Loading, determination peptide sequencing

MALDl instruments, particularly those with automated plate loading, can also be operated successfully as open-access systems. Recently, the MALDl-TOF system in the facility was replaced with a MALDl-TOF/TOF instrument so that interested researchers could undertake peptide sequencing (and other structural determinations) along with standard MALDl-TOF analyses. 

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. 

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