Cell lysis procedure determination

Human peripheral blood-derived DCs take up therapeutic proteins via macropinocytosis or receptor-mediated endocytosis, degrade them proteolyti-cally, and load the resulting peptide fragments onto HLA class II molecules, provided that the latter contain an appropriate epitope and resist downstream quality control by the peptide editor HLA-DM (Vogt et al., 2005). The second step foresees cell lysis, affinity extraction of HLA-peptide complexes, and acid elution of bound peptides. This procedure provides a mixture of both selfepitopes from the DC proteome and potential epitopes derived from the therapeutic protein. To separate mixtures of 1500-2000 distinct peptides with maximal resolution, the so-called Multidimensional Protein Identihcation Technology is employed (Kropshofer and Spindeldreher, 2005). Peptides leaving the HPLC capillary upon separation are sprayed directly into the orifice of an ion trap mass spectrometer for sequence determination. 

A rapid, solvent-free and general synthesis of short- and long-chain substi-tnted zwitterionic meta- and para-amino benzoquinones was reported by Cuccia, Friscic et al. (Scheme 3.96) [64], Yields obtained by ball milling were compared with solution procedure, melt reactions, and mechanosynthesis in a cell lysis mill (Table 3.52). Authors demonstrated that cell lysis mill could be successfully employed to mechanosynthesis of organic compounds, and it is also suitable for coordination-based reactions. In transamination synthesis of meta-aminobenzo-quinones 350, an excess of amine was used, whereas para-aminobenzoquinones 352 are synthesized by condensation of 1,4-benzoquinone and an alkylamine in a 3 2 ratio. After chromatographic workup, it was determined that the yields were comparable for all four methods employed, while the solution reactions are the slowest. 

Determination of changes in optical density of viable cell suspensions has been done both in the presence of osmotic support (71) and in its absence (72). If an osmotic support is used, dilution of the suspending medium results in rapid lysis of osmotically sensitive cells and a corresponding decrease in optical density. This procedure can also be used in combination with microscopic inspection of the treated cells. 

In view of the above discussion, it should be apparent that the localization within the cell of small molecules such as citric-acid-cycle anions is a major problem. There have been two approaches used to determine levels of key intermediates in the cytosol and mitochondria of mammalian cells one is indirect, involving the measurement of whole-cell levels of intermediates, followed by partitioning as judged by a set of calculation the second involves the rapid lysis of isolated cells, followed by their separation via centrifugation through silicone oil. The following is a brief critique of both of these procedures. 

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