Ligand Modification-Free Methods for the Profiling of Protein-Environmental Chemical Interactions.

Adverse health outcomes caused by environmental chemicals are often initiated via their interactions with proteins. Essentially, one environmental chemical may interact with a number of proteins and/or a protein may interact with a multitude of environmental chemicals, forming an intricate interaction network. Omics-wide protein-environmental chemical interaction profiling (PECI) is of prominent importance for comprehensive understanding of these interaction networks, including the toxicity mechanisms of action (MoA), and for providing systematic chemical safety assessment. However, such information remains unknown for most environmental chemicals, partly due to their vast chemical diversity. In recent years, with the continuous efforts afforded, especially in mass spectrometry (MS) based omics technologies, several ligand modification-free methods have been developed, and new attention for systematic PECI profiling was gained. In this Review, we provide a comprehensive overview on these methodologies for the identification of ligand-protein interactions, including affinity interaction-based methods of affinity-driven purification, covalent modification profiling, and activity-based protein profiling (ABPP) in a competitive mode, physicochemical property changes assessment methods of ligand-directed nuclear magnetic resonance (ligand-directed NMR), MS integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS), thermal proteome profiling (TPP), limited proteolysis-coupled mass spectrometry (LiP-MS), stability of proteins from rates of oxidation (SPROX), and several intracellular downstream response characterization methods. We expect that the applications of these ligand modification-free technologies will drive a considerable increase in the number of PECI identified, facilitate unveiling the toxicological mechanisms, and ultimately contribute to systematic health risk assessment of environmental chemicals.

Integrated Protein Solubility Shift Assays for Comprehensive Drug Target Identification on a Proteome-Wide Scale.

Target proteins are often stabilized after binding with a ligand and thereby typically become more resistant to denaturation. Based on this phenomenon, several methods without the need to covalently modify the ligand have been developed to identify target proteins for a specific ligand. These methods usually employ complicated workflows with high cost and limited throughput. Here, we develop an iso-pH shift assay (ipHSA) method, a proteome-wide target identification method that detects ligand-induced protein solubility shifts by precipitating proteins with a single concentration of acidic agent followed by protein quantification via data-independent acquisition (DIA). Using a pan-kinase inhibitor, staurosporine, we demonstrated that ipHSA increased throughput compared to the previously developed pH-dependent protein precipitation (pHDPP) method. ipHSA was found to have high complementarity in staurosporine target identification compared with the improved isothermal shift assay (iTSA) and isosolvent shift assay (iSSA) using DIA instead of tandem mass tags (TMTs) for quantification. To further improve target identification sensitivity, we developed an integrated protein solubility shift assay (IPSSA) by pooling the supernatants yielded from ipHSA, iTSA, and iSSA methods. IPSSA exhibited increased sensitivity in screening staurosporine targets by 38, 29, and 38% compared to individual methods. Increasing the number of replicate experiments further enhanced the sensitivity of target identification. Meanwhile, IPSSA also improved the throughput and reduced the cost compared with previous methods. As a fast and efficient tool for drug target identification, IPSSA is expected to have broad applications in the study of the mechanism of action.

Matrix Thermal Shift Assay for Fast Construction of Multidimensional Ligand-Target Space.

Existing thermal shift-based mass spectrometry approaches are able to identify target proteins without chemical modification of the ligand, but they are suffering from complicated workflows with limited throughput. Herein, we present a new thermal shift-based method, termed matrix thermal shift assay (mTSA), for fast deconvolution of ligand-binding targets and binding affinities at the proteome level. In mTSA, a sample matrix, treated horizontally with five different compound concentrations and vertically with five technical replicates of each condition, was denatured at a single temperature to induce protein precipitation, and then, data-independent acquisition was employed for quick protein quantification. Compared with previous thermal shift assays, the analysis throughput of mTSA was significantly improved, but the costs as well as efforts were reduced. More importantly, the matrix experiment design allowed simultaneous computation of the statistical significance and fitting of the dose-response profiles, which can be combined to enable a more accurate identification of target proteins, as well as reporting binding affinities between the ligand and individual targets. Using a pan-specific kinase inhibitor, staurosporine, we demonstrated a 36% improvement in screening sensitivity over the traditional thermal proteome profiling (TPP) and a comparable sensitivity with a latest two-dimensional TPP. Finally, mTSA was successfully applied to delineate the target landscape of perfluorooctanesulfonic acid (PFOS), a persistent organic pollutant that is hard to perform modification on, and revealed several potential targets that might account for the toxicities of PFOS.

