Lysine 2-hydroxyisobutyrylation (K hib) is a novel posttranslational modification (PTM), which was thought to play a role in active gene transcription and cellular proliferation. Here we report a comprehensive identification of K hib in Proteus mirabilis (P. mirabilis). By combining affinity enrichment with two-dimensional liquid chromatography and high-resolution mass spectrometry, 4735 2-hydroxyisobutyrylation sites were identified on 1051 proteins in P. mirabilis. These proteins bearing modifications were further characterized in abundance, distribution and functions. The interaction networks and domain architectures of these proteins with high confidence were revealed using bioinformatic tools. Our data demonstrate that many 2-hydroxyisobutyrylated proteins are involved in metabolic pathways, such as purine metabolism, pentose phosphate pathway and glycolysis/gluconeogenesis. The extensive distribution of K hib also indicates that the modification may play important influence to bacterial metabolism. The speculation is further supported by the observation that carbon sources can influence the occurrence of K hib. Furthermore, we demonstrate that 2-hy-droxyisobutyrylation on K343 was a negative regulatory modification on Enolase (ENO) activity, and molecular docking results indicate the regulatory mechanism that K hib may change the binding formation of ENO and its substrate 2-phospho-D-glycerate (2PG) and cause the substrate far from the active sites of enzyme. We hope this first comprehensive analysis of nonhistone K hib in prokaryotes is valuable for further functional investigation of this modification.

The development of methods to isolate and enrich low-abundance glycopeptides from biological samples is crucial to glycoproteomics. Herein, we present an easy and one-step surface modification strategy to prepare hydrophilic maltose functionalized Fe3O4 nanoparticles (NPs). First, based on the chelation of the catechol ligand with iron atoms, azido-terminated dopamine (DA) derivative was assembled on the surface of magnetic Fe3O4 nanoparticles by sonication. Second, the hydrophilic maltose-functionalized Fe3O4 (Fe3O4-DA-Maltose) NPs were obtained via copper(I)-catalyzed azide-alkyne cycloaddition (click chemistry). The morphology, structure, and composition of Fe3O4-DA-Maltose NPs were investigated by Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), X-ray powder diffraction (XRD), X-ray photoelectron spectrometer (XPS), and vibrating sample magnetometer (VSM). Meanwhile, hydrophilicity of the obtained NPs was evaluated by water contact angle measurement. The hydrophilic Fe3O4-DA-Maltose NPs were applied in isolation and enrichment of glycopeptides from horseradish peroxidase (HRP), immunoglobulin (IgG) digests. The MALDI-TOF mass spectrometric analysis indicated that the novel NPs exhibited high detection sensitivity in enrichment from HRP digests at concentration as low as 0.05 ng μL(-1), a large binding capacity up to 43 mg g(-1), and good recovery for glycopeptides enrichment (85-110%). Moreover, the Fe3O4-DA-Maltose NPs were applied to enrich glycopeptides from human renal mesangial cells (HRMC) for identification of N-glycosylation sites. Finally, we identified 115 different N-linked glycopeptides, representing 93 gene products and 124 glycosylation sites in HRMC.

Efficient enrichment glycoproteins/glycopeptides from complex biological solutions are very important in the biomedical sciences, in particular biomarker research. In this work, the high hydrophilic polyethylenimine conjugated polymaltose polymer brushes functionalized magnetic Fe3O4 nanoparticles (NPs) denoted as Fe3O4–PEI–pMaltose were designed and synthesized via a simple two-step modification. The obtained superhydrophilic Fe3O4–PEI–pMaltose NPs displayed outstanding advantages in the enrichment of N-linked glycopeptides, including high selectivity (1:100, mass ratios of HRP and bovine serum albumin (BSA) digest), low detection limit (10 fmol), large binding capacity (200 mg/g), and high enrichment recovery (above 85%). The above-mentioned excellent performance of novel Fe3O4–PEI–pMaltose NPs was attributed to graft of maltose polymer brushes and efficient assembly strategy. Moreover, Fe3O4–PEI–pMaltose NPs were further utilized to selectively enrich glycopeptides from human renal mesangial cell (HRMC, 200 μg) tryptic digest, and 449 N-linked glycopeptides, representing 323 different glycoproteins and 476 glycosylation sites, were identified. It was expected that the as-synthesized Fe3O4–PEI–pMaltose NPs, possessing excellent performance (high binding capacity, good selectivity, low detection limit, high enrichment recovery, and easy magnetic separation) coupled to a facile preparation procedure, have a huge potential in N-glycosylation proteome analysis of complex biological samples.

A high level of uric acid may cause hyperuricemia, which further develops into gout, eventually leading to chronic kidney disease. However, the pathogenic mechanism remains largely unknown. To investigate the cause and block the transformation of hyperuricemia to related diseases, it is important to discover the alterations in protein levels between gout patients and non-gout individuals. To date, human blood plasma is still the predominant matrices for clinical analysis. Due to the high abundance, the proteins of plasma samples have strong shielding effects on low abundance proteins, thus, the information on low abundance protein expression is always masked, while the low abundance proteins of human plasma are often of great significance for the diagnosis and treatment of diseases. Therefore, it is very important to separate and analyze the plasma proteins. High-performance liquid chromatography (LC) tandem mass spectrometry (MS)-based proteomics has been developed as a powerful tool to investigate changes in the human plasma proteome. Here, we used LC-MS/MS to detect the differential proteins in the plasmas from simple gout patients, gout with kidney damage patients, and non-gout individuals. We identified 32 obviously differential proteins between non-gout and gout subjects and 10 differential proteins between simple gout and gout with kidney damage patients. These differential proteins were further analyzed to characterize their localization and functions. Additionally, the correlation analysis showed multiple relationships between the abnormal plasma proteins and clinical biochemical indexes, particularly for the immune-inflammatory response proteins. Furthermore, inflammation factors gelsolin (GSN) were confirmed. Our results offer a view of plasma proteins for studying biomarkers of gout patients.

No abstract