Online concentration and separation of basic proteins using a cationic polyelectrolyte in the presence of reversed electroosmotic flow.
We report an online concentration and separation method for basic proteins using poly(diallyldimethylammonium chloride) (PDDA) solutions in the presence of reversed EOF. Using a capillary dynamically coated with 2% PDDA containing 0.1 M NaCl and filled with 1.2% PDDA under neutral conditions (10 mM phosphate, pH 7.0), we have demonstrated the separation of six basic proteins with peak efficiencies ranging from 175 000 to 616 000 plates/m and RSDs of migration time less than 0.4%. Additionally, high-speed separation of six basic proteins (<7 min) was achieved using a short capillary filled with 0.6% PDDA solutions. Under injection of the large-volume sample (210 nL), the LODs at S/N of 3 for basic proteins are down to nanomolar range. For example, the LOD for lysozyme is 1.2 nM, which is a 260-fold sensitivity enhancement compared with conventional injection method. The proposed method has been applied to the stacking of lysozyme in human saliva samples. Without any pretreatment, we also demonstrated the capability of this method to detect low amounts of peptide samples through the stacking of tryptic peptide of myoglobin. The experimental results indicate that our proposed method has great potential for use in clinical diagnosis and proteomics applications.
Yu
Cheng-Ju
CJ
Department of Chemistry, National SunYat-sen University, Kaohsiung, Taiwan.
Tseng
Wei-Lung
WL
D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D009113:Muramidase / Q000032:analysis; D009862:Online Systems; D009995:Osmosis; D010446:Peptide Fragments / Q000032:analysis; D011073:Polyamines / Q000737:chemistry*; D000071228:Polyelectrolytes; D011095:Polyethylenes / Q000737:chemistry*; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D012463:Saliva / Q000201:enzymology; D014357:Trypsin / Q000737:chemistry
D009113:Muramidase; D010446:Peptide Fragments; D011073:Polyamines; D000071228:Polyelectrolytes; D011095:Polyethylenes; D011506:Proteins; D000644:Quaternary Ammonium Compounds; D014357:Trypsin; C041004:poly-N,N-dimethyl-N,N-diallylammonium chloride