P176-M Rapid Purification and Enrichment of Proteins from Crude Solutions Using Reversed Phase Microparticles and Subsequent Generation of Peptide Mass Fingerprints by MALDI-TOF Mass Spectrometry

摘要

The generation of peptide mass fingerprints followed by a database search is a common tool for the mass spectrometric identification of proteins. To provide a high sensitivity, proteins must be efficiently purified and concentrated prior to enzymatic digestion. Common desalting procedures, like ultrafiltration or dialysis, are very time consuming and work best for high protein concentrations. Another critical point is the proteolysis of the investigated protein, which works efficiently only with concentrated protein solutions. Therefore, efficient concentration and simultaneous purification using solid phase extraction (SPE) will be the method of choice to receive pure and highly concentrated protein solutions prior to enzymatic digestion. In this work, we manufactured magnetic reversed phase particles for the efficient purification and simultaneous concentration of protein samples with volumes up to several millilitres. The SPE procedure was compared with dialysis using commercial available microconcentrators with a cut-off membrane. Due to the magnetic core, each washing and elution step could be performed within 15 minutes. Then, the bound protein was digested directly on the beads, resulting in a remarkable increase of protein detection and better mass structural analysis. Useful MOWSE scores were achieved using bovine serum albumin as a model protein with concentrations as low as 50 ng/ml (720 pM). Compared to the dialysis procedure, which needs several hours, the isolation and purification of protein can be performed in minutes with the reversed phase particles.