A Sulfated, Phosphorylated 7 kDa Secreted Peptide Characterized by Direct Analysis of Cell Culture Media Steven W. Taylor,* Chengzao Sun, Amy Hsieh, Nancy L. Andon, and Soumitra S. Ghosh Amylin Pharmaceuticals, Inc., 9360 Towne Centre Drive, San Diego, California 92121 Received July 13, 2007 Abstract: An unusual sulfotyrosine-, phosphoserine- containing motif was mapped on a differentially post- translationally modified 60 residue antimicrobial neuroen- docrine peptide called chrombacin. The study was performed by high resolution FT MS using complemen- tary fragmentation techniques. The peptide was analyzed at low levels directly from cell culture media in contrast to previous reports that required extensive purification and proteolytic digestion. The sulfation site was not previously described nor predicted by informatic analysis of the peptide’s precursor sequence. Keywords: chromogranin • FT-MS • insulinoma • pepti- domics • post-translational modification Introduction Bioactive peptide discovery and structural elucidation has traditionally involved collection of a large amount of source material followed by subfractionation and chromatographic purification monitoring enrichment of a specific activity. Today, the sensitivity of mass spectrometry (MS) allows peptide sequence characterization using much smaller amounts of material (<1–1000 fmole) depending on the peptide’s size, charge, and fragmentation. Microfluidic separations prior to MS greatly enhance the ability to identify peptides even in complex mixtures, and complementary fragmentation tech- niques (e.g., collision induced dissociation (CID) combined with electron capture dissociation (ECD)) aid in obtaining compre- hensive sequence coverage. Recently, the value of “peptidomic” profiling of cell culture models has been demonstrated. 1–3 In the current study, we characterized a large differentially post- translationally modified neuroendocrine peptide called chrom- bacin. Chrombacin was first reported as one of a group of antimicrobial peptides derived from the protein precursor, chromogranin B. 4 Chromogranins are large acidic secretory glycoproteins with a widespread distribution in neuroendocrine tissue and are suggested to be involved in the sorting, process- ing, or packaging of material destined for exocytosis. 5 Chro- mogranins undergo extensive post-translational processing, including cleavage at mono- or dibasic sites (identical to peptide hormone processing) as well as glycosylation, phos- phorylation, sulfation, and C-terminal amidation. In this study we probed chrombacin’s structure at low levels by direct online “Top Down” proteomic analysis of cell culture media and further characterized its microheterogeneity in post-transla- tional modification (Table 1). Our workflow involved neither scale up, purification, nor enzymatic digestion of this large peptide (Scheme 1). Chrombacin displayed an unusual motif consisting of a phosphorylated serine and an unanticipated sulfated tyrosine residue. Experimental Procedures Sample Preparation. Rin-m5F (ATCC) cells were cultured and stimulated with forskolin as has been previously de- scribed, 3 except that RPMI (Invitrogen) media containing forskolin was removed after 5 min and subsequently replaced with forskolin-free RPMI media for remainder of the 1 h incubation. Media were subsequently collected as 900 μL aliquots in prewashed Lo-Bind tubes (Eppendorf) containing 310 μL of acetonitrile, 31 μL of formic acid, and 12.6 μL of 10% TFA in water (resulting in final concentrations of 25%, 2.5%, and 0.1%). Solid-phase extraction of 2 mL aliquots of Rin conditioned media pools was performed over a Michrom peptide MicroTrap, adapted from a previously described pro- tocol. 3 The MicroTrap was prepared by cleaning with 25 μL of 60% acetonitrile, 0.1% formic acid in water followed by 25 μL of 90% acetonitrile, 0.1% formic acid in water. The MicroTrap was then equilibrated with 50 μL of 5% acetonitrile, 0.1% formic acid in water. The conditioned media samples were manually loaded onto the trap at a flowrate of 100–200 μL/min. The trap was washed with 50 μL of 5% acetonitrile, 0.1% formic acid in water. The peptides were eluted with 12 μL of 60% acetonitrile, 0.1% formic acid in water and 12 μL of 90% acetonitrile, 0.1% formic acid in water into an autosampler vial. MALDI-TOF MS MALDI-TOF MS was performed using a Voyager-DE STR (Applied Biosystems, Foster City, CA, USA) in linear delayed extraction mode. Rin samples were applied to a 100-well stainless steel MALDI-TOF target plate (Applied Biosystems) that had been previously coated with methanol-sinapinic matrix solution as described by Gutierrez and co-workers. 6 For spotting on the MALDI-TOF stainless steel target plate, 1 μL of sample was mixed with 1 μL of sinapinic acid matrix solution (10 mg/mL) directly on target. The instrument settings were: positive ion linear mode, manual control, accelerating voltage of 20 000, grid percentage of 93.5, delay time of 500 ns, 500 shots per spectrum, and mass range of 3000-9000 Da with a low mass gate of 3000 Da. Data Explorer, version 4.0 (Applied Biosystems, Foster City, CA, USA), was used for data analyses post acquisition of spectra. Mass spectral processing included mass calibration, baseline correction, and noise filtering. * Corresponding author. E-mail: staylor@amylin.com. 10.1021/pr7006686 CCC: $40.75  2008 American Chemical Society Journal of Proteome Research 2008, 7, 795–802 795 Published on Web 01/09/2008