Abstract: The present invention relates to a method for protein purification and identification. More closely the invention relates to a method for protein pre-fractionation and identification resulting in improved yield of identified proteins. The method for pre-fractionation of protein samples, includes the following steps: a) reducing disulphide bridges (S-S bridges) or protecting cysteines in the proteins in the sample; b) loading the sample onto an ion exchange column; c) eluting the sample; d) collecting each fraction from the column separately in air sealed containers devoid of chromatographic media; e) desalting each fraction on a single RPC (reversed phase chromatography) trap column; f) separating each fraction in a second dimension RPC step to obtain further separated proteins which are collected in fractions; and g) identifying the further separated proteins by MS.

Abstract: The present invention relates to a method for reducing total sample complexity in native or digested biological sample(s), before analysis thereof by mass spectrometry, comprising the following steps: a) selecting a fraction from the entire native or digested biological sample(s) on the basis of pi-value, said fraction comprising native or digested sample representing a subset of or the entire substance population in the sample; b) separating native or digested sample substances from each other; and c) analysing said substances by mass spectrometry. The invention also relates to a system for reducing total sample complexity in the above method, comprising a high capacity charge-selective column coupled to a MDLC work flow path comprising a cation exchange column and a RPC column. The system is followed by a MS/MS instrument.