Review 10.1586/14789450.4.2.161 © 2007 Future Drugs Ltd ISSN 1478-9450 161 www.future-drugs.com Promise of multiphoton detection in discovery and diagnostic proteomics Jasminka Godovac-Zimmermann † , Claire Mulvey, Maria Konstantoulaki, Richard Sainsbury and Larry R Brown † Author for correspondence University College London, Department of Medicine, Centre for Molecular Medicine, 5 University Street, London WC1E 6GF, UK Tel.: +44 207 670 6185 Fax: +44 207 679 0967 j.godovac-zimmermann@ucl.ac.uk KEYWORDS: cancer, immunoassay, multiphoton detection, protein isoform, proteomics, top-down, ultrasensitive Proteomics has lacked adequate methods for handling the complexity (hundreds of thousands of different proteins) and range of protein concentrations (≥10 6 ) of eukaryotic proteomes. New multiphoton-detection methods for ultrasensitive detection of proteins produce 10,000-fold gains in sensitivity and allow highly quantitative, linear detection of 50 zmol (30,000 molecules) to 500 fmol of proteins in complex samples. The potential of multiphoton detection in top-down proteomics analyses is illustrated with applications in monitoring proteomes in very small numbers of cells, in identifying and monitoring complex functional isoforms of cancer-related proteins, and in super-sensitive immunoassays of serum proteins for high-performance detection of cancer. Expert Rev. Proteomics 4(2), 161–173 (2007) Just over a decade after the term ‘proteomics’ was coined to describe a revival in the direct analysis of the proteins contained in cells and organisms, steady progress has led to many recent reviews on advances in proteomics [1–3]. Three major problems still prevent proteomics from reaching its full potential: • The complexity of the protein world is even higher than we expected 10 years ago, since it is now apparent that isoforms of the same protein are common and can have very different biological functions; • This complexity exacerbates a still inade- quate ability to resolve and individually observe important proteins; • The range of concentrations between different proteins in cells or in bodily fluids such as blood can be enormous, this makes observing and quantifying proteins a major challenge. For reasons outlined later and in other papers [4,5], it is important to analyze intact proteins (top-down proteomics), and progress in top- down analysis of protein structure by mass spectrometry (MS) has been promising in recent years [6–8]. Given sufficient amounts of a pro- tein, very complex forms of transcriptional and post-translational modifications can, in principle, be analyzed with current top-down MS methods. This suggests that the major bottleneck in top- down proteomics is our limited ability to observe and quantify intact proteins that are correlated with specific biological functions, especially for low-abundance proteins and for small samples; for example, medically relevant biopsies. New, ultrasensitive, multiphoton-detection (MPD) methods for protein detection have already proved to be very useful in proteomics. Using labeling with 125 I and MPD detection, as little as 50 zmol (30,000 molecules) of protein can be routinely detected. Furthermore, MPD methods are inherently linear with a dynamic range of approximately 10 7 (i.e., amounts of dif- ferent proteins between 50 zmol and 500 fmol) can, in principle, be observed in complex mix- tures. The main thrust of this brief review is to outline how these new MPD methods may help in improving top-down proteomics. Three application areas are considered: • Surveys of cell or tissue proteomes • Discovery of functionally important protein isoforms • Diagnostic monitoring of selected proteins CONTENTS What is multiphoton detection? Need for top-down proteomics Multiphoton detection in discovery proteomics Multiphoton detection in diagnostic proteomics Expert commentary Five-year view Key issues References Affiliations