Journal of Chromatography B, 745 (2000) 197–210 www.elsevier.com / locate / chromb Strategy for qualitative and quantitative analysis in proteomics based on signature peptides Junyan Ji, Asish Chakraborty, Ming Geng, Xiang Zhang, Ahmad Amini, * Minou Bina, Fred Regnier Department of Chemistry, Purdue University, Lafayette, IN 47907, USA Abstract This paper describes a new analytical strategy for identifying proteins in concentration flux based on isotopic labeling peptides in tryptic digests. Primary amino groups in peptides from control and experimental samples were derivatized with acetate and trideuteroacetate, respectively. After mixing samples thus labeled from these two sources, the relative concentration of peptides was determined by isotope ratio analysis with MALDI and ESI mass spectrometry. More than a 100-fold difference in relative concentration could be detected. Simplification of complex tryptic digests prior to mass spectral analysis was achieved by selection of histidine-containing peptides with immobilized metal affinity sorbents or of glycopeptides by lectin columns. Because most of these peptides have sequences that are unique to a single protein, they are a signature of the protein from which they were derived; providing a facile route to protein analysis.  2000 Elsevier Science B.V. All rights reserved. Keywords: Proteomics; Signature peptides; Proteins 1. Introduction will still not understand the dynamics of how living things respond to stimuli. Critical elements of ho- Molecular biology and molecular medicine have meostasis and how systems succumb to diseases will as their focus the explanation of biological phenom- evade us in most cases, unless we also know how ena in terms of molecular structure. This has led to biological systems are regulated. A major component the enormous effort to identify all the molecular of understanding cellular regulation is in being able elements of biological systems and the mechanism to identify proteins in flux in the complex milieu of by which they function. The Human Genome Project the proteome. [1] and proteomics [2] are both examples of efforts Two approaches for examining cellular dynamics to define the molecular elements of biological sys- have been suggested. One is to follow genetic tems and understand how they interact. Within the expression through the synthesis of specific mRNA next 5–10 years it is likely that the world will species [3,4]. This approach assumes that most identify most of the ‘‘molecular players’’ in humans, changes of protein concentration are the result of de domestic plants and animals, some pathogens, and novo protein synthesis. The second is by identifying many common microorganisms. Yet with all this, we changes in specific proteins themselves. It has been found that the correlation between the two is poor [5]. This is not surprising. Changes in protein *Corresponding author. E-mail address: fregnier@purdue.edu (F. Regnier). concentration in response to regulatory stimuli are 0378-4347 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0378-4347(00)00192-4