Many species have been used to model various aspects of human diseases including mental illness. However, the complexity of human biochemistry, anatomy and behavioral factors are not easily modeled in all species and warrant the use of species which have greater degrees of functional equivalence for the measures under investigation. For example, the experimental use of Macaca mulatta (rhesus monkey) has been essential for expanding our knowledge of neurodevelopmental, neurodegenerative and organic human brain diseases, as well as, for normal brain function due in large part to close similarities in neuroanatomy, neurobiochemistry and behavior compared with other species. Moreover, the use of Macaca mulatta has significant translational value for understanding the influence of alterations in gene and protein expression in human disease processes.
One potential obstacle to comprehensive assessments of protein alterations is the relative paucity of available protein annotations for rhesus monkeys. Currently; NCBInr, Swiss-Prot and TrEMBL list 330210, 14991 and 55805 human protein annotations, respectively. However, NCBInr, Swiss-Prot and TrEMBL list only 41968, 297 and 1801 protein annotations for rhesus monkey, respectively. The highly uncharacterized nature of the rhesus monkey proteome makes it difficult to identify proteins, demonstrate differential regulation of proteins and investigate their post-translational modifications. The characterization of proteins of interest from rhesus monkey using the standard database search engines (e.g., MASCOT) has a limitation in that 'broad species database' searches are needed which results in less than optimal protein annotation. This limitation can be overcome in some respects using a de novo sequencing strategy, in which partial or complete amino acid sequence information is obtained using either manual or automated de novo peptide sequence analysis. This approach has been successfully utilized in recent studies to characterize peptides bound to class I MHC molecule HLA-A2.1[1], human skin elastin protein[2] and proteins from unsequenced genome of Halorhodospira halophila[3].
While manual protein sequencing via Edman degradation yields exact amino acid sequence without ambiguity, the procedure is laborious and does not lend itself to high-throughput analysis. It also lacks the sensitivity of mass spectrometry and can be halted by the presence of blocked amino acids. Fortunately, automated software tools have been developed to characterize the amino acid sequences generated from tandem mass spectrometry such as the complex MS/MS fragmentation pattern generated by MALDI-TOF-TOF mass spectrometer. Quality MS/MS spectrum consists of a ladder for y-ions and b-ions peaks. De novo sequencing uses the mass difference between two adjacent ions to deduce the peptide fragment sequence. However, factors such as incomplete fragmentation (whereby not all the y- and b-ions are present in the spectrum), imprecise precursor ion selection due to overlapping peptide fragment masses, low signal-to-noise ratio and unpredicted post-translational modifications (PTMs) complicate the manual de novo sequencing. De novo sequencing enables the analysis of quality MS/MS spectra which fails to generate protein identification after database searches, which is the case for the majority of proteins in rhesus monkeys. In fact, de novo sequencing is the only alternative for study of species with incomplete databases and databases which are not in the public domain [2,4-6]. Several strategies have been utilized more recently for de novo sequencing including chemical derivatization that add acidic, fixed charge or basic moieties[1,7-13]. These have included various derivatization protocols such as sulfonation of the peptide N-terminal group. This derivatization creates a strong acidic group which greatly enhances fragmentation ability of tryptic peptides to produce b and y fragments. The use of 18O incorporation at the C-terminus of peptides during protein hydrolysis has also been applied [14,15]. The current study shows the utility of underivatized peptides using MALDI-TOF-TOF.
To this end, de novo sequencing of peptides isolated from cytosolic fractions of the ventral striatum of rhesus monkeys was performed. The ventral striatum/nucleus accumbens is an integral component of the cortico-straital-palldial-thalamic/mesencephalic circuit which is involved in sensorimotor integration. Furthermore, dysregulation of the ventral striatum has been implicated in a variety of psychiatric disorders, including substance abuse [16,17], schizophrenia [18-20] and depression [21-23]. The use of the ventral striatum tissue and the cytosolic fraction from this region is based on the research interests of our laboratory. However, the de novo sequencing strategy presented here is generalizable to all brain regions, tissues and other protein preparations.
The presented method consists primarily of de novo sequencing of underivatized peptides using the MALDI-TOF-TOF, compared to various derivatizing strategies used recently for various de novo studies. The generated sequences were used to perform homology searches to characterize the protein identification. The current study validates a robust method to confidently characterize proteins from an incomplete sequence database of Macaca mulatta thereby facilitating the use of this animal model in various neuroproteomics studies.
Answers to all your Biology Questions
