Systematic Identification of Lysine 2-hydroxyisobutyrylated Proteins in Proteus mirabilis.

Lysine 2-hydroxyisobutyrylation (Khib) is a novel post-translational modification (PTM), which was thought to play a role in active gene transcription and cellular proliferation. Here we report a comprehensive identification of Khib 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 Khib 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 Khib Furthermore, we demonstrate that 2-hydroxyisobutyrylation on K343 was a negative regulatory modification on Enolase (ENO) activity, and molecular docking results indicate the regulatory mechanism that Khib 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 Khib in prokaryotes is valuable for further functional investigation of this modification.

Identification of hydroxylation at aromatic amino acid residues in yeast kinase using mass spectrometry with affinity enrichment.

RATIONALE: Protein kinases represent the key elements in phosphorylation-based signal transmission. Recent studies suggest that hydroxylation may mediate activities of protein kinases. This paper aims to examine the hydroxylation in protein kinases for improving our understanding of the protein modification. METHODS: We combined affinity-based protein purification with MS analysis for identification of novel hydroxylation at aromatic amino acid residues in yeast kinases. RESULTS: We identified 17 hydroxylation at aromatic amino acid residues (10 at Phe, 1 at Tyr and 6 at Trp) using MS analysis. We further characterized the localization and studied the potential significance of these modifications. CONCLUSIONS: This is a new report on the identification of hydroxylation at aromatic amino acid residues in yeast kinases. This study expands the catalog of hydroxylation in kinases and suggests the potential function of hydroxylation. Copyright © 2016 John Wiley & Sons, Ltd.

The relationship between leukocyte level and hypertension in elderly patients with hyperuricemia.

To evaluate the change of leukocyte level caused by hyperuricemia, and to explore the relationship between leukocyte level and hypertension in elderly patients with hyperuricemia. A cross-sectional study of serum uric acid (UA) level was conducted in 1352 elderly people over 65 years old. The samples were divided into 3 categories according to the tertiles of leukocyte: Tertile 1, leukocyte ≤ 5.2 × 109/L; Tertile 2, leukocyte = 5.3-6.3 × 109/L; Tertile 3, leukocyte ≥ 6.4 × 109/L. Multiple logistic regression models were used for modeling relationships between leukocyte, hyperuricemia and hypertension. Human vascular endothelial cells were treated by different concentrations of UA. The levels of interleukin-1 beta, tumor necrosis factor-α, endothelial nitric oxide synthase, inducible nitric oxide synthase and reactive oxygen species were measured by Western Blot or fluorescence microscope. The levels of leukocyte were higher in elderly patients with hyperuricemia than without hyperuricemia. Hyperuricemia was an independent risk factor of leukocyte in Tertile 3 (odds ratio [OR] = 1.657, 95% confidence interval [CI]: 1.180-2.328). The prevalences of hypertension were higher in elderly patients with hyperuricemia than without hyperuricemia (77.0% vs 63.5%). In the Model 1, hyperuricemia was an independent risk factor of hypertension (OR = 1.536, 95% CI: 1.026-2.302). Leukocyte in Tertile 3 was an independent risk factor of hypertension (OR = 1.333, 95% CI: 1.031-1.724). Expression levels of interleukin-1 beta, inducible nitric oxide synthase and tumor necrosis factor-α were obviously higher in the UA group than the control group, along with the productions of reactive oxygen species. But the expression level of endothelial nitric oxide synthase was obviously lower in the UA group. Hyperuricemia was associated with an increased risk for hypertension. The chronic inflammation caused by hyperuricemia maybe one of important pathogenesis of incident hypertension in patients with hyperuricemia.

Maltose-Functionalized Hydrophilic Magnetic Nanoparticles with Polymer Brushes for Highly Selective Enrichment of N-Linked Glycopeptides.

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.

Click Synthesis of Hydrophilic Maltose-Functionalized Iron Oxide Magnetic Nanoparticles Based on Dopamine Anchors for Highly Selective Enrichment of Glycopeptides.

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.