Novel mass spectrometry based technologies, such as SELDI-TOF-MS, have shown promising results especially when combined with biospecific interactive surfaces. The use of specific antibodies applied on the SELDI chip surface is a powerful tool to selectively identify a desired target in a complex protein mixture. Accurate information about the molecular weight, discrimination between isoforms and possible capture of co-precipitating proteins, represent some of the most attractive information that could be obtained by using this technology.
MCP-4/CCL13 is a chemokine whose concentration in the blood is in the range of 200–300 pg/ml [
3,
7]. This protein is generally easily detected and quantified by ELISA but not by Western Blot analysis [
11]. Matrix assisted laser desorption mass spectrometry (MALDI) analysis of purified recombinant human MCP-4/CCL13 expressed in insect revealed a predominant peak with a mass of about 8575 m/z, which starts with residue 24at QPDAL. Two minor peaks at about 8760 and 9314 m/z, starting at residue 22 and 17 respectively were also detected. The alignment of MCP-4/CCL13 sequence with those of well characterized members of the CC-chemokine family strongly suggested that the recombinant form of m/z 8575, synthesized by
Drosophila cells, is the correct mature protein [
11,
12]. Alternative splicing has not been found [
13]. To date, human MCP-4/CCL13 has not yet been purified from serum or other human fluid/tissue and no evidence of naturally occurring "isoforms" have been reported. By using SELDI immunoaffinity capture chip technology we were able to identify two MCP-4/CCL13 isoforms and evaluate their molecular weight in a complex protein mixture such as serum. The molecular weights of the isoforms we detected in the serum do not correspond to those previously described for the recombinant protein. We detected a major peak at m/z of 8945 instead of 8575, and a minor peak of a m/z of 9113 instead of 8760. The apparent molecular weight of the major serum component is consistent with a peptide starting at residue 20 and extending to residue 98 of the amino acid sequence deduced from cDNA. Thus it would appear possible that the signal peptide is 19 amino acids and not the 16 previously postulated from the recombinant forms (UniProtKB/SWISS-Pro-entry-Q996126) [
15]. Three possible phosphorylation sites (S 44, S 50, T 55) are found in the MCP-4/CCL13 sequence when analyzed by NetPhos 2.0 Server (EXPASY database
http://au.expasy.org/), these are the only post translational modifications potentially occurring given the MCP-4/CCL13 amino acid sequence. It is possible that some of the differences between the isoforms we detected and those observed in the recombinant protein are the result of phosphorylation. We cannot exclude post-translational modification of the serum MCP-4/CCL13 nor speculate on the identity of our 9113 M/Z component. Our assessment of MCP-4/CCL13 isoforms by SELDI immunocapture was purely qualitative (differential quantification between pre and post treatment samples was not consistent). We postulated that in our experimental condition, saturation of antibody crosslinked to the MCP-4/CCL13 protein on the SELDI chip was the limiting factor as previously described by Hess et al. [
10]. In conclusion, this short report establishes for the first time the occurrence of two different isoforms of human MCP-4/CCL13 in the peripheral blood. Furthermore, it demonstrates the versatility of SELDI in rapidly detecting MCP-4/CCL13 isoforms.