For Mass Spectrometry, a calibration mixture of Angiotensin I, ACTH (clip 1-17), and ACTH (clip 18-39) (Applied Biosystems, Foster City, CA) was prepared according to the manufacturer instructions. It was combined with the sample as an internal standard at a concentration of 1 pmol/[tL each protein standard. Tryptic digests were cocrystallized with a matrix of c-cyano-4-hydroxycinnamic acid (Aldrich, St. Louis, MO) and analyzed using an Applied Biosystems Voyager-DE Pro MALDI-TOF mass spectrometer operated in the delayed-extraction, reflector mode. This instrument was equipped with a nitrogen laser delivering pulses of ultraviolet light (337 nm) and spectra from 100 individual laser shots were collected for each sample. An accelerating voltage of 20 kV, grid voltage of 72%, and extraction delay time of 200 nsec were used. Peptide Mass Fingerprinting

     Two Web-based programs dedicated to mass-fingerprinting were used to analyze the resulting MALDI-TOF spectra, MS-Fit (http://prospector.ucsf.edu) and Profound (http://129.85.19.192/profound-bin/ProFound.exe). The obtained peptide mass fingerprint spectra were analyzed by searching the National Center for Biotechnology Information nonredundant (NCBI-nr) protein database with Profound or one or more of the following databases with MS-Fit: Swiss-Prot, Genbank (Genpept), and NCBInr. The following standard parameters were used with both programs: charge state: MH+, protein mass range: 1-100 kDa, all species allowed, full range of pI, one missed cleavage allowed, possible modification of cysteine by acrylamide or carbamidomethylation, possible modification of methionine by oxidation, and peptide mass tolerance of +50 ppm. Possible adjustments to the above parameters included: protein mass range and pI range- narrowed or extended according to the 2D gel information, Salmonella