An RNA tagging approach for system-wide RNA-binding proteome profiling and dynamics investigation upon transcription inhibition.

RNA-protein interactions play key roles in epigenetic, transcriptional and posttranscriptional regulation. To reveal the regulatory mechanisms of these interactions, global investigation of RNA-binding proteins (RBPs) and monitor their changes under various physiological conditions are needed. Herein, we developed a psoralen probe (PP)-based method for RNA tagging and ribonucleic-protein complex (RNP) enrichment. Isolation of both coding and noncoding RNAs and mapping of 2986 RBPs including 782 unknown candidate RBPs from HeLa cells was achieved by PP enrichment, RNA-sequencing and mass spectrometry analysis. The dynamics study of RNPs by PP enrichment after the inhibition of RNA synthesis provides the first large-scale distribution profile of RBPs bound to RNAs with different decay rates. Furthermore, the remarkably greater decreases in the abundance of the RBPs obtained by PP-enrichment than by global proteome profiling suggest that PP enrichment after transcription inhibition offers a valuable way for large-scale evaluation of the candidate RBPs.

An Integrated Strategy for Mass Spectrometry-Based Multiomics Analysis of Single Cells.

Single-cell-based genomics and transcriptomics analysis have revealed substantial cellular heterogeneity among seemingly identical cells. Knowledge of the cellular heterogeneity at multiomics levels is vital for a better understanding of tumor metastasis and drug resistance, stem cell differentiation, and embryonic development. However, unlike genomics and transcriptomics studies, single-cell characterization of metabolites, proteins, and post-translational modifications at the omics level remains challenging due to the lack of amplification methods and the wide diversity of these biomolecules. Therefore, new tools that are capable of investigating these unamplifiable "omes" from the same single cells are in high demand. In this work, a microwell chip was prepared and the internal surface was modified for hydrophilic interaction liquid chromatography-based tandem extraction of metabolites and proteins and subsequent protein digestion. Next, direct electrospray ionization mass spectrometry was adopted for single-cell metabolome identification, and a data-independent acquisition-mass spectrometry approach was established for simultaneous proteome profiling and phosphoproteome analysis without phosphopeptide enrichment. This integrated strategy resulted in 132 putatively annotated compounds, more than 1200 proteins, and the first large-scale phosphorylation data set from single-cell analysis. Application of this strategy in chemical perturbation studies provides a multiomics view of cellular changes, demonstrating its capability for more comprehensive investigation of cellular heterogeneity.

Synthesis of a Highly Azide-Reactive and Thermosensitive Biofunctional Reagent for Efficient Enrichment and Large-Scale Identification of O-GlcNAc Proteins by Mass Spectrometry.

O-linked ß-N-acetylglucosamine (O-GlcNAc) is a ubiquitous post-translational modification of proteins in eukaryotic cells. Despite their low abundance, O-GlcNAc-modified proteins play many important roles in regulating gene expression, signal transduction, and cell cycle. Aberrant O-GlcNAc proteins are correlated with many major human diseases, such as Alzheimer's disease, diabetes, and cancer. Because of the extremely low stoichiometry of O-GlcNAc proteins, enrichment is required before mass spectrometry analysis for large-scale identification and in-depth understanding of their cellular function. In this work, we designed and synthesized a novel thermosensitive immobilized triarylphosphine reagent as a convenient tool for efficient enrichment of azide-labeled O-GlcNAc proteins from complex biological samples. Immobilization of triarylphosphine on highly water-soluble thermosensitive polymer largely increases its solubility and reactivity in aqueous solution. As a result, facilitated coupling is achieved between triarylphosphine and azide-labeled O-GlcNAc proteins via Staudinger ligation, due to the increased triarylphosphine concentration, reduced interfacial mass transfer resistance, and steric hindrance in homogeneous reaction. Furthermore, solubility of the polymer from complete dissolution to full precipitation can be easily controlled by simply adjusting the environmental temperature. Therefore, facile sample recovery can be achieved by increasing the temperature to precipitate the polymer-O-GlcNAc protein conjugates from solution. This novel immobilized triarylphosphine reagent enables efficient enrichment and sensitive detection of more than 1700 potential O-GlcNAc proteins from HeLa cell using mass spectrometry, demonstrating its potential as a general strategy for low-abundance target enrichment.

