Proteomic Analysis of Human Blood Serum Using Peptide Library Beads Lau Sennels,†,‡ Mogjiborahman Salek,† Lee Lomas,§ Egisto Boschetti,| Pier Giorgio Righetti,*,⊥ and Juri Rappsilber*,†,‡ The FIRC Institute for Molecular Oncology Foundation, Via Adamello 16, I-20139 Milan, Italy, Wellcome Trust Centre for Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh EH9 3JR, United Kingdom, Ciphergen Biosystems Inc., Fremont, California 94555, Bio-Rad Laboratories, c/o CEA-Saclay, Gif-sur-Yvette, France, and Polytechnic of Milano, Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Via Mancinelli 7, Milano, Italy Received June 3, 2007
Human serum is thought to contain key information for diagnostics of human disease. However, no single technology is currently nor might ever be available to cope with the complexity and dynamic range of the serum proteome. We here report a large-scale proteomic study of human blood serum using peptide library beads and mass spectrometry. Serum proteins are adsorbed onto polymeric beads coated with a combinatorial library composed of millions of hexameric peptide baits. Analysis of the eluates from this combinatorial library (as obtained with 3 eluants of different strength, able to release 99% of the retentate) via liquid chromatography coupled to high-resolution mass spectrometry resulted in the identification of 1559 proteins or 3869 proteins, respectively, depending on how 95% confidence was estimated. In either case, the analysis showed that ligand beads are able to capture a large number of proteins in a single operation. The ligand bead bound fraction appeared to have a lower dynamic range when compared to the starting material, due to a “normalization” of the protein concentrations in the original mixture. We find that extensive information on the protein composition of complex samples such as serum can be obtained using ligand beads and that these beads enrich the proteomic tool box. Keywords: serum proteins • ligand library • peptide ligands
Introduction There seems to be a general consensus that human blood plasma is the most-important proteome from a clinical/medical point of view. A broad inventory of plasma proteins (both in qualitative and quantitative terms) should provide a foundation for identification of candidate protein markers for disease diagnosis and development of new therapeutic products. Notwithstanding these considerations, the process of discovering and cataloging serum proteins has been painfully slow. Two-dimensional (2D) electrophoresis was able to resolve 40 distinct plasma proteins in 1977,1 but this number had only grown to 60 in 19922 and remained unchanged for quite a few years afterward. Yet, according to Anderson and Anderson,3 the human plasma should contain most, if not all, human proteins, as well as proteins derived from some viruses, bacteria, and * To whom correspondence should be addressed. Juri Rappsilber: Tel, +44 131 651 7057; Fax, +44 131 650 5379; E-mail,
[email protected]. Prof Pier Giorgio Righetti: Tel, +39 02 23993045; Fax, +39 02 23993080; E-mail,
[email protected]. † The FIRC Institute for Molecular Oncology Foundation. ‡ The University of Edinburgh. § Ciphergen Biosystems Inc.. | Bio-Rad Laboratories. ⊥ Polytechnic of Milano. 10.1021/pr070339l CCC: $37.00
2007 American Chemical Society
fungi. Among them, numerous post-translationally modified forms of each protein are present, along with, possibly, millions of distinct clonal immunoglobulin sequences. Perhaps the major cause for this slow discovery rate is the enormous dynamic concentration range, encompassing some 10 orders of magnitude between the least abundant (e.g., interleukins, in concentrations
