Analysis of carbonic anhydrase in human red Blood cells using capillary electrophoresis/ electrospray ionization-mass...

Capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was applied to the analysis of human red blood cells (RBCs) using the split-flow technique for interfacing CE to MS. By using a long (approximately125-cm) and narrow (approximately 15-microm-i.d.) capillary, the four major proteins of the RBC, which are hemoglobin (Hb, alpha- and beta-chains, 900 amol/chain), carbonic anhydrase I (CAI, approximately 7 amol/cell), and carbonic anhydrase II (CAII, approximately 0.8 amol/cell), were separated from each other and detected at low-attomole levels in one run and minimal sample preparation. Under these conditions, the detection limits for CAI and CAII in lysed RBCs were approximately 20 and approximately 44 amol, respectively. The approximately 20-amol detection limit of CAI was confirmed by the CE/ESI-MS analysis of three intact RBCs that had been drawn into the capillary under a microscope. A shorter capillary (approximately 55 cm long) provided faster analysis time but did not separate CAII from the beta-chain of hemoglobin, causing the CAII signal to be masked by the background chemical noise generated by the approximately 1,000 x molar excess of the beta-chain. Under this condition, the CAII detection limit increased to approximately 500 amol. From three methods of sample introduction (injection of lysed blood, injection of intact cells under microscope, and injection of intact cells suspended in saline solution), injection of lysed blood provided the optimum sensitivity. It was found that a background electrolyte (BGE) containing 0.1% acetic acid in water worked best for the analysis of intact cells, while a BGE containing 0.1% acetic acid in water + acetonitrile (50/50 by volume) worked best for the analysis of lysed blood.