APols-Aided Protein Precipitation: A Rapid Method for Concentrating Proteins for Proteomic Analysis

Amphipols (APols) are a newly designed and milder class of detergent. They have been used primarily in protein structure analysis for membrane protein trapping and stabilization. We have recently demonstrated that APols can be used as an alternative detergent for proteome extraction and digestion, to achieve a “One-stop” single-tube workflow for proteomics. In this workflow, APols are removed by precipitation after protein digestion without depleting the digested peptides. Here, we took further advantage of this precipitation characteristic of APols to concentrate proteins from diluted samples. In contrast with tryptic peptides, a decrease in pH leads to the unbiased co-precipitation of APols with proteins, including globular hydrophilic proteins. We demonstrated that this precipitation is a combined effect of acid precipitation and the APols’ protein interactions. Also, we have been able to demonstrate that APols-aided protein precipitation works well on diluted samples, such as secretome sample, and provides a rapid method for protein concentration.     

Investigating the effects of L- to D-amino acid substitution and deamidation on the activity and membrane interactions of antimicrobial peptide anoplin

Isolated from the venom sac of solitary spider wasp, Anoplius samariensis, anoplin is the smallest linear α-helical antimicrobial peptide found naturally with broad spectrum activity against both Gram-positive and Gram-negative bacteria, and little hemolytic activity toward human erythrocytes. Deamidation was found to decrease the peptide's antibacterial properties. In the present work, interactions of amidated (Ano-NH2) and deamidated (Ano-OH) forms of anoplin as well as Ano-NH2 composed of all D-amino acids (D-Ano-NH2) with model cell membranes were investigated by means of Langmuir Blodgett (LB) technique, atomic force microscopy (AFM), X-ray photoemission electron microscopy (X-PEEM) and carboxyfluorescein leakage assay in order to gain a better understanding of the effect of these peptide modifications on membrane binding and lytic properties. According to LB, all three peptides form stable monolayers at the air/water interface with Ano-NH2 occupying a slightly greater area per molecule than Ano-OH. All three forms of the peptide interact preferentially with anionic 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), rather than zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid monolayer. Peptides form nanoscale clusters in zwitterionic but not in anionic monolayers. Finally, membrane lytic activity of all derivatives was found to depend strongly on membrane composition and lipid/peptide ratio. The results suggest that amidated forms of peptides are likely to possess higher membrane binding affinity due to the increased charge.     

Proteomic reactors and their applications in biology

Proteomic analysis requires the combination of an extensive suite of technologies including protein processing and separation, micro-flow HPLC, MS and bioinformatics. Although proteomic technologies are still in flux, approaches that bypass gel electrophoresis (gel-free approaches) are dominating the field of proteomics. Along with the development of gel-free proteomics, came the development of devices for the processing of proteomic samples termed proteomic reactors. These microfluidic devices provide rapid, robust and efficient pre-MS sample procession by performing protein sample preparation/concentration, digestion and peptide fractionation. The proteomic reactor has advanced in two major directions: immobilized enzyme reactor and ion exchange-based proteomic reactor. This review summarizes the technical developments and biological applications of the proteomic reactor over the last decade.