New enhanced diagnostic tools!
The power of commercially available mass spectrometers has increased dramatically in the last decade. Advancements made in instrument development and bioinformatics together with our understanding of the physicochemistry of matter has made it now possible to use stable isotope labeling methods for the study of biological and other natural systems. Furthermore, because of improved computer power it is now possible to acquire large data sets of mass spectra with high-resolution during proteomic studies allowing the analysis of thousands of peptides spanning a dynamic range of several orders of magnitude of protein abundance during one liquid chromatography tandem mass spectrometry experiment (LC-MS/MS).
Quantitative proteomics aims to obtain absolute information about all proteins in a biological sample and stable isotope labeling methods are now a major part of this technology. Since this technique yields information about differences between samples, this approach can be used to compare samples from healthy and diseased people. However, methods used are similar to qualitative proteomics with the difference that quantitative changes in systems can be studied. Usually biological systems, such as organelles, are analyzed to identify all components of the system and to monitor their quantitative changes that may occur during different metabolic phases of the system, such as different stages in the cell cycle.
A Pubmed (http://www.ncbi.nlm.nih.gov/) search for “Stable isotopes” and "stable isotope labelin" retrieved over 15,000 papers reporting the use of stable isotope labeling methods since 1947. This search results indicate that the use of stable isotopes in biological research has become very popular. Because of this our current knowledge that approximately 30,000 human genes appear to code for up to 1 million or more proteins has generated new interest in independent ‘de novo’ protein and peptide sequencing of gene products.
Two methods are available for this task, the classical Edman chemistry based method, or the newer, more recent method which utilizes LC-MS/MS based sequencing. The second method is now considered to be faster and more sensitive.
One popular approach, the use of the absolute quantification method (AQUA) allows targeted quantification of protein and post-translational modifications in complex protein mixtures. The method employs stable isotope-labeled peptides as internal standards.
The AQUA experiment includes two steps: A) method development, and B) application to a biological problem.
During the method development step, peptides from the protein of interest that are easily detected in a mass spectrometer are selected followed by synthesis with stable isotopes such as 13C, 2H or 15N. The abundance of these AQUA peptides used as internal standards and their endogenous counterparts are measured by mass spectrometry with selected reaction monitoring or selected ion monitoring methods. An established AQUA method can be rapidly applied to a wide range of biological samples, from tissue culture cells to human plasma and tissue. After the synthesis of AQUA peptides, the development, optimization and application of the AQUA analyses method to a specific biological problem may be achieved within one week.
Another popular approach is metabolic Stable Isotope Labeling in Amino Acids in Cell Culture (SILAC) which is a technique for mass spectrometric (MS)-based quantitative proteomics.
The Center for Experimental Bioinformatics (CEBI) at the University of Southern Denmark developed and is advocating this technique (Ong SE, et al, MCP, 2002). The key step to SILAC is that differentially labeled samples are mixed early in the experimental process and analyzed together by LC-MS/MS. Since the labeling does not affect the chemical properties of the molecules, they co-elute from the LC column and are analyzed together in the mass spectrometer. The peaks of the peptides correlating to the differentially labeled samples can be very accurately quantified relative to each other, allowing determination of peptide and protein ratios. SILAC labeling typically uses lysine and arginine, which in combination with trypsin digestion results in labeling of every peptide in the mixture (except for the c-terminal peptide of the protein). SILAC experiments involve differential labeling of two to three cell types: cells grown with the natural amino acids, with 2H4-lysine and 13C6-arginine and with 15N213C6-lysine and 15N413C6-arginine. The SILAC method only works well if heavy amino acids are completely incorporated during protein turnover. The use of dialyzed serum is employed so that the added amino acids are the exclusive source. The heavy amino acids create a distinct and known mass difference between the samples only affecting the isotope distribution of the peptides in a minor and predictable way which makes data interpretation and quantification more accurate and robust. The MS scans are used for peptide quantification by allowing multiple scans for each peptide across its elution time. Since protein quantification is based on the median values of multiple peptides a high accuracy of ratio determination is possible. Classical SILAC relies on full incorporation of amino acids into proteins in cultured cells, whereas SILAC can also be used for proteomics of organisms and human tissues by using ‘spike-in’ standards such as AQUA peptides.
The combination of genomic with proteomic methods allows for the identification of gene loci and the investigation of major quantitative trait loci (QTL) in domestic animals and the discovery of new gene loci. Markljung et al. in 2009 used genetic mapping of a QTL for growth and fatness in pigs for their discovery of the transcription factor ZBED6 present in mammals. Strong selection for lean growth in domestic pigs during the last half century has created pigs with increased muscle mass and reduced fat deposition. QTL mapping of an intercross between the European Wild Boar and Large White domestic pig allowed for the identification of the most important locus that has responded to this selective pressure as a paternally expressed QTL colocalized with the gene for insulin-like growth factor 2 (IGF2). The mutation is a single nucleotide substitution in intron 3 of IGF2. The mutation is located in a well-conserved CpG island found in mammals. This study isolated a zinc finger protein of unknown function named ZBED6 and showed that it has a broad tissue distribution which may indicate that ZBED6 affects thousands of other genes that control fundamental biological processes. The group of researchers used a oligonucleotide capture method in combination with quantitative SILAC mass spectrometry to identify the protein by determining the sequence of six unique peptides.
It is thought that system biology, utilizing the use of new improved sequencing technologies, both genomic and proteomic will revolutionize the field of animal genomics as well as all fields of biology, including human medicine.
Biosynthesis Incorporated custom synthesizes SIS Peptides which can be used as internal standards for MRM mass spec analysis. The peptides are synthesized and undergo rigorous purification and characterization and can contain any amino acid residue with the stable isotopes 13C and 15N in place of 12C and 14N, as selected by the customer. All SIS peptides are purified by reversed-phase LC and are accurately quantified by amino acid analysis.
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