Identification of two-dimensionally separated human cerebrospinal fluid proteins by N-terminal sequencing, matrix-assisted laser desorption/ionization-mass spectrometry, nanoliquid chromatography-electrospray ionization-timeof flight-mass spectrometry, and tandem mass spectrometry

Optimal application of biological mass spectrometry (MS) in combination wit h two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) of human ce rebrospinal fluid (CSF) can lead to the identification of new potential bio logical markers of neurological disorders. To this end, we analyzed a numbe r of 2-D PAGE protein spots in a human CSF pool using spot co-localization, N-terminal sequencing, matrix-assisted laser desorption/ionization-mass sp ectrometry (MALDI-MS) and nanoliquid chromatography-electrospray ionization -time of flight-mass spectrometry (nanoLG-ESI-TOF-MS) with tandem MS switch ing. Our constructed CSF master contained 469 spots after image analysis an d processing of 2-D gels. Upon visual inspection of our CSF master with the CSF pattern available on the ExPASy server, it was possible to locate and annotate 15 proteins. N-terminal sequence analysis and MALDI-MS peptide mas s fingerprint analysis of both silver- and Coomassie Brilliant Blue (CBB) G -25O-stained protein spots after in situ trypsin digest not only confirmed nine of the visually annotated spots but additionally resolved the identity of another 13 spots. Six of these proteins were not annotated on the 2-D E xPASy map: complement C3 alpha-chain (1321-1663), complement factor B, cyst atin C, calgranulin A, hemoglobin beta-chain, and beta-2-microglobulin. It was clear that MALDI-MS identification from CBB G-250-stained, rather than from silver-stained, spots was more successful. In cases where no N-termina l sequence and/or no clear MALDI-MS result was available, nanoLC-ESI-TOF-MS and tandem MS automated switching was used to clarify and/or identify thes e protein spots by generating amino acid sequence tags. In addition, enrich ment of the concentration of low-abundant proteins on 2-D PAGE was obtained by removal of albumin and immunoglobulins from the CSF pool using affinity chromatography. Subsequent analysis by 2-D PAGE of the fractionated CSF po ol showed various new silver-stainable protein spots, of which four were id entified by nanoLC-ESI-TOF-MS and tandem MS switching. No significant homol ogy was found in either protein or DNA databases, indicating than these spo ts were unknown proteins.