Signature-peptide approach to detecting proteins in complex mixtures

The objective of the work presented in this paper was to test the concept t hat tryptic peptides may be used as analytical surrogates of the protein fr om which they were derived. Proteins in complex mixtures were digested with trypsin and classes of peptide fragments selected by affinity chromatograp hy, lectin columns were used in this case. Affinity selected peptide mixtur es were directly transferred to a high-resolution reversed-phase chromatogr aphy column and further resolved into fractions that were collected and sub jected to matrix-assisted laser desorption ionization (MALDI) mass spectrom etry. The presence of specific proteins was determined by identification of signature peptides in the mass spectra. Data are also presented that sugge st proteins may be quantified as their signature peptides by using isotopic ally labeled internal standards. Isotope ratios of peptides were determined by MALDI mass spectrometry and used to determine the concentration of a pe ptide relative to that of the labeled internal standard. Peptides in trypti c digests were labeled by acetylation with acetyl N-hydroxysuccinimide whil e internal standard peptides were labeled with the trideuteroacetylated ana logue. Advantages of this approach are that (i) it is easier to separate pe ptides than proteins, (ii) native structure of the protein does not have to be maintained during the analysis, (iii) structural variants do not interf ere and (iv) putative proteins suggested from DNA databases can be recogniz ed by using a signature peptide probe. (C) 2000 Elsevier Science B.V. All r ights reserved.