A Whole Proteome Inventory of Background Photocrosslinker Binding

Affinity‐based protein profiling (AfBPP) is a widely applied method for the target identification of bioactive molecules. Probes containing photocrosslinkers, such as benzophenones, diazirines, and aryl azides, irreversibly link the molecule of interest to its target protein upon irradiation with UV light. Despite their prevalent application, little is known about photocrosslinker‐specific off‐targets, affecting the reliability of results. Herein, we investigated background protein labeling by gel‐free quantitative proteomics. Characteristic off‐targets were identified for each photoreactive group and compiled in a comprehensive inventory. In a proof‐of‐principle study, H8 , a protein kinase A inhibitor, was equipped with a diazirine moiety. Application of this photoprobe revealed, by alignment with the diazirine background, unprecedented insight into its in situ proteome targets. Taken together, our findings guide the identification of biologically relevant binders in photoprobe experiments.     

Quantitative mass spectrometry in proteomics: a critical review

The quantification of differences between two or more physiological states of a biological system is among the most important but also most challenging technical tasks in proteomics. In addition to the classical methods of differential protein gel or blot staining by dyes and fluorophores, mass-spectrometry-based quantification methods have gained increasing popularity over the past five years. Most of these methods employ differential stable isotope labeling to create a specific mass tag that can be recognized by a mass spectrometer and at the same time provide the basis for quantification. These mass tags can be introduced into proteins or peptides (i) metabolically, (ii) by chemical means, (iii) enzymatically, or (iv) provided by spiked synthetic peptide standards. In contrast, label-free quantification approaches aim to correlate the mass spectrometric signal of intact proteolytic peptides or the number of peptide sequencing events with the relative or absolute protein quantity directly. In this review, we critically examine the more commonly used quantitative mass spectrometry methods for their individual merits and discuss challenges in arriving at meaningful interpretations of quantitative proteomic data.     

A Vinyl Sulfone‐Based Fluorogenic Probe Capable of Selective Labeling of PHGDH in Live Mammalian Cells

Chemical probes are powerful tools for interrogating small molecule‐target interactions. With additional fluorescence Turn‐ON functionality, such probes might enable direct measurements of target engagement in live mammalian cells. DNS‐pE (and its terminal alkyne‐containing version DNS‐pE2) is the first small molecule that can selectively label endogenous 3‐phosphoglycerate dehydrogenase (PHGDH) from various mammalian cells. Endowed with an electrophilic vinyl sulfone moiety that possesses fluorescence‐quenching properties, DNS‐pE/DNS‐pE2 became highly fluorescent only upon irreversible covalent modification of PHGDH. With an inhibitory property (in vitro K_i=7.4 μm ) comparable to that of known PHGDH inhibitors, our probes thus offer a promising approach to simultaneously image endogenous PHGDH activities and study its target engagement in live‐cell settings.     

Europium tetracycline as a luminescent probe for nucleoside phosphates and its application to the determination of kinase activity

The determination of enzyme activities and the screening of enzyme regulators is a major task in clinical chemistry and drug development. A broad variety of enzymatic reactions is associated with the consumption of adenosine triphosphate (ATP), including, in particular, phosphorylation reactions catalyzed by kinases, formation of adenosine cyclic monophosphate (cAMP) by adenylate cyclases, and ATP decomposition by ATPase. We have studied the effect of a series of adenosine (ATP, ADP, AMP, cAMP) and guanosine (GTP, GDP) phosphoric esters, and of pyrophosphate (PP) on the fluorescence emission of the europium tetracycline (EuTC) complex. We found that these compounds have strongly different quenching effects on the luminescence emission of EuTC. The triphosphates ATP and GTP behave as strong quenchers in reducing the fluorescence intensity of EuTC to 25 % of its initial value by formation of a ternary 1:1:1 complex. All other phosphate esters showed a weak quenching effect only. The applicability of this fluorescent probe to the determination of the activity of phosphorylation enzymes is demonstrated by means of creatine kinase as a model for non-membrane-bound kinases. In contrast to other methods, this approach does not require the use of radioactively labeled ATP substrates, additional enzymes, or of rather complex immunoassays.     

Cross-linking chemistry and biology: development of multifunctional photoaffinity probes

An efficient method of photoaffinity labeling has been developed based on rationally designed multifunctional photoprobes. Photoaffinity techniques have been used to elucidate the protein structure at the interface of biomolecules by the photochemical labeling of interacting sites. However, the identification of labeled sites within target proteins is often difficult. Novel biotinyl bioprobes bearing a diazirine photophore have contributed significantly to the rapid elucidation of ligand binding sites within proteins, thereby extending conventional photoaffinity methods. This article discusses the synthesis and applications of various photoprobes bearing a biotin, including strategies using cleavable linkages between photophores. The combination of photoaffinity methods with chip technology is also described as a novel entry to rapid affinity-based screening of inhibitors. This review focuses on a rapid and reliable photoaffinity method utilizing diazirine-based multifunctional photoprobes with numerous potential applications in functional proteomics of biomolecular interactions.     

Red‐Shifted Fluorescent Aminated Derivatives of a Conformationally Locked GFP Chromophore

A novel class of fluorescent dyes based on conformationally locked GFP chromophore is reported. These dyes are characterized by red‐shifted spectra, high fluorescence quantum yields and pH‐independence in physiological pH range. The intra‐ and intermolecular mechanisms of radiationless deactivation of ABDI‐BF2 fluorophore by selective structural locking of various conformational degrees of freedom were studied. A unique combination of solvatochromic and lipophilic properties together with “infinite” photostability (due to a dynamic exchange between free and bound dye) makes some of the novel dyes promising bioinspired tools for labeling cellular membranes, lipid drops and other organelles.     

Unravelling the Allosteric Targeting of PHGDH at the ACT-Binding Domain with a Photoactivatable Diazirine Probe and Mass Spectrometry Experiments

The serine biosynthetic pathway is a key element contributing to tumor proliferation. In recent years, targeting of phosphoglycerate dehydrogenase (PHGDH), the first enzyme of this pathway, intensified and revealed to be a promising strategy to develop new anticancer drugs. Among attractive PHGDH inhibitors are the α-ketothioamides. In previous work, we have demonstrated their efficacy in the inhibition of PHGDH in vitro and in cellulo. However, the precise site of action of this series, which would help the rational design of new inhibitors, remained undefined. In the present study, the detailed mechanism-of-action of a representative α-ketothioamide inhibitor is reported using several complementary experimental techniques. Strikingly, our work led to the identification of an allosteric site on PHGDH that can be targeted for drug development. Using mass spectrometry experiments and an original α-ketothioamide diazirine-based photoaffinity probe, we identified the 523Q-533F sequence on the ACT regulatory domain of PHGDH as the binding site of α-ketothioamides. Mutagenesis experiments further documented the specificity of our compound at this allosteric site. Our results thus pave the way for the development of new anticancer drugs using a completely novel mechanism-of-action.