Clinical proteomics

• Clinical proteomics is a rapidly maturing research discipline • The fundamental question s of clinical proteomics are the following:-What proteins or protein isoforms are present in a disease process?-How do those proteins interact?-What are the relative abundance and activation states of disease-related proteins? • This chapter reviews technologies and experimental procedures that enable clinical proteomics research Protein-Building Blocks • Proteins are polymers of amino acids, linked together by peptide bonds. A peptide bond is the amide linkage between a carboxyl group in one amino acid and amino group in another amino acid • Vast numbers of potential amino acid sequences are possible • However, the human genome is made of about 3-4 x 104 genes. Therefore, the proteins produced in the human system are a small subset of the theoretical protein complement • Clinical proteomics focuses on the relatively limited population of clinically relevant proteins transcribed from the genome Protein Structure • The amino acid sequence of a protein is called its primary structure. Out of this sequence arise the intrinsic properties of the protein, such as surface shape, size, and charge. These are important characteristics of a protein, which determine the ultimate function(s) of a protein • Based on the primary structure, a linear chain of amino acids coalesce and fold to form a series of secondary structural elements such as a-helices, p-pleated sheets, and random coils • Tertiary structure is comprised of higher order arrangements of secondary structural motifs. The secondary structural elements fold in such a manner so as to assume a thermodynamically stable conformation • Cysteines can be linked via disulfide bonds, which provide further structural stabil ity • Quaternary structure of a protein refers to the higher order arrangements of tertiary structures • Historically, structural changes in proteins have been linked to diseases. As one example, sickle cell anemia results from of a single amino acid change in an otherwise unaltered primary amino acid sequence. This substitution gives rise to a protein with a different shape than normal hemoglobin and altered higher order protein structures. The aggregation of sickle hemoglobin resulting in the formation of rigid fibers causes red blood cell sickling • Amino acid side chains within proteins are sites for the covalent addition of molecules such as phosphates, sugars, and lipids. These modifications occur after the protein has been translated from mRNA and are termed post-translational modifications (i.e., phosphorylation, glycosylation, and lipidation) • The study of post-translational modified proteins represents a vast and important area of disease pathophysiology research. Certain proteins are phosphorylated or dephosphorylated on specific residues in response to cellular signals. These signaling cascades play an important role in orchestrating cellular growth, migration, and apoptosis, among other functions. In general, post-translational modifications are not detected using genomic approaches Tools Used for Protein Studies: An Overview • Broadly speaking, two classes of protein characteristics have been used to study protein functions-Intrinsic physical properties. These are broadly applicable across a range of protein types and provide information about the protein's mass, charge, or structure. Examples of this type of technology are mass spectrometry, surface plasmon resonance, electrophoresis, ultraviolet (UV) spectroscopy, and chromatography Protein-specific properties. These techniques are protein-specific and provide information about posttranslational modifications as well as presence or absence of specific proteins in complex mixtures. They are usually derived from previously wellcharacterized individual proteins, such as antibodybased detection systems. Examples of approaches used to study protein-specific properties include flow cytometry, immunohistochemistry, enzyme-linked immunosorbent assay, and Western blots Physical Detection Systems • Within a protein, the overall sum of amino acids, the intrinsic qualities and relative abundance of the amino acids provide physical elements suitable for detection by a number of complimentary technologies, including mass spectrometry, chromatography, electrophoresis, surface plasmon resonance, and circular dichroism Mass Spectrometry • Mass spectrometry takes advantage of the behavior of a charged molecule in magnetic fields in order to classify proteins based on mass:charge ratios. This is discussed in further detail later Surface Plasmon Resonance • The biomolecular interactions of unlabeled proteins can be studied using surface plasmon resonance. Proteins are immobilized onto a thin metal film. If ligand binding occurs, the refractive index changes and these changes are detected by an optical sensor. Analyte association! dissociation rate constants may be calculated UV Spectroscopy • By measuring the absorbance of UV light by aromatic side chains of constituent amino acids within proteins, UV spectroscopy enables protein detection and quantitation Circular Dichroism • Circular dichroism spectroscopy uses circularly polarized light of one direction as a quick, low-resolution method for determining protein structure. Within a protein, the relative abundance of secondary structural elements such as a-helices, and B-pleated sheets can be measured Electrophoresis • Standard one-dimensional (lD) gel electrophoresis provides a means for protein separation. The movement of proteins through a solution in a polymeric matrix based on the application of an electrical field represents the primary technology. Differences in migration are based primarily on size of the protein • In two-dimensional electrophoresis, another level of separation, isoelectric potential, is used to further separate protein species in addition to size-based separation. In the first step, a pH gradient permits movement of the constituent proteins until they reach their isoelectric point. In the second step, the separation occurs at right angles to the first dimension and is based on protein size as in ID electrophoresis Capillary Electrophoresis • Electrophoresis can also be performed on protein samples within a capillary tube, which provides high-resolution protein separations when voltage is applied to the system Chromatography • Proteins can be separated by passing them over a resin that partitions the molecules between the liquid or gas phase and the bound resin. Types of chromatography include size exclusion, ion exchange, and high-pressure liquid chromatography Specific Affinity Techniques for Protein Detection • A molecule or macromolecular structure that selectively interacts with a protein can be utilized as an affinity system for specific protein detection. Affinity reagents that can be used to isolate specific proteins include metals, carbohydrates, proteins, and nucleic acids • A commonly used type of affinity reagent is an antibody that binds to a specific protein. Validation of a reagent's sensitivity and specificity must be performed for each antibody • Other uses of antibody reagents for detection of specific proteins within clinical samples include immunohistochemistry, immuno-flow cytometry, Western blots, and enzyme-linked immunosorbent assay Hybrid Technologies • Many times, a system for protein characterization and detection represents the fusion of several technologic approaches. A physical detection method, such as electrophoresis, can be paired with an antibody probing step, as is the case with a Western blot. Affinity chromatography using antibodies immobilized to a chromatography resin represents another example Limitations of Classical Protein Detection Tools as Clinical Tools • Proteomic tests in a clinical setting must be rapidly performed using very limited clinical material. Classical proteomic tools have had limited applicability due to the need for relatively large sample volume and time constraints.