Cytocompatible Modification of Thermoresponsive Polymers on Living Cells for Membrane Proteomic Isolation and Analysis

Abstract

Efficient strategies for enriching and separating proteins are important and challenging for membrane proteomics. Many existing methods are caught in the dilemma of preserving maximal membrane proteins while avoiding the contamination of cytoplasmic proteins and organelles. Here, we report a polymer anchoring strategy for the selective preparation of membrane proteins through cell surface-initiated atom transfer radical polymerization. The cytocompatible polymerization strategy enables thermoresponsive poly(N-isopropylacrylamide) (pNIPPAm) chains to be grown from a specific protein on the surface of living cells. The polymer tagged membrane protein could be easily separated and enriched by thermoprecipitation. This method led to the identification of 1825 proteins of which 1036 (71.7%) were specific membrane proteins in E. coli. The separated proteins were identified by 2-DE and mass spectrometry. Among the 12 protein spots from the gel slice, eight were identified as outer membrane proteins. The described strategy opens up a new avenue for membrane protein enrichment and separation and may expedite the future development of membrane proteomics.

Aglycone sterics-selective enzymatic glycan remodeling

“…Having demonstrated the modulation of aglycone-steric selectivity by polymer length adjustment, we then proceeded to integrate the external-stimuli responsiveness of polymer shell to achieve superimposed regulation of GO activity. Given the thermal sensitivity of PNs ( Heredia et al., 2005 ; Chen and Hoffman, 1993 ; Stayton et al., 1995 ; Boyer et al., 2007 ; De et al., 2008 ; Mackenzie and Francis, 2013 ; Gobbo et al., 2018 ; Wu et al., 2019 ), we firstly investigated the combined effect of polymer shielding and temperature elevation on the catalytic activity of GO and GO-PN (1∼3) towards GM1 or Gal at 25 or 37°C. For native GO, the elevation of temperature only led to a minimal increase of activity, regardless of substrates used ( Figures 5 A and 5B).…”

“…To demonstrate our strategy, we first tagged bis[2-(2′-bromoisobutyryloxy)ethyl]disulfide (BiBOEDS) onto the amine groups of GO via disulfide exchange using N -succinimidyl3-(2-pyridyldithio)propionate (SPDP)-based linkage chemistry ( Scheme S1 ) ( Wu et al., 2019 ), and yielded GO-initiator conjugate (GO-Br, I 0 ) with a BiBOEDS-to-GO molar ratio of approximately 4:1 ( Figure S1 ). We then chose N -isopropylacrylamide (NIPAm, M 0 ) as the model monomer, and grafted it from GO-Br using N , N , N ′, N ”, N ″-pentamethyldiethylenetriamine (PMDETA) as the ligand, and L-ascorbic acid (Vc) as the reducing agent, to generate Cu I , the catalyst of ATRP, from Cu II ( Scheme 1 A) ( Wu et al., 2019 ). By adjusting the feed molar ratio and polymerization time, a set of GO-poly( N -isopropylacrylamide) (GO-PN) composites, GO-PN (1∼15), with different degrees of polymerization, were successfully prepared ( Tables S1 and S2 ), as evidenced by the phase and color change during the polymerization reaction.…”

“…By adjusting the feed molar ratio and polymerization time, a set of GO-poly( N -isopropylacrylamide) (GO-PN) composites, GO-PN (1∼15), with different degrees of polymerization, were successfully prepared ( Tables S1 and S2 ), as evidenced by the phase and color change during the polymerization reaction. To verify the grafting of PNs from GO and investigate whether GO-PNs possess length adjustability ( Theodorou et al., 2020 ), thermo sensitivity ( Heredia et al., 2005 ; Cummings et al., 2013 ; Chen and Hoffman, 1993 ; Stayton et al., 1995 ; Boyer et al., 2007 ; De et al., 2008 ; Mackenzie and Francis, 2013 ; Wu et al., 2019 ), and chemical responsiveness ( Boyer et al., 2007 ; Wu et al., 2019 ; Liu et al., 2007 ), we chose GO-PN (1∼3) (M 0 /I 0 /PMDETA/Cu II /Vc = 15000/1/59/59/59, 1-, 2-, 3-h polymerization) as the model enzyme-polymer composites.…”

“…NIPAm (200 mg), PMDETA (1.2 mg), CuCl 2 ⋅2H 2 O (1.2 mg) were dissolved with H 2 O (2 mL) and methanol (MeOH) (1 mL) ( Wu et al., 2019 ) to obtain a blue monomer solution in a Schlenk flask at NIPAm/PMDETA/CuCl 2 = 255/1/1 (molar ratio). The flask was sealed carefully, frozen with liquid nitrogen, and deoxygenated with nitrogen for 15 min 200 μL Vc (3.1 mg/mL for Entries 4∼6; 3.7 mg/mL for Entries 7∼9; 4.3 mg/mL for Entries 10∼12; 6.2 mg/mL for Entries 1∼3 and 16∼18; 9.3 mg/mL for Entries 13∼15, Tables S1 and S2 ) was then added into the thawed monomer solution under nitrogen atmosphere, deoxygenated for 15 min, and mixed with 1 mL deoxygenated GO-Br solution (2 mg/mL).…”

Dealing with the complexity of conjugated and self‐assembled polymer‐nanostructures using field‐flow fractionation

“…The application of thermoresponsive poly( N ‐isopropylacrylamide) polymers is another strategy to create smart PCBs. The goal of this proteomic approach is to obtain cytocompatibility by modification of the surface of living cells 81 . The successful conjugation via disulfide (SH‐) bonds was validated by microscopy (SEM); chemo‐selective separation by SDS‐PAGE; LC‐MS/MS and MALDI.…”