Representations of Permutation Groups I
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1. Introduction The human body contains a broad spectrum of proteins in intracellular and extracellular compartments. Differential expression of proteins and peptides in disease has been the focus of numerous studies (1–4), which aimed to understand disease pathogenesis, to establish therapeutic targets and relevant diagnostics markers. Blood is a good example of a body fluid which is convenient to obtain and which contains large numbers of various proteins. However, more than 99% of the plasma protein fractions are made of approximately 20 major proteins such as haemoglobin, M. Soloviev (ed.), Peptidomics, Methods in Molecular Biology 615, DOI 10.1007/978-1-60761-535-4 20, © Humana Press, a part of Springer Science+Business Media, LLC 2010
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albumin and globulins (5). Therefore, the remaining ∼1% of the plasma proteins should be investigated if disease-related proteins are sought. In fact, biologically important proteins such as hormones, cytokines and chemokines account for only minor parts of the plasma proteins and considerable efforts were spent to detect and identify such low-abundance proteins. Peptides produced by processing and/or degradation of proteins with exopeptidases or specific endopeptidases represent an alternative to protein targets. Some such protein-derived peptides can have biological functions and be disease-related markers. Until recently it has been difficult to detect directly such short peptides in body fluids; however, the dramatic improvement of mass spectrometry made it possible. Many such peptides or their parent proteins have been reported to be associated with pathogenesis of a particular disease and/or to be useful disease markers in cases of cancers (6, 7), diabetes (8, 9), neural diseases (10) and collagen diseases (2). For example, in the field of autoimmune diseases, we reported that complement C3fdes-arginine peptide, detected predominantly in systemic sclerosis (SSc) sera, enhanced proliferation of vascular endothelial cells (2). Mass spectrometry is now universally applied to study various types of body fluids including blood (2, 11) urine (12, 13), and cerebrospinal fluid (14, 15). In this chapter, we describe protocols for peptidomic analysis of serum peptides. These include 1) collection of peripheral blood from patients 2) separation of sera from the blood 3) purification of peptides from the serum samples 4) detection of individual peptides 5) pattern recognition and clustering (bioinformatics) and 6) sequence identification of the peptides of interest. Biological and pathological functions of the identified peptides and their usefulness as disease markers can be elucidated using synthetic peptides and their parent proteins.
2. Materials 2.1. Collection of Peripheral Blood Samples and Separation of Sera
1. 5-ml syringes, 21 G needles and blood-collecting tubes (Terumo, Tokyo, Japan)
2.2. Purification of Serum Peptides
1. Magnetic bead-based hydrophobic interaction chromatography 18 (MB-HIC18, Bruker Daltonics, Ettlingen, Germany). The kit con
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