ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 352 (2006) 77–86 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.02.001 De novo proteomic sequencing of a monoclonal antibody raised against OX40 ligand Victoria Pham a , William J. Henzel a , David Arnott a , Sarah Hymowitz b , Wendy N. Sandoval a , Bao-Tran Truong b , Henry Lowman c , Jennie R. Lill a,¤ a Department of Protein Chemistry, Genentech, South San Francisco, CA 94080, USA b Department of Protein Engineering, Genentech, South San Francisco, CA 94080, USA c Department of Antibody Engineering, Genentech, South San Francisco, CA 94080, USA Received 28 October 2005 Available online 21 February 2006 Abstract De novo sequencing of a full-length monoclonal antibody raised against OX40 ligand is described. Using a combination of overlap- ping complementary proteolytic and chemical digestions, with analysis by mass spectrometry and Edman degradation, both the heavy and light chains were fully sequenced. Particular attention was paid to those modiWcations that could be susceptible to degradation in the complementarity determining region and Fc region. An overview of the protocol is described, and suggestions for improvements to aid in such sequencing projects in the future are discussed.  2006 Elsevier Inc. All rights reserved. Keywords: OX40 Ligand; Mass spectrometry; Edman degradation; De novo sequencing Over the past decade, murine [1], chimeric [2], and fully humanized [3] monoclonal antibodies were used as thera- peutics for the successful treatment of both malignant [4,5] and autoimmune [6,7] diseases. Initial research into the eYcacy of antibodies for neutralization activity, and hence viability for drug candidates, typically begins with mono- clonal antibody production in hybridomas, that is, cells resulting from the fusion of B cells with myeloma cells [8]. Although immortalized, hybridomas are often genetically unstable, and this lack of stability is a concern in both the laboratory and clinical setting. Mutations in the comple- mentarity determining region (CDR) 1 or Fc region of an antibody can alter antigen speciWcity and/or eVector func- tion, both of which are essential for correct immunological functioning. Although eVorts to forgo the use of hybrido- mas in the initial production of monoclonal antibodies were investigated [9], hybridomas remain the paradigm for nonclinical monoclonal antibody production. Along with alterations in sequence resulting from genetic instability of hybridomas, other modiWcations at the protein level [1] (i.e., resulting from prolonged storage of the antibody) are also of concern. At the primary * Corresponding author. Fax: +1 650 225 5945. E-mail address: jlill@gene.com (J.R. Lill). 1 Abbreviations used: CDR, complementarity determining region; MS/MS, tandem mass spectrometry; MALDI, matrix-assisted laser desorption/ioniza- tion; OX40L, OX40 ligand; TNF, tumor necrosis factor; DTT, dithiothreitol; BME, -mercaptoethanol; NIPIA, n-isopropyl iodoacetamide; CAPS, 3-(cy- clohexylamino)-1-propanesulfonic acid; Lys-N, lysine-speciWc endopeptidase; PGAP, pyroglutamate aminopeptidase; TFA, triXuoroacetic acid; DITC, diisothiocyanate; CHCA, -cyano-4-hydroxycinnaminic acid; PBS, phosphate-buVered saline; BSA, bovine serum albumin; RT, room temperature; HRP, horseradish peroxidase; SDS, sodium dodecyl sulfate; PVDF, polyvinylidene diXuoride; RP–HPLC, reverse-phase HPLC; MALDI–TOF, MALDI time- of-Xight; CID, collision-induced dissociation; ELISA, enzyme-linked immunosorbent assay; PITC, phenylisothiocyanate; ATZ, anilinothiazolinone; NTCB, 2-nitro-5-thiocyanobenzoic acid; CDAP, 1-cyano-4-dimethylaminopyridinium tetraXuoroborate.