Mass spectrometry-based proteomics is rapidly becoming an essential tool for biologists. The power of proteomics is usually revolutionizing biological research. Many scientists are wondering how to harness this emerging technology for their own studies. One of the most common uses of mass spectrometry based-proteomics is the identification of individual proteins from samples made up of many proteins. This is especially useful for identifying users of purified protein complexes. Traditionally a worm geneticist could identify genes acting in the same pathway by mutagenic or RNAi-based screens for animals with a mutant phenotype. However such genetic screens are limited by an failure to determine if the gene products are interacting actually. Generally a biochemical purification process that isolates a small selection of proteins is required to identify actually interacting components of a complex although in practice the final sample often contains non-specific ‘background’ proteins that are not members of the complex. With the development of efficient protein separation technologies the extreme sensitivity of the modern mass spectrometer can aid in the identification of true complex members. Understanding the relationship between methods utilized for complex purification protein identification and complex determination is usually of key importance in designing a successful experiment. You will find three crucial decisions to be made when designing an experiment. 1) How will the protein complex be isolated? 2) How will proteins be recognized? 3) How will users of the complex be distinguished from non-specific background proteins? After a conversation of these questions we will examine published work to see how different methods impact results. 2 Isolation of the protein complex by co-immunoprecipitation (co-IP) This question focuses on the biochemical purification actions used to isolate the protein complex. The worm lysate contains thousands of proteins. The goal of purification is usually to generate a sample containing only the complex of interest. In this regard the worm poses a challenge. Even though genome and proteome of is usually smaller then those of a mammal proteomic analysis may not be any less difficult. With larger animals you can reduce the variety of proteins in a XL647 sample by surgically removing a tissue of interest. However when we lyse an adult worm all 959 somatic cells and the germ cells are present. (Think about your response to a colleague grinding up an entire adult mouse to study kidney proteins.) This reduces the relative amount of desired complex and increases the number of background proteins that need to be removed. Harvesting synchronized worm cultures can help reduce complexity. The groups studying the kinetochore during early development enriched for embryos by dissolving synchronized adults in bleach and collecting the bleach-resistant embryos[1-2]. Techniques such as gel filtration and differential centrifugation may be used to enrich for organelles. Membrane-associated or DNA binding complexes present specific challenges to complex purification [4-5]. A popular way to isolate a protein complex from worm lysate is usually by co-immunoprecipitation. The co-IP can be XL647 performed in a single step or as part of a tandem purification. An antibody targeting a known member of the complex is bound to a bead and incubated with lysate in order to extract the target from the solution along with the other members of the complex. Rabbit polyclonal to HPCAL4. The antibody utilized for the co-IP can identify the protein of interest or a protein tag that has been genetically fused to the protein of interest. The advantage of using an antibody against the protein of interest is that the protein expression is not altered; the disadvantages are mostly practical e.g. more time required to synthesize and/or purify the peptide or protein antigen and then to immunize animals and harvest the antibodies. These polyclonal antibodies are limited in supply and unique to each XL647 immunized animal. Also one cannot be certain that the antibodies will be specific enough for useful separation. Alternatively a molecular “tag” can be added to a protein of interest expressed from a transgene and purified using an antibody against the tag. The tag can be utilized for a single-step purification or a multistep or tandem purification. Many different tags have been used in biologists have come to expect (observe section 5.1 Identifying XL647 a kinetochore regulatory complex). There are several caveats to tagging proteins. Gene expression may not be regulated under the same promoter.