Mechanical stress induced protein precipitation method for drug target screening.

The process of protein precipitation can be used to decipher the interaction of ligand and protein. For example, the classic Thermal Proteome Profiling (TPP) method uses heating as the driving force for protein precipitation, to discover the drug target protein. Under heating or other denature forces, the target protein that binds with the drug compound will be more resistant to precipitation than the free protein. Similar to thermal stress, mechanical stress can also induce protein precipitation. Upon mechanical stress, protein will gradually precipitate along with protein conformational changes, which can be exploited for the study of the ligand-protein interaction. Herein, we proposed a Mechanical Stress Induced Protein Precipitation (MSIPP) method for drug target deconvolution. Its streamlined workflow allows in situ sample preparation on the surface of microparticles, from protein precipitation to digestion. The mechanical stress was generated by vortexing the slurry of protein solution and microparticle materials. The mechanical stress induced protein precipitate was captured by the microparticles, which guarantees the MSIPP method to be scalable and user-friendly. The MSIPP method was successfully applied to four drug compounds, Methotrexate, Raltitrexed, SHP099, Geldanamycin and a pan-inhibitor of protein kinases, Staurosporine. Besides, DHFR was demonstrated to be a target of Raltitrexed, which has not been revealed by any other modification-free drug target discovery method yet. Thus, MSIPP is a complementary method to other drug target screening methods.

Rapid Enzyme-Mediated Biotinylation for Cell Surface Proteome Profiling.

Cell surface is the primary site for sensing extracellular stimuli. The knowledge of the transient changes on the surfaceome upon a perturbation is very important as the initial changed proteins could be driving molecules for some phenotype. In this study, we report a fast cell surface labeling strategy based on peroxidase-mediated oxidative tyrosine coupling strategy, enabling efficient and selective cell surface labeling within seconds. With a labeling time of 1 min, 2684 proteins, including 1370 (51%) cell surface-annotated proteins (cell surface/plasma membrane/extracellular), 732 transmembrane proteins, and 81 cluster of differentiation antigens, were identified from HeLa cells. By comparison with the negative control experiment using quantitative proteomics, 500 (68%) out of the 731 significantly enriched proteins (p-value < 0.05, ≥2-fold) in positive experimental samples were cell surface-annotated proteins. Finally, this technology was applied to track the dynamic changes of the surfaceome upon insulin stimulation at two time points (5 min and 2 h) in HepG2 cells. Thirty-two proteins, including INSR, CTNNB1, TFRC, IGF2R, and SORT1, were found to be significantly regulated (p-value < 0.01, ≥1.5-fold) after insulin exposure by different mechanisms. We envision that this technique could be a powerful tool to analyze the transient changes of the surfaceome with a good time resolution and to delineate the temporal and spatial regulation of cellular signaling.

Comparative evaluation of MAX-Ti3AlC2 and MXene-Ti3C2 as affinity chromatographic materials for highly selective enrichment of phosphopeptides.

MAX and MXene have received considerable attention owing to their outstanding performance in fields like battery and catalysis. However, their possible biomedical applications have rarely been considered, especially the affinity chromatographic applications in proteomics. In this work, considering the large number of exposed metal sites, small binding potential resistance and fast mass transfer speed, layered ternary carbides MAX-Ti3AlC2 and MXene-Ti3C2 with a two-dimensional nanostructure were successfully explored for the first time as affinity chromatography stationary phases for the specific capture of phosphopeptides from complex biological samples. Helium ion microscopy, transmission electron microscopy, atomic force microscopy, X-ray diffraction spectra, X-ray photoelectron spectroscopy and zeta potential measurement results confirmed that the MXene-Ti3C2 was well exfoliated from the pristine MAX-Ti3AlC2. Ti3AlC2 showed better enrichment specificity than MXene-Ti3C2. The detection limit of Ti3AlC2 was as low as 5 fmol. Even when the molar ratio of BSA to ß-casein tryptic digests increased to 1000 : 1, two characteristic phosphopeptides with a relatively clear background could be detected after enrichment. After five cycles of repeated use, the enrichment specificity of Ti3AlC2 still remains. Furthermore, 91 and 830 unique phosphopeptides from 23 and 525 phosphoproteins were identified from milk and BEL7402 cells, respectively. Among them, 27 and 170 phosphopeptides, 12 and 56 phosphoproteins identified from milk and BEL7402 cells were not detected with commercial TiO2 after three independent replicates, which have great potential in providing complementary coverage of phosphoproteome. This work opens up new applications of Ti3AlC2 and MXene-Ti3C2, and will play more important role for phosphorylated proteomics in biomedicine.

