Expression, purification and mass spectrometric analysis of LIM mineralization protein-1 in human lung epithelial cells

LIM mineralization protein-1 (LMP-1) is a novel osteoin-ductive protein that has been cloned and shown to induce bone formation both in vitro and in vivo. Detection and evaluation of the possible presence of carbohydrate structures in LMP-1 is an important regulatory consideration for the therapeutic use of recombinantly expressed protein. The sequence of LMP-1 contains a highly conserved N-terminal PDZ domain and three C-terminal LIM domains. The sequence analysis of LMP-1 predicts two potential N-glycosylation sites and several O-glycosylation sites. Here, we report the cloning and overexpression of LMP-1 in human lung carcinoma (A549) cells. Even though our group already reported the sequence of LMP-1 cDNA, we undertook this work to clarify whether or not the overexpressed protein undergoes any glycosylation in vivo. The expressed full-length recombinant protein was purified and subjected to chemical analysis and internal sequencing. The absence of any hexosamines (N-acetyl glucosamine or N-acetyl galactosamine) in chemical composition analysis of LMP-1 protein revealed that there is little or no post-translational glycosylation of the LMP-1 polypeptide in lung carcinoma cells (A549). We performed in-gel trypsin digestion on purified LMP-1, and the resulting peptide digests were analyzed further using matrix-assisted laser desorption and ionization mass spectrometry for peptide mass finger printing, which produced several exact matches with the corresponding LMP-1 peptides. Separation by high performance liquid chromatography and purification of the desired peptides followed by N-terminal sequencing resulted in many exact LMP-1 matches for several purified peptides, thus establishing the identity of the purified protein as LMP-1.