Reversible Lysine Derivatization Enabling Improved Arg‑C Digestion, a Highly Specific Arg‑C Digestion Using Trypsin

The bottom-up proteomics approach has become an important strategy in diverse areas of biological research, and the enzymatic digestion is essential for this technology. Endopeptidase Arg-C catalyzing the hydrolytic cleavage of peptide bonds C-terminal to arginine could be an important protease in bottom-up proteomics. However, it has been seldom applied due to its low specificity and high cost. In this report, the reversible amine derivatization method (citraconylation and decitraconylation) was introduced and optimized toward a real Arg-C digestion using trypsin. Combination of the reversible derivatization and trypsin digestion (termed iArg-C digestion for improved Arg-C digestion) resulted in 64.2% more peptide identification (11 925 ± 199 vs 7262 ± 59) and significantly higher cleavage specificity (95.6% vs 73.6%) than the conventional Arg-C digestion. Comparison of iArg-C digestion with the widely used trypsin and Lys-C digestion revealed that iArg-C performed slightly better than Lys-C although not comparable to trypsin. Therefore, the well-established iArg-C digestion method is a promising approach for proteomics studies and could be used as the prior alternative digestion method to trypsin digestion in order to achieve higher proteome coverage. Data are available via ProteomeXchange with identifier PXD007994

Enzyme and Chemical Assisted N‑Terminal Blocked Peptides Analysis, ENCHANT, as a Selective Proteomics Approach Complementary to Conventional Shotgun Approach

Shotgun (bottom-up) approach has been widely applied in large-scale proteomics studies. The inherent shortages of shotgun approach lie in that the generated peptides often overwhelm the analytical capacity of current LC–MS/MS systems and that high-abundance proteins often hamper the identification of low-abundance proteins when analyzing complex samples. To reduce the sample complexity and relieve the problems caused by abundant proteins, herein we introduce a modified selective proteomics approach, termed ENCHANT, for enzyme and chemical assisted N-terminal blocked peptides analysis. Modified from our previous Nα-acetylome approach, ENCHANT aims to analyze three kinds of peptides, acetylated protein N-termini, N-terminal glutamine and N-terminal cysteine containing peptides. Application of ENCHANT to HeLa cells allowed to identify 3375 proteins, 19.6% more than that by conventional shotgun approach. More importantly, ENCHANT demonstrated an excellent complementarity to conventional shotgun approach with the overlap of 34.5%. In terms of quantification using data independent acquisition (DIA) technology, ENCHANT quantified 23.9% more proteins than conventional shotgun approach with the overlap of 27.6%. Therefore, our results strongly suggest that ENCHANT is a promising selective proteomics approach, which is complementary to conventional shotgun approach in both qualitative and quantitative proteomics studies. Data are available via ProteomeXchange with identifier PXD007863

Lys-C/Arg-C, a More Specific and Efficient Digestion Approach for Proteomics Studies

Nowadays, bottom-up approaches are predominantly adopted in proteomics studies, which necessitate a proteolysis step prior to MS analysis. Trypsin is often the best protease in choice due to its high specificity and MS-favored proteolytic products. A lot of efforts have been made to develop a superior digestion approach but hardly succeed, especially in large-scale proteomics studies. Herein, we report a new tandem digestion using Lys-C and Arg-C, termed Lys-C/Arg-C, which has been proven to be more specific and efficient than trypsin digestion. Reanalysis of our previous data (<i>Anal. Chem.</i> <b>2018</b>, <i>90</i> (3), 1554–1559) revealed that both Lys-C and Arg-C are trypsin-like proteases and perform better when considered as trypsin. In particular, for Arg-C, the identification capacity is increased to 2.6 times and even comparable with trypsin. The good complementarity, high digestion efficiency, and high specificity of Lys-C and Arg-C prompt the Lys-C/Arg-C digestion. We systematically evaluated Lys-C/Arg-C digestion using qualitative and quantitative proteomics approaches and confirmed its superior performance in digestion specificity, efficiency, and identification capacity to the currently widely used trypsin and Lys-C/trypsin digestions. As a result, we concluded that the Lys-C/Arg-C digestion approach would be the choice of next-generation digestion approach in both qualitative and quantitative proteomics studies. Data are available via ProteomeXchange with identifier PXD009797

Enhancing the Power of Liquid Chromatography–Mass Spectrometry-Based Urine Metabolomics in Negative Ion Mode by Optimization of the Additive

