Hydrophobic Tagging-Assisted N‑Termini Enrichment for In-Depth N‑Terminome Analysis

The analysis of protein N-termini is of great importance for understanding the protein function and elucidating the proteolytic processing. Herein, we develop a negative enrichment strategy, termed as hydrophobic tagging-assisted N-termini enrichment (HYTANE) to achieve a global N-terminome analysis. The HYTANE strategy showed a high efficiency in hydrophobic tagging and C18 material-assisted depletion using bovine serum albumin (BSA) as the sample. This strategy was applied to N-termini profiling from <i>S. cerevisiae</i> cell lysates and enabled the identification of 1096 protein N-termini, representing the largest N-terminome data set of <i>S. cerevisiae</i>. The identified N-terminal peptides accounted for 99% of all identified peptides, and no deficiency in acidic, histidine (His)-containing, and His-free N-terminal peptides was observed. The presented HYTANE strategy is therefore a highly selective, efficient, and unbiased strategy for the large scale N-terminome analysis. Furthermore, using the HYTANE strategy, we identified 329 cleavage sites and 291 substrates of caspases in Jurkat cells, demonstrating the great promise of HYTANE strategy for protease research. Data are available via ProteomeXchange with identifier PXD004690

Enzymatic Reactor with Trypsin Immobilized on Graphene Oxide Modified Polymer Microspheres To Achieve Automated Proteome Quantification

Protein digestion and isotope labeling are two critical steps in proteome quantification. However, the conventional in-solution protocol unavoidably suffers from disadvantages such as time-consuming, low labeling efficiency, and tedious off-line manual operation, which might affect the quantification accuracy, reproducibility, and throughput. To address these problems, we developed a fully automated proteome quantification platform, in which an ultraperformance immobilized microreactor (upIMER) with graphene-oxide-modified polymer microspheres as the matrix was developed, to achieve not only the simultaneous protein digestion and ¹⁸O labeling, but also the online integration with nano-high-pressure liquid chromatography–electrospray ionization-tandem mass spectrometry (nanoHPLC–ESI-MS/MS). Compared to the conventional off-line protocols, such a platform exhibits obviously improved digestion and ¹⁸O labeling efficiency (only 8% peptides with missed cleavage sites, 99% labeling efficiency, and 2.5 min reaction time), leading to the increased quantification coverage, accuracy, precision and throughput. All the results demonstrated that our developed fully automated platform should provide new opportunities to improve the accuracy, reproducibility, and throughput for proteome quantification

Gold-Coated Nanoelectrospray Emitters Fabricated by Gravity-Assisted Etching Self-Termination and Electroless Deposition

To improve the stability and sensitivity of nanoelectrospray for liquid chromatography-mass spectroscopy (LC-MS) analysis, we present a new method to fabricate gold-coated emitters. Via gravity-assisted etching self-termination, the emitter with a tapered outer surface and a straight inner surface is prepared with good reproducibility, without the need of fluid introduced to protect internal surface during etching. Followed by electroless deposition, the emitter is further coated with gold film homogeneously, by which the relative standard deviation (RSD) value of total ion current in 160 h is <5%, showing good stability. Compared to that obtained by a commercial emitter, the identified protein number from 2 μg HeLa cell digests is increased over 10%, contributed by the stable electrospray and improved signal intensity of peptides. Furthermore, the integrated gold-coated emitter is prepared at the end of the ultranarrow-bore packed column (inner diameter of 25 μm), and 218 proteins are identified from 2 ng HeLa cell digests. All of these results demonstrate the great promise of such emitters for use in ultrasensitive proteome analysis

In-Depth Proteomic Quantification of Cell Secretome in Serum-Containing Conditioned Medium

Secreted proteins play key roles during cellular communication, proliferation, and migration. The comprehensive profiling of secreted proteins in serum-containing culture media is technically challenging. Most studies have been performed under serum-free conditions. However, these conditions might alter the status of the cells. Herein, we describe an efficient strategy that avoids the disturbance of serum by combining metabolic labeling, protein “equalization,” protein fractionation, and filter-aided sample preparation, called MLEFF, enabling the identification of 534 secreted proteins from HeLa conditioned media, including 31 cytokines, and growth factors. This MLEFF strategy was also successfully applied during a comparative secretome analysis of two human hepatocellular carcinoma cell lines with differentially metastatic potentials, enabling the quantification of 61 significantly changed proteins involved in tumor invasion and metastasis

In-Depth Proteomic Quantification of Cell Secretome in Serum-Containing Conditioned Medium

Secreted proteins play key roles during cellular communication, proliferation, and migration. The comprehensive profiling of secreted proteins in serum-containing culture media is technically challenging. Most studies have been performed under serum-free conditions. However, these conditions might alter the status of the cells. Herein, we describe an efficient strategy that avoids the disturbance of serum by combining metabolic labeling, protein “equalization,” protein fractionation, and filter-aided sample preparation, called MLEFF, enabling the identification of 534 secreted proteins from HeLa conditioned media, including 31 cytokines, and growth factors. This MLEFF strategy was also successfully applied during a comparative secretome analysis of two human hepatocellular carcinoma cell lines with differentially metastatic potentials, enabling the quantification of 61 significantly changed proteins involved in tumor invasion and metastasis

3‑Carboxybenzoboroxole Functionalized Polyethylenimine Modified Magnetic Graphene Oxide Nanocomposites for Human Plasma Glycoproteins Enrichment under Physiological Conditions

Boronate affinity materials have been successfully used for the selective recognition of glycoproteins. However, by such materials, the large-scale glycoproteins enrichment from human plasma under physiological conditions is rarely reported. In this work, 3-carboxybenzoboroxole (CBX) functionalized polyethylenimine (PEI) modified magnetic graphene oxide nanocomposites were synthesized. Benefitting from the low p<i>K</i>_a value of CBX (∼6.9) and PEI dendrimer-assisted multivalent binding, the Freundlich constant (<i>K</i>_F) for the adsorption of horseradish peroxidase (HRP) was 3.0–7.3 times higher than that obtained by previous work, displaying the high enrichment capacity. Moreover, PEI could improve the hydrophilicity of nanocomposites and reduce nonglycoprotein adsorption. Therefore, such nanocomposites were successfully applied to the analysis of human plasma glycoproteome under physiological conditions, and the identified glycoproteins number and recognition selectivity was increased when compared to the results obtained by previous boronic acid-functionalized particles (Sil@Poly­(APBA-<i>co</i>-MBAAm)) under common alkaline condition (137 vs 78 and 67.8% vs 57.8%, respectively). In addition, thrombin (F2), an important plasma glycoprotein, labile under alkaline conditions, was specifically identified by our method, demonstrating the great promise of such nanocomposites in the deep-coverage glycoproteome analysis