Peptides from the anterior and posterior portions of 6-day regenerated S. mediterranea were extracted into acidified acetone, which was used to extract peptides in a previous study on
elucidating the prohormone complement of planarians.23 Combining the anterior and posterior portions allows peptides from both the cephalic ganglia and blastema to be extracted at once. To achieve an in-depth coverage of the peptidome, two-stage LC analysis was conducted on the peptide extract. All fractions from first stage LC were profiled via MALDI-TOF MS, and only those with putative peptide signals were further subjected to second-stage LC-ESI MS/MS analysis. During the extraction process, an oily phase and some undissolved residue were observed. These two parts were desalted via solid phase extraction and also profiled using MALDI- TOF MS. Peptide signals were observed in the undissolved residue, which was therefore also subjected to LC-ESI MS/MS analysis. Obtained MS/MS spectra were searched against a previously established S. mediterranea protein database.23
Peptidomics results were back-referenced to the imaging data set to identify the markers revealed from MSI (Table 6.1, 6.2). Identification results from this effort are presented in Table 6.4. Not all MSI markers were detected during peptidomics analysis, likely a result of ionization differences between ESI and MALDI. For ions that are characteristic of the cephalic ganglia (Table 6.1), MS/MS analysis identified most of these as peptides derived from known S. mediterranea prohormones, which is expected since these prohormones are generally located toward the cephalic ganglia.23 Among these peptides, the peptide derived from secreted peptide prohormone 4 (m/z
1223.9; SPP-4 [33-42]) appears not to have recovered to the intact level even after 12 days of regeneration (Table 6.1, 6.4). Indeed, while peptides from other prohormones, such as EYE53-1, are not significantly different between intact and 12-day regenerated planarians, SPP-4 [33-42] remains at significantly lower intensity even 12 days after amputation (P=0.03). EYE53-1 is necessary for proper visual system function.46 This pattern suggests that although most structure
and chemical differences, such as the visual system, are recovered after 12 days of regeneration, chemical differences can still be observed and therefore not all functions may have been restored.
Peptide markers that do not match entries in the protein database were also detected. In particular, all the peptide markers for the blastema (Table 6.2) do not match entries in the database, which may be because the database was constructed from intact planarians and not regenerating ones like those used in this study. Because there were no matches against the protein database, obtained de novo peptide sequence tags were searched against the planarian transcriptome, and their BLAST matches against sequences from other organisms were examined. Sequences presented in Table 6.4 are complete sequences produced via the PEAKS software package. However, because each sequence contains residues with varying amount of support from the associated MS/MS spectrum, it is typically difficult to find BLAST results that match the entire
de novo sequence. The presented BLAST results therefore focuses on matching residues with local
confidence above 80%, as indicated by the PEAKS algorithm. Focusing on high confident residues reduced impact on the search results by residues that are weakly supported by MS/MS. For example, sequences containing a single cysteine that is modified with half a disulfide bond are unexpected, as two cysteine residues are required to make a disulfide bond, and these samples were not treated to reduce disulfide bonds prior to analysis. In all such sequences, the modified cysteine residue has a low confidence score and were not focused on for the search. BLAST searching in this manner revealed proteins involved in a variety of functions. These include sequences with similarities to histone and other DNA and RNA binding domains. Although these proteins should normally be present in intact planarians, they likely play an important role during regeneration for synthesizing new proteins to replace the missing tissue. Other detected peptides contain similarities to proteins involved in cellular trafficking, such as ANTH-like domain and
regulator of vacuolar protein sorting (Vps). Sequences without BLAST matches were detected as well and are entered in Table 6.4 with their FASTA description from the transcriptome database. One of these contain a characteristic signal peptide near the N-terminus, suggesting that it may be destined for the secretory pathway.
One of the markers associated with the blastema has sequence similarity to histone H4. Histone is a protein that helps compact DNA to form chromosomes, plays a role in regulating DNA transcription, and is involved with DNA replication during mitosis.47 In S. mediterranea,
histone H4 RNA is used as a marker for neoblasts.3 Unlike other cells in planarians, neoblasts contain cytoplasmic chromatoid bodies, and ~80% of all chromatoid bodies contain histone H4 mRNA.3 The exact function of chromatoid bodies is not known. However, it has been speculated that chromatoid bodies help regulate histone synthesis in neoblasts. During regeneration, neoblasts proliferate at the base of the blastema, but do not enter the actual blastema region until becoming descendants.48 Once differentiated to a target cell type, neoblasts lose their chromatoid bodies along with the associated histone RNA. Ion images show histone H4 signal increases toward the base of the blastema in 3-day regenerated S. mediterranea (Figure 6.10), where neoblasts are proliferating. This localization pattern corroborates increased histone synthesis during neoblast proliferation and may be evidence of unique regulation of histone synthesis in S. mediterranea.
6.4.5 Peptidomics Analysis of Regenerating Planarians. Beyond identifying markers