Identifying Proteins in Zebrafish Embryos Using Spectral Libraries Generated from Dissected Adult Organs and Tissues

Abstract

Spectral libraries provide a sensitive and accurate method for identifying peptides from tandem mass spectra, complementary to searching genome-derived databases or sequencing de novo. Their application requires comprehensive libraries including peptides from low-abundant proteins. Here we describe a method for constructing such libraries using biological differentiation to "fractionate" the proteome by harvesting adult organs and tissues and build comprehensive libraries for identifying proteins in zebrafish (Danio rerio) embryos and larvae (an important and widely used model system). Hierarchical clustering using direct comparison of spectra was used to prioritize organ selection. The resulting and publicly available library covers 14 164 proteins, significantly improved the number of peptide-spectrum matches in zebrafish developmental stages, and can be used on data from different instruments and laboratories. The library contains information on tissue and organ expression of these proteins and is also applicable for adult experiments. The approach itself is not limited to zebrafish but would work for any model system.

Differentiating samples and experimental protocols by direct comparison of tandem mass spectra

“…This is most likely due to the use of a less robust chromatography and electrospray method (lower flow rate, nanoelectrospray). We know from all other work that the setup with the Eksigent chromatography systems, Bruker standard ESI source and amaZon ion trap yields highly reproducible spectra . Unknown bacterial isolates can be putatively identified by comparing their distance in the compareMS2 metric based on shared spectral content from a number of reference datasets from well‐characterised isolates of known species or lineage .…”

“…In celebration of the 50th anniversary of the seminal paper on molecular phylogenetics by Zuckerkandl, Jones and Pauling, we applied this new method in a partial reproduction of the original study of the evolutionary relationship between human and non‐human primates using patterns of tryptic peptides – replacing paper chromatography by tandem mass spectrometry. In planning the acquisition of tandem mass spectrometry data for a zebrafish spectral library, we used the software developed for the primate study, compareMS2, to measure the overlap between different tissue preparations of adult zebrafish and prioritize their analysis . The resulting spectral library is now available from NIST .…”

A full-body transcriptome and proteome resource for the European common carp

“…Peptide separation and MS/MS measurement were performed as described previously [37]. In summary, to separate all peptides from each individual gel lane, ten μL of each sample was loaded and desalted on C18 PepMap 300 Å precolumn (Thermo Scientific) and separated by reversed-phase liquid chromatography using two identical 150 mm 0.3 mm–i.d.…”

Species and tissues specific differentiation of processed animal proteins in aquafeeds using proteomics tools

“…Recently methods based on the direct use of spectral libraries have been suggested [33,34] for unbiased species [20,35,36] and tissue specific [37] classification even of processed samples. To assess if such Fig.…”

“…This commonly involves gel-based or gel-free separation of proteins followed by mass spectrometric identification of proteins and peptides of interest through peptide mass fingerprinting [20]. Complementary to this, recently methods based on the direct use of spectral libraries have been suggested [33,34] that allow for unbiased species [20,35,36] and tissue specific [37] classification even of processed samples.…”