Fluorescent modification of proteins of interest (POI) in living cells is desired to study their behaviour and functions in their natural environment. In a perfect setting it should be easy to perform, inexpensive, efficient and site-selective. Although multiple chemical and biological methods have been developed, only a few of them are applicable for cellular studies thanks to their appropriate physical, chemical and biological characteristics. One such successful system is a tetracysteine tag/motif and its selective biarsenical binders (e.g. FlAsH and ReAsH). Since its discovery in 1998 by Tsien and co-workers, this method has been enhanced and revolutionized in terms of its efficiency, formed complex stability and breadth of application. Here, we overview the whole field of knowledge, while placing most emphasis on recent reports. We showcase the improvements of classical biarsenical probes with various optical properties as well as multifunctional molecules that add new characteristics to proteins. We also present the evolution of affinity tags and motifs of biarsenical probes demonstrating much more possibilities in cellular applications. We summarize protocols and reported observations so both beginners and advanced users of biarsenical probes can troubleshoot their experiments. We address the concerns regarding the safety of biarsenical probe application. We showcase examples in virology, studies on receptors or amyloid aggregation, where application of biarsenical probes allowed observations that previously were not possible. We provide a summary of current applications ranging from bioanalytical sciences to allosteric control of selected proteins. Finally, we present an outlook to encourage more researchers to use these magnificent probes. This journal is @en

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.@en