Untargeted liquid chromatography–mass spectrometry (LC-MS)-based metabolomics studies are usually carried out in both positive and negative ion modes; however, it is frequently ignored that the optimal conditions in positive ion mode and negative ion mode are often not the same. We carried out a systematic investigation on urine samples to evaluate the additive effects in negative ion mode. It was found that the widely used conditions, 0.1% formic acid (FA) and NH₄Ac at different pH, are far from the optimum for untargeted urine metabolomics studies. Compared to 0.1% FA, the use of 1 mM acetic acid (HAc) resulted in almost three times as many detected peaks (401 vs 148) and around five times the size of the peak area (33.55 × 10⁶ vs 6.47 × 10⁶). The remarkable improvement can be explained by two factors: (i) a significantly enhanced ionization efficiency due to the combination of an appropriate pH at around 4.0–4.5, the reducibility of H⁺, and the high gas-phase basicity of Ac^– and (ii) a reproducible LC separation due to an acceptable buffering capacity. Our study revealed the importance and necessity of additive optimization, which can be of benefit in related metabolomics studies

Enhancing the Power of Liquid Chromatography–Mass Spectrometry-Based Urine Metabolomics in Negative Ion Mode by Optimization of the Additive

Untargeted liquid chromatography–mass spectrometry (LC-MS)-based metabolomics studies are usually carried out in both positive and negative ion modes; however, it is frequently ignored that the optimal conditions in positive ion mode and negative ion mode are often not the same. We carried out a systematic investigation on urine samples to evaluate the additive effects in negative ion mode. It was found that the widely used conditions, 0.1% formic acid (FA) and NH₄Ac at different pH, are far from the optimum for untargeted urine metabolomics studies. Compared to 0.1% FA, the use of 1 mM acetic acid (HAc) resulted in almost three times as many detected peaks (401 vs 148) and around five times the size of the peak area (33.55 × 10⁶ vs 6.47 × 10⁶). The remarkable improvement can be explained by two factors: (i) a significantly enhanced ionization efficiency due to the combination of an appropriate pH at around 4.0–4.5, the reducibility of H⁺, and the high gas-phase basicity of Ac^– and (ii) a reproducible LC separation due to an acceptable buffering capacity. Our study revealed the importance and necessity of additive optimization, which can be of benefit in related metabolomics studies

Systematic Optimization of C‑Terminal Amine-Based Isotope Labeling of Substrates Approach for Deep Screening of C-Terminome

It is well-known that protein C-termini play important roles in various biological processes, and thus the precise characterization of C-termini is essential for fully elucidating protein structures and understanding protein functions. Although many efforts have been made in the field during the latest 2 decades, the progress is still far behind its counterpart, N-termini, and it necessitates more novel or optimized methods. Herein, we report an optimized C-termini identification approach based on the C-terminal amine-based isotope labeling of substrates (C-TAILS) method. We optimized the amidation reaction conditions to achieve higher yield of fully amidated product. We evaluated different carboxyl and amine blocking reagents and found the superior performance of Ac-NHS and ethanolamine. Replacement of dimethylation with acetylation for Lys blocking resulted in the identification of 232 C-terminal peptides in an <i>Escherichia coli</i> sample, about 42% higher than the conventional C-TAILS. A systematic data analysis revealed that the optimized method is unbiased to the number of lysine in peptides, more reproducible and with higher MASCOT scores. Moreover, the introduction of the Single-Charge Ion Inclusion (SCII) method to alleviate the charge deficiency of small peptides allowed an additional 26% increase in identification number. With the optimized method, we identified 481 C-terminal peptides corresponding to 369 C-termini in <i>E. coli</i> in a triplicate experiments using 80 μg each. Our optimized method would benefit the deep screening of C-terminome and possibly help discover some novel C-terminal modifications. Data are available via ProteomeXchange with identifier PXD002409

Intake of Hydrolyzed Casein is Associated with Reduced Body Fat Accretion and Enhanced Phase II Metabolism in Obesity Prone C57BL/6J Mice

<div><p>The amount and form of dietary casein have been shown to affect energy metabolism and lipid accumulation in mice, but the underlying mechanisms are not fully understood. We investigated 48 hrs urinary metabolome, hepatic lipid composition and gene expression in male C57BL/6J mice fed Western diets with 16 or 32 energy% protein in the form of extensively hydrolyzed or intact casein. LC-MS based metabolomics revealed a very strong impact of casein form on the urinary metabolome. Evaluation of the discriminatory metabolites using tandem mass spectrometry indicated that intake of extensively hydrolyzed casein modulated Phase II metabolism associated with an elevated urinary excretion of glucuronic acid- and sulphate conjugated molecules, whereas glycine conjugated molecules were more abundant in urine from mice fed the intact casein diets. Despite the differences in the urinary metabolome, we observed no differences in hepatic expression of genes involved in Phase II metabolism, but it was observed that expression of <i>Abcc3</i> encoding ATP binding cassette c3 (transporter of glucuronic acid conjugates) was increased in livers of mice fed hydrolyzed casein. As glucuronic acid is derived from glucose and sulphate is derived from cysteine, our metabolomic data provided evidence for changes in carbohydrate and amino acid metabolism and we propose that this modulation of metabolism was associated with the reduced glucose and lipid levels observed in mice fed the extensively hydrolyzed casein diets.</p></div