Chapter 1. Isolation, identification, and production of posttranslationally modified chemokines.

Chemokines attract cells during the development of lymphoid tissues, leukocyte homing, and pathologic processes such as cancer and inflammation. Limited posttranslational modification of chemokines may significantly alter the glycosaminoglycan and/or receptor binding properties and signaling potency of these chemotactic proteins. To compare the in vitro and in vivo biologic activities of posttranslationally modified chemokine isoforms, considerable amounts of pure chemokine isoforms are required. This chapter describes a number of chromatographic techniques that are useful for the isolation of natural, posttranslationally modified chemokines from primary human cell cultures. In addition, combination of immunologic assays and biochemical techniques such as automated Edman degradation and mass spectrometry are used for the identification of modifications. Alternate methods for the generation of specific chemokine isoforms are discussed such as modification of chemokines by specific enzymes and total chemical syntheses and folding of chemokine isoforms. In particular, in vitro processing of chemokines by the protease aminopeptidase N/CD13 and citrullination or deamination of chemokines by peptidyl arginine deiminases (PAD) are described as methods for the confirmation or generation of posttranslationally modified chemokine isoforms.