[Large-scale enrichment and identification of human urinary N-glycoproteins/N-glycopeptides].

N-Glycosylation of proteins, an important post-translational modification in eukaryotic cells, plays an essential role in the regulation of cell adhesion, migration, signal transduction, and apoptosis. Abnormal changes in protein glycosylation are closely related to the occurrence of many critical diseases, including diabetes, tumors, and neurological, kidney, and inflammatory diseases. A non-invasive type of liquid biopsy, urine sampling has the advantage of reducing the complexity of proteomic analysis. This facilitates the design of large-scale and continuous or multi-time point sampling strategies. However, the dynamic range of urinary protein abundance is relatively large, owing to individual differences and physiological conditions. Currently, there is a lack of specialized research on individual differences, physiological fluctuations, and physiological abundance ranges of urinary N-glycoproteins in large healthy populations. Therefore, it is difficult to accurately distinguish individual differences and normal physiological fluctuations from changes caused by disease; this poses a great challenge in disease marker research. Liquid chromatography-mass spectrometry (LC-MS) is an analytical technique widely used for the large-scale profiling of proteomes in biological systems, and the enrichment of N-glycopeptides is a prerequisite for their detection by MS.In this study, we established an approach based on hydrophilic interaction chromatography (HILIC) by optimizing the activation, cleaning, and elution processes of the enrichment method, for instance through the optimization of particle size and solvent composition, and investigated the identification number, selectivity, and stability of N-glycoprotein/N-glycopeptide enrichment under different experimental conditions. We found that N-glycoproteins and N-glycopeptides were highly enriched in a trifluoroacetic acid system with 5-µm filling particles in the HILIC column. On this basis, we analyzed the levels of N-glycoproteins/N-glycopeptides in urine samples. The consistency of N-glycoprotein/N-glycopeptide levels in urine samples taken from the same healthy person for five consecutive days was investigated by correlation analysis. This analysis revealed that the urinary N-glycoproteome of the same healthy person was relatively stable over a short period of time. Next, urinary samples from 20 healthy male volunteers and 20 healthy female volunteers were enriched for N-glycoproteins/N-glycopeptides, which were profiled by MS through qualitative and quantitative analyses. Screening and functional analysis of differential proteins were then carried out. A total of 1016 N-glycoproteins and 2192 N-glycopeptides were identified in the mid-morning urine samples of the 40 healthy volunteers. A label-free quantitation strategy was used to investigate the fluctuation range of the physiologically abundant urinary N-glycopeptides. The abundance of urinary N-glycopeptides spanned across approximately five orders of magnitude. Subsequently, gender differences in the N-glycosylation levels of urinary proteins were also explored in healthy people. Functional analysis of the N-glycoproteins that exhibited gender differences in abundance was performed. Based on multivariate statistical analysis, 206 differentially expressed proteins (p<0.05, fold change (FC)> 4) were identified. In females, we found 175 significantly down-regulated N-glycoproteins and 31 significantly up-regulated N-glycoproteins with respect to males. The expression levels of N-glycopeptides between the two groups suggested a clear gender difference. To investigate the biological processes and functions of these proteins, gene ontology (GO) analysis was performed on the N-glycoproteins/N-glycopeptides differentially expressed between males and females. Metabolic pathway analysis was also carried out based on the kyoto encyclopedia of genes and genomes (KEGG). Differentially expressed N-glycoproteins were mostly associated with platelet degranulation, extracellular region, and ossification. The top three relevant pathways were glycan biosynthesis and metabolism, metabolism of cofactors and vitamins, and lipid metabolism. Overall, sex may be an important factor for urinary N-glycoproteome differences among normal individuals and should be considered in clinical applications. This study provides relevant information regarding the function and mechanisms of the urinary glycoproteome and the screening of clinical biomarkers.

