springer verlag n y 1988 4 ion exchange chromatography of the mixture of modified peptides the selection of peptides is accomplished by a strong cation exchange chromatography at ph 2 4 the acidic conditions include the use of formic acid trifluoroacetic acid or any other buffer system which gives the desired ph of work this chromatographic step readily separates non charged from single and double charged peptides the charged peptides are retained by the exchanger while nhnr peptides are collected in the flow through 5 regeneration of u03b1 amino terminal groups and u03b5 amino groups of lysines this is an optional step which greatly depends on the amino group modifier initially employed if maleic or citraconic anhydride were used as modifying agents the free amino groups could be restored by timed incubation in the same acid conditions employed during the cation exchange chromatography for the case of reagents used in the peptide synthesis for the transitory protection of amino groups the protective groups could generally be removed using basic conditions if the modifier agent has a photosensitive properties it could be eliminated by light irradiation of modified peptides the method of this invention as specifically exemplified employs steps of washing peptides on reverse phase columns to remove undesired materials from the peptide sample the determination of differences in concentration of one or more proteins in different samples is achieved through the mass spectra analysis of isolated nhnr peptides isotopically labeled in three different ways a the proteins are extracted from tissues or cell cultures grown in media with two isotopic version of.