At-line coupling of magnetic-nanoparticle-based extraction with gel isoelectric focusing for protein analysis.
Sample preparation is a crucial step for protein analysis. Functionalized magnetic nanoparticle (MNP)-based extraction has been developed to be a useful sample preparation technique for proteomic analysis. In this paper, we present a strategy for at-line coupling of MNP-based extraction (MNE) with gel isoelectric focusing (IEF). The key to the at-line combination is to use an anolyte or a catholyte as the desorbing agent. Thus, functionalized MNPs can be facilely at-line coupled with gel IEF, provided that the extraction/desorption process is pH-controlled. MNPs extracted with target proteins are added to the sample well, which can function as a natural adapter. Once a focusing electric field has been applied across the gel, proton ions migrating from the anolyte or hydroxide ions migrating from the catholyte can act as a desorbing agent, releasing the proteins from the MNE probes. The released proteins are consequently focused into distinct bands where the local pH equals their pI values. The at-line combination was well demonstrated with three types of functionalized nanoparticles: (1) phenylboronic acid functionalized MNPs for extracting glycoproteins through boronate affinity; (2) carboxyl-functionalized MNPs for extracting positively charged proteins through a weak cation exchange mechanism; and (3) amino-functionalized MNPs for extracting negatively charged proteins through a weak anion exchange mechanism. The at-line combination exhibited several significant advantages, including selectivity, sensitivity, and speed.
Key Lab of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China.
Peng
P
Dou
Zhen
Z
Liu
D000327:Adsorption; D000818:Animals; D002417:Cattle; D007525:Isoelectric Focusing / Q000379:methods*; D008280:Magnetics; D053758:Nanoparticles / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D052616:Solid Phase Extraction / Q000295:instrumentation / Q000379:methods*
D011506:Proteins