the layered fine-control prevalent in the purine metabolic pathway to fulfill requisite purine demands. Examples of enzymes that turn-on only under allosteric control, are regulated via long-distance communication that facilitates transient conduits have additionally been explored.

Metal-based anticancer agents occupy a distinct chemical space due to their particular coordination geometry and reactivity. Despite the initial DNA-targeting paradigm for this class of compounds, it is now clear that they can also be tuned to target proteins in cells, depending on the metal and ligand scaffold. Since metallodrug discovery is dominated by phenotypic screenings, tailored proteomics strategies were crucial to identify and validate protein targets of several investigative and clinically advanced metal-based drugs. Here, such experimental approaches are discussed, which showed that metallodrugs based on ruthenium, gold, rhenium and even platinum, can selectively and specifically target proteins with clear-cut down-stream effects. Target identification strategies are expected to support significantly the mechanism-driven clinical translation of metal-based drugs.

Mass spectrometry-based phosphoproteomics is currently the leading methodology for the study of global kinase signaling. The scientific community is continuously releasing technological improvements for sensitive and fast identification of phosphopeptides, and their accurate quantification. To interpret large-scale phosphoproteomics data, numerous bioinformatic resources are available that help understanding kinase network functional role in biological systems upon perturbation. Some of these resources are databases of phosphorylation sites, protein kinases and phosphatases; others are bioinformatic algorithms to infer kinase activity, predict phosphosite functional relevance and visualize kinase signaling networks. In this review, we present the latest experimental and bioinformatic tools to profile protein kinase signaling networks and provide examples of their application in biomedicine.

DNA N6-methyladenine modification (6mA) is a predominant epigenetic mark in prokaryotes but rarely present in multicellular metazoa. The analytical technologies have been developed for sensitive detection of 6mA, including ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS) and single molecule real-time sequencing (SMRTseq). However, it remains challenging to detect 6mA at global level and/or in the context of sequence in multicellular metazoa (including mammals). This mini-review brings insights into current dilemma and potential solutions for the identification and quantifications of 6mA in mammals.