We invented a new method for highly efficient and specific enrichment of glycopeptides using two different nanomaterials synergistically. One is boronic-acid-functionalized Fe3O4 nanoparticles, enriching glycopeptides through formation of cyclic boronate esters between the boronic acid groups and the cis-diol groups on glycopeptides. The other nanomaterial is conventional poly(methyl methacrylate) nanobeads, which have strong adsorption toward nonglycopeptides. By optimizing the proportion of these two materials, extremely high sensitivity and selectivity are achieved in analyzing the standard glycopeptides/nonglycopeptides mixture solutions. Since the washing step is not necessary for these conditions, the enrichment process is simplified and the recovery efficiency of target glycopeptides reaches 90%. Finally, this approach is successfully applied to analyze human serum with the sample volume as little as 1 μL, in which 147 different N-glycosylation peptides within 66 unique glycoproteins are identified. All these performances by the synergistic enrichment are much better than employing one specific enrichment agent alone.

C-termini of proteins often play an important role in various biological processes. The determination of the protein C-terminus is crucial because it provides not only distinct functional annotation but also a way to monitor the proteolysis-modified proteins. In this study, an isotopic labeling approach based on oxazolone chemistry was developed to achieve the identification and quantification of C-termini. Aminolysis reagents such as arginine selectively react with the α-carboxyl group at the peptide C-terminus via an oxazolone-like intermediate. Side chain carboxyl groups do not participate in this reaction. When an isotopic mixture consisting of 50% arginine ((0)Arg) and 50% C6-arginine ((6)Arg) was introduced to react with C-terminus of protein and followed by proteolysis, the C-terminal peptide could be directly recognized in the mass spectrum due to its unique isotopic paired peaks, and the sequence could be interpreted in MS2. Besides, the incorporation of an additional basic amino acid in the C-terminal peptide greatly enhanced the signal intensity for C-termini detection. Moreover, the isotopic arginine labeling strategy could be applied for relative C-termini quantitation. Our method showed an excellent correlation of the measured ratios to theoretical ratios and high reproducibility within 2 orders of magnitude of the dynamic range. The correlation coefficients (R(2)) were higher than 0.99, with the coefficients of variation (CVs) ranging from 1.16 to 10.91%. Finally, the approach was used to analyze the C-termini from Thermoanaerobacter tengcongensis , which was cultured under different temperatures. As a result, 68 C-termini have been identified, and 53 of them were quantified in total using our strategy. In addition, 24 neo-C-terminal peptides have also been discovered.

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Selective capture of protein C-termini is still challenging in view of the lower reactivity of the carboxyl group relative to amino groups and difficulties in site-specifically labeling the carboxyl group on the C-terminus rather than that on the side chains of acidic amino acids. For highly efficient purification of C-terminus peptides, a novel positive enrichment approach based on the oxazolone chemistry has been developed in this study. A bifunctional group reagent containing biotin and arginine was incorporated into the C-terminus of protein. Together with a streptavidin affinity strategy, the C-terminal peptides could be readily purified and analyzed by mass spectrometry (MS). Unlike the negative enrichment approach, C-terminal peptides, other than non-C-terminal peptides, were captured directly from the peptide mixture in this new method. The labeling efficiency (higher than 90%), enrichment selectivity (purifying C-terminal peptides from mixtures of non-C-terminal peptides at a 1:50 molar ratio), and ionization efficiencies in MS were dramatically improved. Moreover, the highly efficient identification of C-terminal peptides was further achieved by defining biotin as the 21st amino acid and optimizing the database search strategy. We have successfully identified 183 C-terminal peptides from Thermoanaerobacter tengcongensis using this creative method, which affords a highly selective and efficient purification approach for C-terminomics study.

Aims: Evidence linking serum uric acid (sUA) and bone mineral density (BMD) in adolescents is very limited. To the best of our knowledge, only one report has focused on the relationship between sUA and BMD in adolescents. Thus, this study aimed to determine the association between sUA and total BMD in adolescents aged 12-19 years.Methods: A cross-sectional study was conducted on a sample composed of non-institutionalized US population from the National Health and Nutrition Examination Survey. Weighted multivariate linear regression models were used to evaluate the association between sUA and total BMD. Subgroup analyses were further performed.Results: sUA positively correlated with total BMD in the multiple regression model after adjusting for potential confounders. However, in the subgroup analyses stratified by sex, age, or race/ethnicity, the association between sUA and total BMD followed an inverted U-shaped curve in female adolescents, adolescents aged 16-19 years, and other race/ethnicity. Conclusions: Our results suggested that the correlation between sUA level and total BMD differed by sex. The increased sUA level would be beneficial to bone health in adolescents with low sUA levels, but for female adolescents, a higher sUA level (turning point, 3.9 mg/dL) may have an adverse effect on bone health.