Speaker: Oleg Chertov, IRSP, SAIC-Frederick, NCI-FCRDC, Frederick, MD

Topic: The Use of a Proteomics Approach (High Through-Put Protein Analysis Based on Mass Spectrometry) for the Identification and Functional Studies of Neutrophil Granule Proteins

Place: Building 426, Conference Room, NCI-Frederick, Frederick, MD

Time: Thursday, September 21, 2000, at 2:00 PM

Abstract: For several years the Laboratory of Molecular Immunoregulation has been studying neutrophil dependence of mononuclear cells infiltration into sites of inflammation. Previously we identified a-defensins (JBC, 1996; 271:2935) and cathepsin G (JExpMed, 1997; 186:739) as neutrophil polypeptides responsible for attraction of T lymphocytes and monocytes. We identified these polypeptides by amino acid sequencing of chemotactically active HPLC fractions obtained from neutrophil granule lysate. We expected that other neutrophil granule proteins, which we were unable to purify to homogeneity in sufficient quantity, might also contribute to this activity. The availability of new highly sensitive mass-spectroscopic methods for identification of proteins made it possible to try to identify all or at least the most proteins in these HPLC fractions. So we prepared neutrophil granules, lysed them and subjected the obtained protein mixture to RP-HPLC. Further analysis was performed by Dr. Joseph Wooters from Genetics Institute (Cambridge, MA). Briefly, the fractions were separated by SDS-PAGE and silver stained. A total of 165 gel bands were excised and digested with trypsin using the Abimed DigestPro robot. A tryptic digest was injected onto a microcapillary C18 column (75 mm x 10 cm) eluting directly into a Finnigan LcQ ion trap mass spectrometer. Typically 500 to 900 collision spectra were collected in a single LC-MS-MS experiment. LcQ datafiles were searched against the nonredundant protein database using the SEQUEST search algorithm running on a PC cluster. In all more than 150 different proteins were assigned including all 19 proteins recently identified in neutrophil granules using 2D gel-electrophoresis. Detected proteins could be separated into three main groups: 1) known proteins, 2) proteins represented by ESTs and 3) proteins absent in available data bases. So what to do with this treasure of information? Analyze. For example, in one of the chemotactically active fractions an antimicrobial peptide LL-37 (cathelicidin) was detected. We checked the chemotactic activity of corresponding synthetic peptide and found that it is indeed chemotactic for monocytes, T cells and neutrophils. Then we identified its receptor, which appeared to be formylpeptide receptor-like 1 receptor (FPRL1). Now we can propose that neutrophil granule peptide LL37 as well as a-defensins along with their antibacterial activity are also involved in the recruitment of the immune cell subsets that potentially contribute to the initiation of adaptive immunity. Thus these peptides appear to mobilize both innate and adaptive immune responses (Yang et al, J.Exp.Med, 2000, in press). More examples will be presented during seminar.