TuP-15

IDENTIFICATION OF PROTEOLYTIC FRAGMENTS OF PEPTIDE HORMONES BY LC-MS AND LC-MS-MS

Anthony M. Treston*, Richard H. Snider**, Eric S. Nylen**, Michael J. Kelley#, and Kenneth L. Becker§**

*BPRB, National Cancer Institute, Rockville MD 20850-3300

**Metabolism Division, Veterans Affairs Medical Center, Washington DC

#NCI-Navy Med. Oncol. Branch, National Cancer Institute, Bethesda MD 20889

§Division of Endocrinology, George Washington University, Washington DC, USA.

Peptide hormones are growth factors for human lung cancer cells. Possible degradative pathways are therefore potential targets for therapies aimed at reducing tumor growth rates. Neutral endopeptidase (NEP, EC 3.4.24.11; known as CALLA, CD10) is a metallopeptidase located on the cell surface which cleaves bioactive peptides, including gastrin releasing peptide (GRP) and atrial natriuretic hormone [1,2]. Inhibition of NEP results in increased levels of GRP and more rapid cell growth in lung cancer cell lines producing GRP. These cell lines also secrete other peptides including products of the CALC1 gene, calcitonin (CT) and katacalcin (KAT) [3].

NEP was provided by R. Bridenbraugh (Genentech). Endoproteolytic digests of synthetic CT (Ct=CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGA-amide) and KAT (KAT=DMSSDLERDHRPHVSMPQNAN-amide) peptides (Peninsula) was carried out in an 100 µL reaction mix containing 20 nmol of peptide, 400 ng NEP, 5 mMHEPES, 0.15 M NaCl. Reactions were carried out for two time intervals (30min and 4 h) to yield both partial and complete digestion products. The peptides were analyzed using a C18 reversed phase HPLC cloumn (Axxiom, Thompson Instruments) with a gradient from 0-50% acetonitrile in 0.1 M ammonium sulfate/0.1%TFA. Several fragments were identified by comparison of UV peak retention volumes to standards (previously identified peptides). Unidentified peptides were collected by hand and the fractions freeze-dried.

The resulting solid (usually 0.3-1.5 nmol peptide with 0.1-0.4 mmol ammonium sulfate) was dissolved in 100-200 µL of water/0.1% trifluoroacetic acid immediately before analysis by LC-MS. To desalt the sample, 50 µL was injected onto a 2.1 mm Vydac C18 column at 100 µL/min. The effluent was diverted from the mass spectrometer source from t+2 min to t+7 min to allow salt to be eluted. The column effluent was split 1/20 (Accurate flow splitter, LC Packings,) and 5 µL/min was introduced into the source of a PE-SCIEX API 111-plus triple quadrupole mass spectrometer (the balance was collected and freeze-dried for further analyses). Initial analyses were carried out as Q1 scans from 300-1200 amu at orifice potentials of 60 or 100V. Mass spectral resolution was ca. 1000. Peptides were eluted with a linear 1%/min acetonitrile gradient. Closely-eluting polymers (detergent) were identified in all samples, but did not interfere with the mass analysis of the peptides. Most peptides were identifiable on the basis of their mass in Q1 scan mode. Allpeptides were re-analyzed by LC-MS-MS using Ar/He collision gas at a CGT of 300-350 mTorr and collision energies of 40-120 V.

We have unequivocally identified 3 endoprotease-generated peptides in digests of CT and 3 peptide in digests of KAT using discrete mass and LC-MS-MS sequence information. Digests of additional peptides are undergoing analysis.

  1. Nadel, J.A. Am. Rev. Resp. Dis. 145:S48, 1992.
  2. Johnson, B. et al. Am Rev. Resp. Dis 132:565, 1985.
  3. Kelley, M.J .et al. Cancer Lett 81:19, 1994.