Comprehensive analysis of lysine crotonylation modification in patients with chronic renal failure

Background Post-translational modifications (PTMs) are at the heart of many cellular signaling events, which changes the function of protein. Crotonylation, one of the most important and common PTMs, plays a crucial role in the regulation of various biological processes. However, no study has evaluated the role of lysine crotonylation modification in chronic renal failure (CRF) patients. Methods Here, we comparatively evaluated the crotonylation proteome of normal controls and chronic renal failure patients using liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with highly sensitive immune-affinity purification. Results A total of 1109 lysine modification sites were identified, of which 772 sites were up-regulated and 69 sites were down-regulated. This suggested that crotonylation modification maintains high levels in the patients with chronic renal failure. Gene ontology(GO) enrichment analysis showed that the crotonylated proteins were significantly enriched in the platelet alpha granule lumen, platelet degradulation, and cell adhesion molecule binding. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG)-based functional enrichment analysis in the Kyoto encyclopedia showed that crotonylated protein was enriched in CD36, which is closely linked to renal failure. Conclusions This is the first report of the global crotonylation proteome in chronic renal failure patients. Crotonylation of histone and non-histone may play important roles in delaying the continuous deterioration of renal function in patients with chronic renal failure.

Small Mass but Strong Information: Diagnostic Ions Provide Crucial Clues to Correctly Identify Histone Lysine Modifications

(1) Background: The proteomic analysis of histones constitutes a delicate task due to the combination of two factors: slight variations in the amino acid sequences of variants and the multiplicity of post-translational modifications (PTMs), particularly those occurring on lysine residues. (2) Methods: To dissect the relationship between both aspects, we carefully evaluated PTM identification on lysine 27 from histone H3 (H3K27) and the artefactual chemical modifications that may lead to erroneous PTM determination. H3K27 is a particularly interesting example because it can bear a range of PTMs and it sits nearby residues 29 and 31 that vary between H3 sequence variants. We discuss how the retention times, neutral losses and immonium/diagnostic ions observed in the MS/MS spectra of peptides bearing modified lysines detectable in the low-mass region might help validate the identification of modified sequences. (3) Results: Diagnostic ions carry key information, thereby avoiding potential mis-identifications due to either isobaric PTM combinations or isobaric amino acid-PTM combinations. This also includes cases where chemical formylation or acetylation of peptide N-termini artefactually occurs during sample processing or simply in the timeframe of LC-MS/MS analysis. Finally, in the very subtle case of positional isomers possibly corresponding to a given mass of lysine modification, the immonium and diagnostic ions may allow the identification of the in vivo structure.

Comprehensive Analysis of Lysine Crotonylation Modification in Patients with Chronic Renal Failure

Background： Post-translational modifications (PTMs) are at the heart of many cellular signaling events, which changes the function of protein. Crotonylation, one of the most important and common PTMs, plays a key role in the regulation of various biological processes. However, no study has evaluated the role of lysine crotonylation modification and chronic renal failure patients. Methods: Here, we comparatively evaluated the crotonylation proteome of normal controls and chronic renal failure patients using liquid chromatography tandem mass spectrometry (LC-MS/MS) coupled with highly sensitive immune-affinity purification.Results: A total of 1109 lysine modification sites were identified, of which 772 sites were up-regulated and 69 sites were down-regulated; this suggests that crotonylation modification maintains high levels in the patients’ kidneys with chronic renal failure. Gene ontology enrichment analysis showed that the crotonylated proteins were significantly enriched in the platelet alpha granule lumen, platelet degradulation, and cell adhesion molecule binding. In addition, KEGG-based gene and genomic functional enrichment analysis in Kyoto encyclopedia showed that battoacyl protein was enriched in CD36, which has an important relationship with renal failure.Conclusion: This is the first report on the global crotonylation proteome of chronic renal failure patients. crotonylation of histone and non-histone may play an important role in delaying the continuous deterioration of renal function in patients with chronic renal failure.

The Effect of Pore Size Distribution and l-Lysine Modified Apatite Whiskers (HAP) on Osteoblasts Response in PLLA/HAP Foam Scaffolds Obtained in the Thermally Induced Phase Separation Process

In this research, we prepared foam scaffolds based on poly(l-lactide) (PLLA) and apatite whiskers (HAP) using thermally induced phase separation technique supported by the salt leaching process (TIPS-SL). Using sodium chloride having a size of (a) 150–315 μm, (b) 315–400 μm, and (c) 500–600 μm, three types of foams with different pore sizes have been obtained. Internal structure of the obtained materials has been investigated using SEM as well as μCT. The materials have been studied by means of porosity, density, and compression tests. As the most promising, the composite prepared with salt size of 500–600 μm was prepared also with the l-lysine modified apatite. The osteoblast hFOB 1.19 cell response for the scaffolds was also investigated by means of cell viability, proliferation, adhesion/penetration, and biomineralization. Direct contact cytotoxicity assay showed the cytocompatibility of the scaffolds. All types of foam scaffolds containing HAP whiskers, regardless the pore size or l-lysine modification induced significant stimulatory effect on the cal-cium deposits formation in osteoblasts. The PLLA/HAP scaffolds modified with l-lysine stimulated hFOB 1.19 osteoblasts proliferation. Compared to the scaffolds with smaller pores (150–315 µm and 315–400 µm), the PLLA/HAP foams with large pores (500–600 µm) promoted more effective ad-hesion of osteoblasts to the surface of the biomaterial.

Site-selective lysine conjugation methods and applications towards antibody–drug conjugates

In this feature article we discuss developments in site-selective lysine modification methodologies and their application towards the synthesis of antibody–drug conjugates; including our recent work on a cysteine-to-lysine transfer (CLT) protocol.