An agarose-based gel-concentration system for microsequence and mass spectrometric characterization of proteins...

An agarose-based concentration gel system is described for eluting and concentrating proteins previously purified either in one-dimensional or two-dimensional gels. Using the technique, proteins can be concentrated from about 1 ml into volumes as small as 10 microliters. After the proteins have been melted out of the agarose gels, they can be digested with proteases, producing peptide patterns similar to those observed with in-solution digestions. The overall peptide recovery, calculated from the amount of protein loaded on the primary separating gel to the collection of fragments after HPLC, is at least 70% of the peptide yields obtained with digests of the same amount of protein in free solution. These results are routinely obtained with 50 pmol amounts (referring to amounts of protein initially loaded on the primary gel). Proteins can also be analysed by a combination of microsequencing and on-line electrospray mass spectrometry, allowing their identification by peptide mass fingerprinting.