Selenium-based metabolic oligosaccharide engineering strategy for quantitative glycan detection.

Metabolic oligosaccharide engineering (MOE) is a classical chemical approach to perturb, profile and perceive glycans in physiological systems, but probes upon bioorthogonal reaction require accessibility and the background signal readout makes it challenging to achieve glycan quantification. Here we develop SeMOE, a selenium-based metabolic oligosaccharide engineering strategy that concisely combines elemental analysis and MOE,enabling the mass spectrometric imaging of glycome. We also demonstrate that the new-to-nature SeMOE probes allow for detection, quantitative measurement and visualization of glycans in diverse biological contexts. We also show that chemical reporters on conventional MOE can be integrated into a bifunctional SeMOE probe to provide multimodality signal readouts. SeMOE thus provides a convenient and simplified method to explore the glyco-world.

Quantifying Turnover Dynamics of Selenoproteome by Isotopic Perturbation.

Selenium, as an essential trace element of life, is closely related to human health and is required to produce selenoproteins, a family of important functional proteins in many living organisms. All selenoproteins contain a special amino acid, selenocysteine, which often serves as their active-site residue, and the expression and activity of selenoproteins are fine-tuned. However, the turnover dynamics of selenoproteome has never been systematically investigated, especially in a site-specific manner for selenocysteines. In the current work, we developed a chemical proteomic strategy named "SElenoprotein Turnover Rate by Isotope Perturbation (SETRIP)" to quantitatively monitor the turnover dynamics of selenoproteins at the proteomic level. The kinetic rates and half-lives of nine selenoproteins were accurately measured by combining Na274SeO3 metabolic labeling with pulse-chase chemoproteomics. The half-lives of selenoproteins were measured to range from 6 to 32 h with the housekeeping selenoprotein glutathione peroxidases (GPX4) showing a faster turnover rate, implying that the hierarchy regulation also exists in the turnover of selenoproteins in addition to expression and activity. Our study generated a global portrait of dynamic changes in the selenoproteome and provided important clues to study the roles of selenium in biology.

An accelerated and optimized algorithm of selenium-encoded isotopic signature targeted profiling for global selenoproteome analysis.

Selenoproteins play crucial roles including protection and recovery from oxidative stress in organisms. Direct profiling of selenoproteins in proteomes is challenging due to their extremely low abundance. We have developed a computational algorithm termed selenium-encoded isotopic signature targeted profiling (SESTAR) to increase the sensitivity of detecting selenoproteins in complex proteomic samples. In this chapter, we briefly described the basic algorithm of SESTAR. We then introduced SESTAR++, an updated version of SESTAR, with accelerated computation speed and lowered false positive rate. We also provided a detailed workflow to apply SESTAR++ to proteomic profiling of selenoproteins, including the instruction of running the software and implementing it in a targeted profiling mode.

Quantitative Chemoproteomic Profiling with Data-Independent Acquisition-Based Mass Spectrometry.

Activity-based protein profiling (ABPP) has emerged as a powerful and versatile tool to enable annotation of protein functions and discovery of targets of bioactive ligands in complex biological systems. It utilizes chemical probes to covalently label functional sites in proteins so that they can be enriched for mass spectrometry (MS)-based quantitative proteomics analysis. However, the semistochastic nature of data-dependent acquisition and high cost associated with isotopically encoded quantification reagents compromise the power of ABPP in multidimensional analysis and high-throughput screening, when a large number of samples need to be quantified in parallel. Here, we combine the data-independent acquisition (DIA) MS with ABPP to develop an efficient label-free quantitative chemical proteomic method, DIA-ABPP, with good reproducibility and high accuracy for high-throughput quantification. We demonstrated the power of DIA-ABPP for comprehensive profiling of functional cysteineome in three distinct applications, including dose-dependent quantification of cysteines' sensitivity toward a reactive metabolite, screening of ligandable cysteines with a covalent fragment library, and profiling of cysteinome fluctuation in circadian clock cycles. DIA-ABPP will open new opportunities for in-depth and multidimensional profiling of functional proteomes and interactions with bioactive small molecules in complex biological systems.

A Dimethyl-Labeling-Based Strategy for Site-Specifically Quantitative Chemical Proteomics.

Activity-based protein profiling (ABPP) has emerged as a powerful functional chemoproteomic strategy which enables global profiling of proteome reactivity toward bioactive small molecules in complex biological and/or pathological processes. To quantify the degree of reactivity in a site-specific manner, an isotopic tandem orthogonal proteolysis (isoTOP)-ABPP strategy has been developed; however, the high cost and long workflow associated with the synthesis of isotopically labeled cleavable tags limit its wide use. Herein, we combined reductive dimethyl labeling with TOP-ABPP to develop a fast, affordable, and efficient method, termed "rdTOP-ABPP", for quantitative chemical proteomics with site-specific precision and triplex quantification. The rdTOP-ABPP method shows high accuracy and precision, good reproducibility, and better capacity for site identification and quantification and is highly compatible with many commercially available cleavable tags. We demonstrated the power of rdTOP-ABPP by profiling the target of (1 S,3 R)-RSL3, a canonical inducer for cell ferroptosis, and provided the first global portrait of its proteome reactivity in a quantitative and site-specific manner.