[Frontiers in Bioscience 17, 1804-1815, January 1, 2012] 1804 Processing-independent analysis of peptide hormones and prohormones in plasma Jens Peter Goetze 1 , Ingrid Hunter 1,2 , Solvej Koelvraa Lippert 1 , Linda Bardram 3 , Jens Frederik Rehfeld 1 1 Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark, 2 Department of Small Animal Clinical Sciences, University of Copenhagen, Denmark, 3 Department of Gastrointestinal Surgery, Rigshospitalet, Copenhagen, Denmark TABLE OF CONTENTS 1. Abstract 2. Introduction 3. The posttranslational phase of gene expression 3.1. Endoproteolytical cleavage 3.2. Exoproteolytic trimming 3.3. Amino acid derivatizations 4. Gut and heart: examples of posttranslational processing 4.1. Gastrin 4.2. Chromogranin 4.3. Cardiovascular peptides 5. Processing-independent analysis (PIA) – or how to quantitate proteins irrespective of the degree and nature of the posttranslational processing 6. Pitfalls of PIA measurements 7. Two examples of the clinical use of PIA measurements 7.1. Progastrin 7.2. ProBNP 8. Perspective 9. References 1. ABSTRACT Peptide hormones are post-translationally matured before they reach a structure in which they can fulfill their biological functions. The prohormone processing may encompass a variety of endoproteolytic cleavages, N- and C-terminal trimmings, and amino acid derivatizations. The same prohormone can be variably processed in different cell types and, in addition, diseased cells often change the processing of a given precursor. The translational process is often either increased or decreased in diseased cells, which renders the ensuing modifications of the prohormone incomplete. Consequently, a variable mixture of precursors and processing-intermediates accumulates in plasma. In order to exploit disturbed posttranslational processing for diagnostic use and at the same time provide an accurate measure of the translational product, a simple analytical principle named “processing-independent analysis” (PIA) was designed. PIA-methods quantitate the total mRNA product irrespective of the degree of processing. PIA- methods have now been developed for a number of prohormones and proteins, and their diagnostic potential appears promising in diagnosis of cardiovascular disease and in several malignancies. 2. INTRODUCTION The mapping and identification of the human genome has resulted in a paradigmatic shift in diagnosis, therapy and prevention of disease. The understanding of the pathogenesis of diseases at an exact molecular and cellular level and the implementation of molecular biology with its vast toolbox of technologies has led to the concept of molecular medicine. In the last decades, particularly DNA techniques have attracted attention with an exponential increase in the number of recognized mutated and deleted disease genes. More recently, DNA-array technologies have shown disease-specific patterns of gene expression that influence biomedical research, drug development and, not least, diagnosis. In the wake of gene diagnostics, new and improved diagnosis at protein level has now also been established under headings such as post- or functional genomics and proteomics. Early on, advanced mass spectrometry of proteins and peptides was surrounded by a great diagnostic promise (1). The rapid development of protein-array techniques was also followed with great expectations (2, 3). Nevertheless, the proteomic era in peptide biomarker research has largely failed and left the