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

Abstract

A novel combination of methods, two-dimensional liquid-phase electrophoresis (2D-LPE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), have been used for the analysis of intact brain-specific proteins in cerebrospinal fluid (CSF). 2D-LPE is especially useful for isolating proteins present in low concentrations in complex biological samples. The proteins are separated in the first dimension by liquid-phase isoelectric focusing (IEF) in the Rotofor cell and in the second dimension by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in the Preparative cell. The removal of SDS by chloroform/methanol/water, followed by sample preparation with the addition of n-octylglucoside, easily interfaced 2D-LPE with MALDI-TOFMS for analysis of intact proteins. Further characterization by proteolytic digestion is also demonstrated. The knowledge of both the molecular weights of the protein and of the proteolytic fragments obtained by peptide mapping increases specificity for protein identification by searching in protein sequence databases. Two brain-specific proteins in human CSF, cystatin C and transthyretin, were isolated in sufficient quantity for determination of the mass of the whole proteins and their tryptic digest by MALDI-TOFMS. This approach simplified the interface between electrophoresis and MALDI-TOFMS.

Serum Transthyretin Monomer as a Possible Marker of Blood-to-CSF Barrier Disruption

“…Note the appearance of a distinct spot after BBB disruption. This spot corresponded to a protein of approximate molecular weight of 14 kDa and a pI of 5.5. tein in epithelial cells of the choroid plexus (Puchades et al, 1999;Hiraoka et al, 2000).…”

Mass spectrometry: A tool for the identification of proteins separated by gels

“…Preparative IEF [74,75] or IPG-IEF [76,77] have been combined with MS to obtain molecular masses. MS-based techniques reveal additional structural information by determining the mass of an intact protein [69±73].…”

Liquid phase interfacing and miniaturization in matrix-assisted laser desorption/ionization mass spectrometry

“…Some of the more recent approaches utilize solution based IEF as the first micropreparative step followed by SDS electrophoresis [40][41][42] or reversed phase separation [43,44] and MALDI-MS analysis of the collected fractions. On the protein level the prefractionation by the solution IEF and MALDI-TOF analysis of the resulting fractions can be used as a crude substitute for 2-D PAGE before further analysis.…”