Highly Chemo-, Regio- and Stereoselective Cysteine Modification of Peptides and Proteins with Ynamides

Chemoselective modification of peptides and proteins has wide applications in chemical biology and pharmaceutical development. An efficient cysteine (Cys) precise modification protocol via rationally designed β-addition of ynamides is reported. The strong electron-withdrawing triflyl group on the nitrogen atom of ynamides plays a crucial role for controlling the chemo-, stereo- and regioselectivities of this protocol. Another substituent of the terminal ynamides offers a handle for functionality diversification. This Cys modification with ynamides proceeds efficiently in a slightly basic aqueous media (pH 8) to provide a series of Z-isomer of the corresponding conjugated products with excellent stereoselectivity (> 99%) and superior stability. All the reactive peptide side chain functional groups such as amino, carboxyl, primary amide, and hydroxyl groups, as well as the unprotected imidazole and indole rings are compatible. This method displays a broad substrates scope including linear and cyclic peptides and proteins. The potential application of this method in peptide and protein chemical biology was exemplified by Cys-bioconjugation with ynamides containing different functional molecules, including drug, fluorescent and affinity tags. In addition, this strategy is also compatible with click chemistry (performed in one pot), which remarkably extends the toolbox for further applications. The chemoselective biotinylation of ubiquitin(G47C) variant with a biotinylated ynamide, as well as the regioselective modification of Cys14 and Cys38 in bovine pancreatic trypsin inhibitor (BPTI) were accomplished under the optimized conditions and in high yield, without perturbation of disulfide bonds.

Glycan Labeling and Analysis in Cells and In Vivo

As one of the major types of biomacromolecules in the cell, glycans play essential functional roles in various biological processes. Compared with proteins and nucleic acids, the analysis of glycans in situ has been more challenging. Herein we review recent advances in the development of methods and strategies for labeling, imaging, and profiling of glycans in cells and in vivo. Cellular glycans can be labeled by affinity-based probes, including lectin and antibody conjugates, direct chemical modification, metabolic glycan labeling, and chemoenzymatic labeling. These methods have been applied to label glycans with fluorophores, which enables the visualization and tracking of glycans in cells, tissues, and living organisms. Alternatively, labeling glycans with affinity tags has enabled the enrichment of glycoproteins for glycoproteomic profiling. Built on the glycan labeling methods, strategies enabling cell-selective and tissue-specific glycan labeling and protein-specific glycan imaging have been developed. With these methods and strategies, researchers are now better poised than ever to dissect the biological function of glycans in physiological or pathological contexts.

The C29–C34 parts of antitumor macrolide aplyronine A serve as versatile actin-affinity tags

Anticancer drug development inspired by natural products based on protein–protein interactions (PPI) is a promising strategy. We developed structurally-simplified C29–C34 side-chain analogs of aplyronine A (ApA), an antitumor marine macrolide....

Detectability of biotin tags by LC-MS/MS

ABSTRACTThe high affinity of biotin to streptavidin has made it one of the most widely used affinity tags in proteomics. Early methods used biotin for enrichment alone and mostly ignored the biotin labeled peptide. Recent advances in labeling led to an increase in biotinylation efficiency and shifted the interest to detection of the site of biotinylation. This increased confidence in identification and provides additional structural information yet it requires efficient release of the biotinylated protein/peptide and sensitive separation and detection of biotinylated peptides by LC-MS/MS. Despite its long use in affinity proteomics the effect of biotinylation on the chromatographic, ionization, and fragmentation behaviour and ultimate detection of peptides is not well understood. To address this we compare two commercially-available biotin labels EZ-Link Sulfo-NHS-Biotin and Sulfo-NHS-SS-Biotin, the latter one containing a labile linker to efficiently release biotin to determine the effects of peptide modification on peptide detection. We describe an increase of hydrophobicity and charge reduction with increasing number of biotin labels attached. Based on our data we recommend gradient optimization to account for more hydrophobic biotinylated peptides and include singly charged precursors to account for charge reduction by biotin.

Affinity Tags for Protein Purification

The affinity tags are unique proteins/peptides that are attached at the N- or C-terminus of the recombinant proteins. These tags help in protein purification. Additionally, some affinity tags also serve a dual purpose as solubility enhancers for challenging protein targets. By applying a combinatorial approach, carefully chosen affinity tags designed in tandem have proven to be very successful in the purification of single proteins or multi-protein complexes. In this mini-review, the key features of the most commonly used affinity tags are discussed. The affinity tags have been classified into two significant categories, epitope tags, and protein/domain tags. The epitope tags are generally small peptides with high affinity towards a chromatography resin. The protein/domain tags often perform double duty as solubility enhancers as well as aid in affinity purification. Finally, protease-based affinity tag removal strategies after purification are discussed.

Method for purification of collagen: A systematic review

Collagen has diverse applications in various industries and thus a various method of purifications has been developed over the years to produce high yield and high purity of collagen to fulfill collagen market demand. The objective of this qualitative systematic review is to summarize the current literature on the existing purification methods for collagen from different sources. Toward this end, three databases were searched and articles were screened for all original articles explaining the purification of collagen regardless of their main sources. After the comprehensive screening, out of 715 articles, 12 articles were chosen and were further reviewed. The analysis of the technical details, theory, advantages, and disadvantages of the techniques used are reported in this study. We found four types of purification methods that commonly used to purify various types of collagen from mammals, marine, bacteria, and recombinant collagen. The correlations between the prevalence of the method and the efficiency of collagen purification were also identified. It highlighted that many factors should be considered before choosing any method of purification such as types, sources and structure of the collagen itself. Affinity chromatography commonly used for purification of recombinant collagen as they have been genetically modified and appended by affinity tags whereas conventional purification method is preferable for non-recombinant collagen from mammalian and marine sources.