PMC

Peptides resulting from processing of prohormones at non-dibasic residues were analyzed by removal of peptides with flanking dibasic residues from the data set, followed by analyses of resultant peptides’ N- and C-termini and flanking residues within parent prohormones. Such data are represented by LOGO maps as follows:
(a) Analyses of N-termini of endogenous peptides derived from prohormones by non-basic residue cleavages. P1-P15 residues flanking the N-termini of peptides within parent prohormones are illustrated.
(b) Analyses of C-termini of peptides derived from prohormones by non-basic residue cleavages. P1’-P15’ residues flanking the C-termini of peptides within proproteins are illustrated.

Peptides derived from human proSAAS () were studied by analyzing the low molecular weight soluble pool of human chromaffin secretory vesicles without protease digestion, with trypsin, or with V8 protease digestion followed by nano-LC-MS/MS analyses. Data for identification of peptides is provided in –, –, with Inspect analyses. Peptides were mapped to proSAAS, illustrated by colored lines: colored lines: QTOF-no enzyme (purple), QTOF-trypsin (bright violet), QTOF-V8 (lavender), Trap-no enzyme (olive), Trap-trypsin (turquoise blue), Trap-V8 (bright green). ProSAAS peptide domains are indicated (SAAS, PEN-LEN, PEN, and LEN regions) found in humans proSAAS () and in other species ().

Endogenous peptides derived from human proenkephalin (PE) () are illustrated with respect to their location within PE. Peptides were identified by ion-trap and QTOF MS/MS, combined with InsPecT (Ins) and Spectrum Mill (SM) bioinformatic analyses of MS/MS data at 1% FDR. Identification of (Leu)enkephalin (*) was found at 5% FDR. MS/MS peptide identifications are summarized in , , , with InsPecT or Spectrum Mill analyses. Peptides identified under each of these conditions were mapped to PE, illustrated by colored lines: QTOF MS/MS data analyzed by InsPect (Ins, orange) or Spectrum Mill (SM, yellow), and ion-trap (Trap) analyzed by insPect (Ins, green) or SM (olive). Within PE, the active enkephalin neuropeptides sequences are shown in yellow. Dibasic cleavage sites are highlighted by boxes; in addition, monobasic residues within PE are shown. Hyphens at the end of some lines indicate peptides that were split between two lines in the figure.

The low molecular weight soluble pool of the isolated human secretory vesicles was subjected to digestion with trypsin or V8 protease, and then identified by LC-MS/MS in the ion trap MS instrument with bionformatic analyses by InsPect. MS/MS identifications of peptides are provided in - and summarized in .
(a) Peptides. The Venn diagram illustrates the numbers of common and unique peptides identified among the three conditions of no protease, trypsin digestion, and V8 digestion. A total of 330 unique peptides were identified among all enzyme conditions. Results are the averages from triplicate experiments
(b) Proteins. The Venn diagram illustrates the numbers of common and unique parent proproteins represented by peptides identified under conditions of no protease, trypsin digestion, and V8 digestion. Results are the averages from triplicate experiments. A total of 23 proteins were identified (list of proteins is in ).

To enhance identification of peptides, the soluble low molecular weight soluble fraction of human chromaffin granules was digested with trypsin or V8 protease for nano-LC-MS/MS identification of peptides. Data are provided in – and - with InsPecT analyses. Peptides derived from proenkephalin (PE) were mapped to PE, illustrated by colored lines: QTOF-no enzyme (purple), QTOF-trypsin (bright violet), QTOF-V8 (lavender), Trap-no enzyme (olive), Trap-trypsin (turquoise blue), Trap-V8 (bright green). Active enkephalin neuropeptides within PE are shown in yellow, and dibasic cleavage sites are highlighted by boxes. Phosphorylated peptides are indicated (light blue with phosphorylation site indicated by dark blue square). Hyphens at the end of some lines indicate peptides that were split between two lines in the figure.

Peptides derived from human proNPY (NPY, neuropeptide Y) () were studied by analyzing the low molecular weight soluble pool of human chromaffin secretory vesicles without protease digestion, with trypsin, or with V8 protease digestion followed by nano-LC-MS/MS analyses. Data for identification of peptides is provided in –, –, with Inspect analyses. Peptides derived from proNPY are mapped, illustrated by colored lines: colored lines: QTOF-no enzyme (purple), QTOF-trypsin (bright violet), QTOF-V8 (lavender), Trap-no enzyme (olive), Trap-trypsin (turquoise blue), Trap-V8 (bright green). Phosphorylated peptides are indicated (light blue with phosphorylation site indicated by dark blue square). ProNPY domains of NPY neuropeptide and the C-terminal peptide are indicated. The signal sequence is also shown since one identified peptide apparently included several residues at the C-terminal end of the putative signal sequence.

Endogenous peptides in the low molecular weight (MW) pool of human chromaffin secretory vesicles were identified by nano-LC-MS/MS (MS/MS data shown in ). Analyses of peptide sequences indicated their derivation from prohormonesw. Amino acid sequences of 15 residues flanking the N- and C-termini of peptides within their precursors were analyzed and depicted by LOGO maps.
(a) Amino acid sequences flanking N-termini of endogenous peptides derived from prohormones. The N-termini of peptides are indicated by the arrow, with amino acid sequences flanking the N-termini illustrated within parent prohormones illustrated by web LOGO maps (). The x-axis indicates residues at the P1 to P15 positions relative to observed cleavage sites at P1-↓P1’ residues. The relative frequency of the single amino acid symbols at each position is illustrated (y-axis).
(b) Amino acid sequences flanking C-termini of endogenous peptides derived from prohormones. The C-termini of peptides are indicated by the arrow, with residues of respective prohormones flanking the C-termini at P1’-P15’ positions illustrated by web LOGO maps.