Microparticle-Assisted Precipitation Screening Method for Robust Drug Target Identification.

While thermal proteome profiling (TPP) shines in the field of drug target screening by analyzing the soluble fraction of the proteome samples treated at high temperature, the counterpart, the insoluble precipitate, has been overlooked for a long time. The analysis of the precipitate is hampered by the inefficient sample processing procedure. Herein, we propose a novel method, termed microparticle-assisted precipitation screening (MAPS), for drug target identification. The MAPS method exploits the principle that drug-bound proteins will be more resistant to thermal unfolding similar to the classic TPP method, but the process of protein precipitation is assisted by microparticles. Upon heating, proteins unfold and aggregate on the surface of the microparticles. The introduction of a microparticle simplifies the whole sample preparation workflow. The proteins that precipitate on the microparticles are subjected to washing, alkylation, and digestion. The whole sample preparation is processed conveniently on the surface of the microparticles without any transfer. With the assistance of microparticles, sample loss is minimized. The MAPS method is compatible with minute amounts of initial proteins. MAPS was applied to screen the targets of several well-studied drugs and the known target proteins were successfully identified with high confidence and specificity. To investigate the specificity of the method, MAPS was applied to screen the targets of the pan-kinase inhibitor, staurosporine, and 32 protein kinases (specificity of 80%) were identified using only 20 µg of initial proteins of each sample. MAPS is an unbiased robust method for drug target screening, filling the vacancy of stability-based target screening using a precipitate.

Integrated Microstructured Photonic Fiber as a Bifunctional Robust Frit and Efficient Electrospray Emitter of a Packed Column for Capillary Liquid Chromatography-Tandem Mass Spectrometry Analysis of Complex Biological Samples.

Although capillary liquid chromatography married with tandem mass spectrometry (cLC-MS/MS) has become a powerful technique for proteomics and metabolomics research, it is still a great challenge to fabricate durable capillary-based analytical columns coupling continuous nanoflow (<1 000 nL/min) electrospray ionization (ESI) with MS, owing to the issue of clogging and fragile of emitters. Here, we proposed a simple approach to integrate microstructured photonic fibers (MPFs) into wide bore capillaries with 150 µm i.d., serving as an integral bifunctional frit or/and ESI emitter of packed columns. Two kinds of MPFs containing 126 homogeneous microchannels with different inner diameter, 3.2 µm for MPF-1 and 2.6 µm for MPF-2, were explored for preparation. The octadecylsilicate (ODS) silica-packed column using MPF-1 as a frit exhibited the lowest plate heights of 14.2-19.7 µm for five alkylbenzenes at the velocity of 1.5 mm/s, which were slightly lower than those of packed column with porous polymer monolith (PPM)-based frit by cLC coupling with ultraviolet (UV) detection. Additionally, the packed columns with integral MPF frit-emitters were further applied in analysis of a complex biological sample of digest of Hela cells by cLC-MS. An average of 7109 unique peptides could be identified in a single analysis by using MPF-1 emitter, and 7110 unique peptides were identified by using the MPF-2 emitter, which were superior to the identified result of packed column with an integral tapered tip emitter (6894 peptides). It is obvious that this novel integral MPF-based frit-emitter does not easily suffer from the issues of cracking owing to the silica cladding around independent microchannels (>100), which always encumbers both independent and integral tapered tip emitters for cLC-MS.

Pseudotargeted MS Method for the Sensitive Analysis of Protein Phosphorylation in Protein Complexes.

In this study, we presented an enrichment-free approach for the sensitive analysis of protein phosphorylation in minute amounts of samples, such as purified protein complexes. This method takes advantage of the high sensitivity of parallel reaction monitoring (PRM). Specifically, low confident phosphopeptides identified from the data-dependent acquisition (DDA) data set were used to build a pseudotargeted list for PRM analysis to allow the identification of additional phosphopeptides with high confidence. The development of this targeted approach is very easy as the same sample and the same LC-system were used for the discovery and the targeted analysis phases. No sample fractionation or enrichment was required for the discovery phase which allowed this method to analyze minute amount of sample. We applied this pseudotargeted MS method to quantitatively examine phosphopeptides in affinity purified endogenous Shc1 protein complexes at four temporal stages of EGF signaling and identified 82 phospho-sites. To our knowledge, this is the highest number of phospho-sites identified from the protein complexes. This pseudotargeted MS method is highly sensitive in the identification of low abundance phosphopeptides and could be a powerful tool to study phosphorylation-regulated assembly of protein complex.