Amphibian Skin Secretomics:  Application of Parallel Quadrupole Time-of-Flight Mass Spectrometry and Peptide Precursor cDNA Cloning to Rapidly Characterize the Skin Secretory Peptidome of Phyllomedusa hypochondrialis azurea: Discovery of a Novel Peptide Family, the Hyposins

Abstract

This study reports the variety of peptides present in the skin secretory peptidome of Phyllomedusa hypochondrialis azurea. Peptide structures, along with post-translational modifications, were elucidated by QTOF MS/MS analysis, cDNA sequencing, or a combination of both. Twenty-two peptides, including 19 novel structures, were identified from six different structural classes, including tryptophyllins, dermorphins, and a novel group of peptides termed hyposins. The study demonstrates the power of this combined approach to mine the rich peptidome compliment of the amphibian defensive skin secretome.

Identification of Biofilm Matrix-Associated Proteins from an Acid Mine Drainage Microbial Community

“…This database contains proteins derived from the 5-way (53) and UBA (34) (53), and common contaminants such as keratin and trypsin (36 proteins). The output data files were then filtered and sorted with the DTASelect algorithm (44) using parameters reported previously to give a false-discovery rate of Ͻ5% (34,44,52). The high-accuracy mass measurements of the LTQ-Orbitrap allowed better than Ϯ10 ppm for ϳ80 to 85% of identified peptides.…”

“…All MS data, including spectral counts, sequence counts, sequence coverage, and normalized spectral abundance factor (NSAF) (34,44,52) values, as well as links to all identified spectra can be found at: http://compbio.ornl.gov/amd_eps/.…”

Automated Mass Spectrometry–Based Functional Assay for the Routine Analysis of the Secretome

“…Although the principle is simple, this method is not very successful, simply because many eukaryotic cells secrete proteins that are intensely posttranslationally modified. [40][41][42][43][44][45][46][47] Therefore, a peak in the MALDI-MS spectrum does not represent a protein or a peptide but rather a posttranslationally modified protein (or peptide). [45][46][47] However, even if the differences detected are not at the "naked" protein level, the differences observed may still be detected and the proteins responsible for them can be identified.…”

“…For example, secreted proteins are glycosylated through both N-and O-glycosidic bonds, and if the peptide backbone is occupied by oligosaccharides, then most likely they will not be identified in a proteomic analysis. 40,[45][46][47][70][71][72] As such, if the glycosylated protein has a low molecular mass, then the chance of being identified decreases. Therefore, the immediate consequence is false-negative data.…”

Antimicrobial peptides from the skin of the Asian frog, Odorrana jingdongensis: De novo sequencing and analysis of tandem mass spectrometry data

“…It is impossible to discriminate the isobaric I/L based on the data of the low energy CID MS/MS [17]; it could be achieved by the high energy collision-induced dissociation (HCD) MS/MS; however, the characteristic losses fragment ions (d or w ions) for the discrimination of I/L in HCD are not commonly observed [30,39,48,49]. Thus, it has been accepted to assign I/L indirectly based on the known sequences of the cDNA clones in the same species [29,50] or on the known sequences of analogues in the same or others species [39,48,49]. This assignment in our study is based on the interspecific distribution of the identical AMPs is common in this genus as described above.…”