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 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.
Puchades
M
M
Department of Clinical Neuroscience, Experimental Neuroscience Unit, Göteborg University, Sahlgrenska University Hospital/Mölndal, SE-431 80 Mölndal, Sweden.
Westman
A
A
Blennow
K
K
Davidsson
P
P
D015153:Blotting, Western; D002556:Cerebrospinal Fluid Proteins / Q000134:cerebrospinal fluid*; D055316:Cystatin C; D015891:Cystatins / Q000134:cerebrospinal fluid; D004586:Electrophoresis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D010449:Peptide Mapping; D011228:Prealbumin / Q000134:cerebrospinal fluid; D012997:Solvents; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin
C525030:CST3 protein, human; D002556:Cerebrospinal Fluid Proteins; D055316:Cystatin C; D015891:Cystatins; D011228:Prealbumin; D012997:Solvents; D014357:Trypsin