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1.
Fig. 2

Fig. 2. From: Investigating Endogenous Peptides and Peptidases using Peptidomics.

Posttranslational modifications found on bioactive peptides. Some modifications, such as N-terminal acetylation or C-terminal amidation, are common, while others, such as serine octanoylation are rare.

Arthur D. Tinoco, et al. Biochemistry. ;50(35):7447-7461.
2.
Fig. 4

Fig. 4. From: Investigating Endogenous Peptides and Peptidases using Peptidomics.

The key steps in any peptidomics experiment include sample preparation and peptide isolation, processing of the sample (including isotopic labeling), and finally detection and data analysis. While these steps vary in subtle ways from experiment-to-experiment the overall workflow is consistent for most peptidomics experiments.

Arthur D. Tinoco, et al. Biochemistry. ;50(35):7447-7461.
3.
Fig. 11

Fig. 11. From: Investigating Endogenous Peptides and Peptidases using Peptidomics.

Peptidomics revealed an unappreciated physiological pathway for the renal catabolism of proline containing peptides that interlinks aminopeptidase (AP) and dipeptidyl peptidase 4 (DPP4) activities. These experiments demonstrate the value of peptidomics in understanding peptide processing and characterizing the biochemical and physiological functions of enzymes.

Arthur D. Tinoco, et al. Biochemistry. ;50(35):7447-7461.
4.
Fig. 3

Fig. 3. From: Investigating Endogenous Peptides and Peptidases using Peptidomics.

Peptide hormone signaling regulates physiology and can be harnessed for therapeutic gain. DPP4 inhibitors extend the half-life of the bioactive form of the insulinotropic hormone GLP-1. In doing so these inhibitors increase insulin secretion and improve physiological glucose tolerance, which is of tremendous value in the treatment of diabetes.

Arthur D. Tinoco, et al. Biochemistry. ;50(35):7447-7461.
5.
Fig. 12

Fig. 12. From: Investigating Endogenous Peptides and Peptidases using Peptidomics.

A peptidomics-based approach to identify physiologically relevant proteolytic pathways that process peptide hormones. In this approach, peptidomics identification of fragments of bioactive peptides enables physiological-relevant proteolytic pathways to be identified. Application of this approach to the peptide hormone PHI(1-27) identified a previously unappreciated pathway for the C-terminal proteolysis of this peptide. Interestingly, this pathway was also shown to regulate the activity of PHI(1-27) in a cell-based glucose-stimulated insulin secretion (GSIS) assay.

Arthur D. Tinoco, et al. Biochemistry. ;50(35):7447-7461.
6.
Fig. 9

Fig. 9. From: Investigating Endogenous Peptides and Peptidases using Peptidomics.

Identification of endogenous substrates of peptidases relies on peptidomics. While there are subtle differences in the methods for quantitation and data analysis the overall workflow for these experiments is similar. In this approach, comparison of the peptidomes of mice that differ in the activity of a particular peptidase/protease can reveal peptides regulated by the enzyme. These peptides will include substrates and/or products of the enzyme, which in turn, can be used to infer biochemical and biological function of the peptidase/protease.

Arthur D. Tinoco, et al. Biochemistry. ;50(35):7447-7461.
7.
Fig. 8

Fig. 8. From: Investigating Endogenous Peptides and Peptidases using Peptidomics.

Neuropeptidomics of honeybees as they arrive (A) or depart (D) a food source (pollen (P) or nectar (N)) demonstrated that there are specific neuropeptides that are associated with food gathering in honeybees. By correlating changes in these peptide levels with phenotypes associated with forging, these studies provided new insights into the functions of these peptides, and revealed the ability of peptidomics to correlate changes in peptide levels with complex behaviors.

Arthur D. Tinoco, et al. Biochemistry. ;50(35):7447-7461.
8.
Fig. 6

Fig. 6. From: Investigating Endogenous Peptides and Peptidases using Peptidomics.

