Strategy for purification and mass spectrometry identification of SELDI peaks corresponding to low-abundance plasma and serum proteins.

Abstract

Analysis by SELDI-TOF-MS of low abundance proteins makes it possible to select peaks as candidate biomarkers. Our aim was to define a purification strategy to optimise identification by MS of peaks detected by SELDI-TOF-MS from plasma or serum, regardless of any treatment by a combinatorial peptide ligand library (CPLL). We describe 2 principal steps in purification. First, choosing the appropriate sample containing the selected peak requires setting up a databank that records all the m/z peaks detected from samples in different conditions. Second, the specific purification process must be chosen: separation was achieved with either chromatographic columns or liquid-phase isoelectric focusing, both combined when appropriate with reverse-phase chromatography. After purification, peaks were separated by gel electrophoresis and the candidate proteins analyzed by nano-liquid-chromatography-MS/MS. We chose 4m/z peaks (9400, 13571, 13800 and 15557) selected for their differential expression between two conditions, as examples to explain the different strategies of purification, and we successfully identified 3 of them. Despite some limitations, our strategy to purify and identify peaks selected from SELDI-TOF-MS analysis was effective.

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MALDI MS

MALDI (matrix-assisted laser desorption/ionization), a laser-based soft ionization method has proven to be one of the most successful ionization methods for mass spectrometric analysis and investigation of large molecules.[1,2] In addition analysis of post source decay (decay of proteins following the ionization) in many cases allows rapid sequencing of proteins. Thus MALDI has gained a crucial importance for protein analysis.
Its constituting feature is that the sample is embedded in a chemical matrix (ca. 1000 x molar excess) that greatly facilitates the production of intact gas-phase ions from large, nonvolatile, and thermally labile compounds such as proteins, oligonucleotides, synthetic polymers. and large inorganic compounds. The matrix plays a key role in this technique by absorbing the laser light energy and causing a small part of the target substrate to vaporize.

The most important applications of MALDI mass spectrometry are (in decreasing order of importance): peptides and proteins, synthetic polymers, oligonucleotides, oligosaccharides, lipids, inorganics.

In its current state, MALDI is primarily based on the laser desorption of solid matrix-analyte deposits [6]. The technique suffers from some disadvantages such as low shot-to-shot reproducibility, short sample life time and strong dependence on the sample preparation method.

MALDI MS-based systems have been developed to rapidly characterize microorganisms, such as intact  proteins in the 5K-20K Da range in a top-down proteomics. In such experiments, proteins are identified either by conducting a homology search of extracted sequence tags or by comparison of MS/MS spectra with fragmentations predicted from protein sequences in existing protoeme database.
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