Analysis of intact proteins from cerebrospinal fluid by matrix-assisted laser desorption/ionization mass spectrometry after two-dimensional liquid-phase electrophoresis

A novel combination of methods, two-dimensional liquid-phase electrophoresi s (2D-LPE) and matrix-assisted laser desorption/ionization time-of-flight m ass spectrometry (MALDI-TOFMS), have been used for the analysis of intact b rain-specific proteins in cerebrospinal fluid (CSI), 2D-LPE is especially u seful for isolating proteins present in low concentrations in complex biolo gical samples, The proteins are separated in the first dimension by liquid- phase isoelectric focusing (IEF) in the Rotofor cell and in the second dime nsion by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PA GE) in the Preparative cell. The removal of SDS by chloroform/methanol/wate r, followed by sample preparation with the addition of n-octylglucoside, ea sily interfaced 2D-LPE with MALDI-TOFMS for analysis of intact proteins, Fu rther characterization by proteolytic digestion is also demonstrated. The k nowledge of both the molecular weights of the protein and of the proteolyti c fragments obtained by peptide mapping increases specificity for protein i dentification by searching in protein sequence databases. Two brain-specifi c proteins in human CSF, cystatin C and transthyretin, were isolated in: su fficient quantity for determination of the mass of the whole proteins and t heir tryptic digest by MALDI-TOFMS, This approach simplified the interface between electrophoresis and MALDI-TOFMS. Copyright (C) 1999 John Wiley & So ns, Ltd.