Peptidomics of highly basic peptides from QGP-1 cells revealed the production of a series of IFGBP-5 peptides. While this protein was predicted to be secreted, it had not previously been hypothesized to produce bioactive peptides. The structure of the antimicrobial-IFGBP-5 (AMP-IFGBP-5) was highly charged and reminiscent of known defensin antimicrobial peptides. Subsequent antimicrobial and antifungal assays, demonstrated that AMP-IFGBP-5 has antimicrobial and antifungal activity equal to or better than that of defensins. Lastly, AMP-IFGBP-5 was present in the gut, which suggests that this peptide might play a role in innate immunity or regulation of the microbiome.

Arthur D. Tinoco, et al. Biochemistry. ;50(35):7447-7461.
9.
Fig. 7

Fig. 7. From: Investigating Endogenous Peptides and Peptidases using Peptidomics.

There are two primary approaches used for quantitative proteomics: stable isotope labeling methods and label-free approaches. Isotopic labeling relies on the chemical labeling of the peptidome using stable isotope variants of the same reagent. Analysis of the MS for each peptide should reveal a heavy and light labeled version of the peptide and the ratio of these peptides enables quantitation. By contrast, in label-free approaches samples are run sequentially and the peak intensities are used to determine changes in the concentration of the peptide between two samples.

Arthur D. Tinoco, et al. Biochemistry. ;50(35):7447-7461.
10.
Fig. 5

Fig. 5. From: Investigating Endogenous Peptides and Peptidases using Peptidomics.

Discovery of novel bioactive peptides using structural features of the peptide as a guide. A number of bioactive peptides contain C-terminal amides, which result from enzymatic production in the secretory pathway. Analysis of peptides secreted by TT cells using a peptidomics approach led to the discovery of the neuroendocrine regulatory peptides (NERPs). These peptides were later shown to co-localize with arginine vasopressin (AVP), a hormone involved in blood pressure, and physiological studies demonstrated that NERPs control AVP release in vivo. In doing so, this approach demonstrates the utility of peptidomics in accelerating the discovery of novel bioactive peptides.

Arthur D. Tinoco, et al. Biochemistry. ;50(35):7447-7461.
11.
Fig. 10

Fig. 10. From: Investigating Endogenous Peptides and Peptidases using Peptidomics.

Peptidomics of Prep revealed that Prep regulates endogenous levels of CGRP(20-37). Follow up in vitro experiments demonstrated the Prep is able to process the shorter CGRP(20-37) but not the longer, full-length, peptide CGRP(1-37). This length preference enables Prep to participate in the catabolism of CGRP without regulating the bioactive form of the molecule (i.e., CGRP(1-37)), even though the cut sites are identical within the two molecules. This data supports a model of CGRP proteolysis in the nervous system where the full-length CGRP is processed by unknown enzymes followed by Prep processing of the proline-containing CGRP(20-37) fragment.

Arthur D. Tinoco, et al. Biochemistry. ;50(35):7447-7461.
12.
Fig. 1

Fig. 1. From: Investigating Endogenous Peptides and Peptidases using Peptidomics.

The key steps in the production and regulation of bioactive peptides. A prepropeptide is produced from mRNA encoding bioactive peptides. This peptide enters the secretory pathway through the ER and Golgi, before being packaged into secretory vesicles. In the trans Glogi as well as the secretory vesicles the prohormone encounters subtilisin-like proteases, called prohormone convertases, that process the prohormone to generate a mature form of the bioactive peptide. During this maturation process these peptides can also obtain additional posttranslational modifications, such as C-terminal amidation and N-terminal acetylation. Stimulation of cells can lead to secretion of the peptides through fusion of the vesicles with the plasma membrane. Once released the peptides can bind to receptors, typically G-protein coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs), to elicit a cellular or physiological response. Once released, peptides can also undergo proteolysis to regulate their activity.

Arthur D. Tinoco, et al. Biochemistry. ;50(35):7447-7461.

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