CE-microreactor-CE-MS-MS for protein analysis

Abstract

Capillary electrophoresis-mass spectrometry (CE-MS) provides a powerful system that combines the speed and automation capabilities of CE with the detection and identification capabilities of MS. Integrating on-line enzymatic microreactor digestion with a CE-MS system reduces sample handling and allows fast digestions of small protein samples.In this thesis work, an on-line CE-microreactor-CE-MS-MS instrument is developed that can separate proteins in a first dimension, digest them inside a pepsin-immobilized microreactor, separate peptides in a second dimension, and detect the peptides with MS. The development begins from the MS end by coupling capillary free zone electrophoresis (CZE) to a linear ion trap mass spectrometer with a sheath flow interface, and works its way forward to the protein separation.Two CE-MS interfaces are tested and a sheath flow configuration is chosen for further instrument development because it provides robust electrosprays and does not cause band broadening. The CZE separation is optimized using bulk solution digested cytochrome C. Peptide-capillary wall interactions are reduced by coating capillaries with poly(vinyl alcohol) and by using high ionic strength buffers. The information dependent acquisition (IDA) capability of the mass spectrometer is used to obtain peptide fragment mass spectra that are submitted to a protein database search engine to identify cytochrome C.Pepsin-immobilized monolithic capillary microreactors are prepared and their homogeneity and digestion efficiency are investigated in both off-line and on-line experiments with CZE-ESI-MS. On-line coupling is achieved with a Plexiglas-glass interface. The microreactors are rather heterogeneous, yet cytochrome C was still successfully digested within 2 min. Hydroquinone (and p-benzoquinone) is added to buffers to alleviate CE current instabilities due to the electrolysis of water.The instrument is modified to a 2D CE-UV-microreactor-CE-ESI-MS-MS system by adding a protein separation capillary in front of the peptide separation. The protein separation capillary end that is coupled to the peptide separation dimension has an ∼ 1 cm microreactor integrated in it. A cytochrome C and myoglobin mixture is separated and identified using this 2D system. Only myoglobin is detected in a 6 protein sample, possibly because the other proteins contain disulfide bonds and/or are pepsin-resistant.