Characterization by Electrospray Mass Spectrometry of Human Ca2+–sensitive Cytosolic Phospholipase A2 Produced in Baculovirus–infected Insect Cells

Abstract

The 85-kD cytosolic phospholipase A2 (cPLA2) is a novel receptor-regulated phospholipase that is thought to initiate the production of inflammatory lipid mediators. Since cPLA2 is present only in minute amounts (less than 0.01% of total cellular protein) in various cells and tissues, we have used the baculovirus expression system to produce sufficient quantities of cPLA2 for structural and functional analysis. The cDNA for cPLA2 was cloned into a baculovirus expression vector and, upon infection of Spodoptera frugiperda Sf-21 cells with the recombinant virus, cPLA2 was produced at high levels (9% of total cellular soluble protein). Gel electrophoresis and immunoblot analysis demonstrated that the recombinant protein has properties indistinguishable from cPLA2 present in human monocytic U937 cells. Structural analysis of recombinant cPLA2, using electrospray mass spectrometry in conjunction with automated sequence analysis, confirmed the expected sequence and revealed two post-translational modifications of the protein, phosphorylation on at least one site, and acetylation of the N-terminal serine residue after removal of the initiating methionine. In spite of the presence of six potential N-glycosylation sites, there is no evidence that any of them is glycosylated. The baculovirus expression system should prove useful for production of cPLA2, and electrospray mass spectrometry is a rapid and accurate method for the analysis of post-translational modifications.

Increased Sensitivity of Tryptic Peptide Detection by MALDI-TOF Mass Spectrometry Is Achieved by Conversion of Lysine to Homoarginine

“…The column effluent was passed through a splitting T valve with 98% of the flow passed through a UV absorbance detector and 2% introduced to an electrospray mass spectrometer. Mass spectra were collected on the column effluent on an Sciex API III triple quadrapole instrument as previously described (8).…”

Comparative vaccine efficacy of different isoforms of recombinant protective antigen against Bacillus anthracis spore challenge in rabbits

“…A follow-on study that defines the identity and extent of post-translational modifications for amino acids in rPA by LC/MS/MS will be described separately (Powell et al, in preparation). A reduction or complete loss of in vitro or in vivo biological activities has been reported for a variety of deamidated proteins [27,28], while others appear unaffected [29]. It is therefore important to establish methods for evaluating the effects of deamidation on the biological activities and antigenicities of vaccine candidate polypeptides.…”

Biological Activity of Recombinant Bovine Interferon .TAU. Using an Autographa california Nuclear Polyhedrosis Virus Expression System

“…In recent years, recombinant IFNτ has been produced using bacteria-or yeast-expression systems [1,13]. Insect cells have similarities with mammalian cells in regard to post-translational events such as proteolytic processing [14], phosphorylation of serine residues [15,16], C-terminal amination and glycosylation [17]. These properties of insect cells are thought to be useful for the production of recombinant mammalian proteins by baculovirusinsect cell systems, because the proteins produced are expected to be structurally and biologically equivalent to the native proteins.…”