De novo protein sequence analysis of Macaca mulatta

Abstract

Background: Macaca mulatta is one of the most utilized non-human primate species in biomedical research offering unique behavioral, neuroanatomical, and neurobiochemcial similarities to humans. This makes it a unique organism to model various diseases such as psychiatric and neurodegenerative illnesses while also providing insight into the complexities of the primate brain. A major obstacle in utilizing rhesus monkey models for human disease is the paucity of protein annotations for this species (~42,000 protein annotations) compared to 330,210 protein annotations for humans. The lack of available information limits the use of rhesus monkey for proteomic scale studies which rely heavily on database searches for protein identification. While characterization of proteins of interest from Macaca mulatta using the standard database search engines (e.g., MASCOT) can be accomplished, searches must be performed using a 'broad species database' which does not provide optimal confidence in protein annotation. Therefore, it becomes necessary to determine partial or complete amino acid sequences using either manual or automated de novo peptide sequence analysis methods.

Results: The recently popularized MALDI-TOF-TOF mass spectrometer yields a complex MS/MS fragmentation pattern difficult to characterize by manual de novo sequencing method on a proteomics scale. Therefore, PEAKS assisted de novo sequencing was performed on nucleus accumbens cytosolic proteins from Macaca mulatta. The most abundant peptide fragments 'b-ions and y-ions', the less abundant peptide fragments 'a-ions' as well as the immonium ions were utilized to develop confident and complete peptide sequences de novo from MS/MS spectra. The generated sequences were used to perform homology searches to characterize the protein identification.

Conclusion: The current study validates a robust method to confidently characterize the proteins from an incomplete sequence database of Macaca mulatta, using the PEAKS de novo sequencing software, facilitating the use of this animal model in various neuroproteomics studies.

Figure 1

Representative 2D gels of Macaca mulatta protein sample stained with SyproRuby™. The polypeptide molecular mass scale in kDa is depicted on the y-axis while the x-axis shows the pI range. The proteins were resolved in 4–7 linear pH gradient (Immobiline DryStrips; 240 × 3 × 0.5) and 8–15% gradient SDS-PAGE (2400 × 2000 × 1 mm). The results of the proteins that were identified (indicated by arrows) by in-gel trypsin digestion and MALDI-TOF-TOF followed by de novo sequencing are elaborated in Table 1.

Figure 2

Representative De novo analysis of a MALDI-TOF-TOF spectrum. (A) The x- and y-axes show the mass to charge (m/z) ratio and the % abundance of the precursor ion fragments, respectively. The MS/MS spectrum was analyzed by PEAKS de novo sequencing software to generate 'RSALQAAHDAVAQEGQCR'. (B) The table details peptide fragments 'b-ions, y-ions and a-ions' and the neutral losses of water and ammonia for b-ions and y-ions as well as the immonium ions to develop confident and complete peptide sequence de novo from MS/MS spectrum. (C) The SPIDER homology search of this peptide resulted in sequence from Ubiquitin carboxyl-terminal hydrolase isozyme L1 as 'NEAIQAAHDAVAQEGQCR'.

Figure 3

Schematic of the methodology for compilation of protein database for Macaca mulatta from De Novo analysis of MALDI-TOF-TOF spectra.