A triarylphosphine-trimethylpiperidine reagent for the one-step derivatization and enrichment of protein post-translational modifications and identification by mass spectrometry.

We report a new reagent that is capable of both chemical derivatization and selective enrichment of azide-labeled PTM peptides for sensitive identification by mass spectrometry (MS). Facile sample recovery, enhanced ionization and fragmentation in MS of the enriched PTM peptides are achieved, which leads to the identification of 3293 O-GlcNAc peptides and the location of 1706 sites in HeLa cells and efficiently expands the current mapping scale.

Development a hydrazide-functionalized thermosensitive polymer based homogeneous system for highly efficient N-glycoprotein/glycopeptide enrichment from human plasma exosome.

As one of the most common post-translational modifications, protein N-glycosylation precipitates in many important biological processes and has closely correlations with the occurrence and progression of multiple diseases. Plasma exosomes secreted by cells contain various bioactive N-glycoproteins which may serve as potential biomarkers for early disease diagnosis and treatment. However, the protein N-glycosylation profile in human plasma exosome is largely unknown, due to the technical challenges in glycoprotein identification. Signals of the rare N-glycoproteins/N-glycopeptides are severely suppressed by the abundant coexisting non-glycosylated counterparts in mass spectrometry analysis. Therefore, specific enrichment of N-glycoprotein/glycopeptide is a prerequisite for large scale N-glycosylation profiling. In this work, we developed a hydrazide functionalized thermosensitive polymer for efficient enrichment and in-depth identification of protein N-glycosylation in human plasma exosome by mass spectrometry. The polymer chains completely dissolve in the enrichment system to form a homogeneous solution. Therefore, efficient covalent coupling between the N-glycoprotein/glycopeptide and the polymer chain is achieved, due to the reduced interfacial mass transfer resistance and the densely packed accessible functional groups on the polymer chains. Furthermore, the thermosensitive polymer can be easily precipitated and recovered by simply rising the system temperature to above 34 °C. As a result, 329 N-glycosylation sites corresponding to 180 N-glycoproteins were enriched and identified from plasma exosomes of glioma patients and healthy subjects using the thermosensitive polymer. By quantitative comparison, we found 26 N-glycoproteins significantly changed between the glioma patients and the healthy subjects, demonstrating the potential of this new strategy for N-glycoproteome research of plasma exosome and biomarker discovery.

A fast sample processing strategy for large-scale profiling of human urine phosphoproteome by mass spectrometry.

Liquid biopsies using body fluids have gained much attention in recent years due to their multiple advantages in clinical diagnosis, such as less/non-invasive collection, suitability for longitudinal disease monitoring, and better representation of tumor heterogeneity. As an attractive choice for liquid biopsy, urine proteins and their post-translational modifications (PTMs) have the potential to offer significant insights into physiological variations and pathological changes in the human body. However, due to the intrinsically large variability of urine proteins and their PTMs among different individuals, there is a high demand for strategies for high-throughput analysis of a large number of samples to obtain a comprehensive view and a reliable reference interval of the urine proteome. In this work, we proposed a new urine phosphoproteome sample processing strategy that combines fast protein extraction, efficient multiple immobilized-proteases digestion, and tandemly connected centrifugal tips device-based facile phosphopeptide enrichment & fractionation. This strategy is capable of paralleled sample processing with an approximate five-fold reduction in processing time and is therefore particularly suitable for handling a large number of urine samples. Totally, we identified 4196 phosphosites in human urine proteins by mass spectrometry in replicated tests, a number which is dozens of times larger than those previously reported. Therefore, this strategy may have great potential in urine-based phosphoprotein biomarker screening and drug response studies.