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We have applied a recently developed label-free mass spectrometry based peptidomic approach to identify and quantify a variety of endogenous peptides from rat nucleus accumbens following withdrawal in naloxone-precipitated, morphine-dependent rats of two separate strains. We focused on maturated, partially processed and truncated peptides derived from the peptide precursors proenkephalin, prodynorphin and preprotachykinin. The expression of several identified peptides was dependent on strain and was affected during morphine withdrawal.

The combination of surface plasmon resonance and mass spectrometry is emerging as a sensitive tool for the elucidation of protein-protein interactions. With the use of surface plasmon resonance-mass spectrometry, peptides, and brain extracts, we now report a novel interaction between the voltage-gated sodium channel type X alpha-subunit and caveolin-1, the central protein controlling caveolae formation. Surface plasmon resonance binding analyses show that this interaction involves amino acids 85-103 of voltage-gated sodium channel type X alpha-subunit and amino acids 81-100 of caveolin-1, a known scaffolding domain of caveolin-1. It is anticipated that the surface plasmon resonance-mass spectrometry approach utilized in this study will be important for the elucidation of protein-protein network analysis in native tissues including the brain.

FKBP-12, a 12 kDa FK506-binding protein (neuroimmunophilin), acts as a receptor for the immunosuppressant drug FK506. Neuroimmunophilins, including FKBP-12, are abundant in the brain and have been shown to be involved in reversing neuronal degeneration and preventing cell death. In this report, we have utilized several analytical techniques, such as in situ hybridization, Western blotting, two-dimensional gel electrophoresis, and liquid chromatography electrospray tandem mass spectrometry to study the transcriptional expression as well as protein levels of FKBP-12 in the unilateral 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease. The FKBP-12 protein was also detected directly on brain tissue sections using mass spectrometry profiling. We found increased levels of FKBP-12 mRNA and protein in the dorsal and middle part of the 6-OHDA lesioned striatum. Thus, these studies clearly demonstrate that FKBP-12 is increased in the brain of a common animal model of Parkinson's disease (PD). Additionally, we have identified potential binding partners to FKBP-12 that may be implicated in the pathophysiology of Parkinson's disease, such as alpha-enolase, 14-3-3 zeta/delta, pyruvate kinase isozymes, and heat shock protein 70, using surface plasmon resonance sensor technology in combination with mass spectrometry. In conclusion, these data strongly suggests that FKBP-12 is altered in an experimental model of PD.

Differential quantification of proteins and peptides by LC-MS is a promising method to acquire knowledge about biological processes, and for finding drug targets and biomarkers. However, differential protein analysis using LC-MS has been held back by the lack of suitable software tools. Large amounts of experimental data are easily generated in protein and peptide profiling experi- ments, but data analysis is time-consuming and labor- intensive. Here, we present a fully automated method for scanning LC-MS/MS data for biologically significant peptides and proteins, including support for interactive confirmation and further profiling. By studying peptide mixtures of known composition, we demonstrate that peptides present in different amounts in different groups of samples can be automatically screened for using statistical tests. A linear response can be obtained over almost 3 orders of magnitude, facilitating further profiling of peptides and proteins of interest. Furthermore, we apply the method to study the changes of endogenous peptide levels in mouse brain striatum after administra- tion of reserpine, a classical model drug for inducing Parkinson disease symptoms.

The cyanobacterial toxin β-N-methylamino-l-alanine (BMAA) is suggested to play a role in neurodegenerative disease. We have previously shown that although the selective uptake of BMAA in the rodent neonatal striatum does not cause neuronal cell death, exposure during the neonatal development leads to cognitive impairments in adult rats. The aim of the present study was to characterize the changes in the striatal neuropeptide systems of male and female rat pups treated neonatally (postnatal days 9-10) with BMAA (40-460 mg/kg). The label-free quantification of the relative levels of endogenous neuropeptides using mass spectrometry revealed that 25 peptides from 13 neuropeptide precursors were significantly changed in the rat neonatal striatum. The exposure to noncytotoxic doses of BMAA induced a dose-dependent increase of neurosecretory protein VGF-derived peptides, and changes in the relative levels of cholecystokinin, chromogranin, secretogranin, MCH, somatostatin and cortistatin-derived peptides were observed at the highest dose. In addition, the results revealed a sex-dependent increase in the relative level of peptides derived from the proenkephalin-A and protachykinin-1 precursors, including substance P and neurokinin A, in female pups. Because several of these peptides play a critical role in the development and survival of neurons, the observed neuropeptide changes might be possible mediators of BMAA-induced behavioral changes. Moreover, some neuropeptide changes suggest potential sex-related differences in susceptibility toward this neurotoxin. The present study also suggests that neuropeptide profiling might provide a sensitive characterization of the BMAA-induced noncytotoxic effects on the developing brain.

PMCID: PMC3539432; NIHMSID: NIHMS430346.-- et al., A first research development progress report of the Chromosome 19 Consortium with members from Sweden, Norway, Spain, United States, China and India, a part of the Chromosome-centric Human Proteome Project (C-HPP) global initiative, is presented (http://www.c-hpp.org). From the chromosome 19 peptide-targeted library constituting 6159 peptides, a pilot study was conducted using a subset with 125 isotope-labeled peptides. We applied an annotation strategy with triple quadrupole, ESI-Qtrap, and MALDI mass spectrometry platforms, comparing the quality of data within and in between these instrumental set-ups. LC-MS conditions were outlined by multiplex assay developments, followed by MRM assay developments. SRM was applied to biobank samples, quantifying kallikrein 3 (prostate specific antigen) in plasma from prostate cancer patients. The antibody production has been initiated for more than 1200 genes from the entire chromosome 19, and the progress developments are presented. We developed a dedicated transcript microarray to serve as the mRNA identifier by screening cancer cell lines. NAPPA protein arrays were built to align with the transcript data with the Chromosome 19 NAPPA chip, dedicated to 90 proteins, as the first development delivery. We have introduced an IT-infrastructure utilizing a LIMS system that serves as the key interface for the research teams to share and explore data generated within the project. The cross-site data repository will form the basis for sample processing, including biological samples as well as patient samples from national Biobanks. © 2012 American Chemical Society., This work was supported by grants from the Swedish Academy of Pharmaceutical Sciences who is the core founder our consortium, Swedish Research Council, the Swedish Foundation for Strategic Research, Vinnova, Ingabritt & Arne Lundbergs forskningsstiftelse, the Crafoord Foundation and by Thermo Fisher Scientific for mass spectrometry instrument support. T.E.F. is supported by the Mobilitas Program sponsored by the European Union Social Fund and administered by the Estonian Science Foundation. We gratefully acknowledge financial support to M.F. from Health Institute Carlos III of Spain (ISCIII, FIS PI02114) and M.G.-G. is supported by a PhD scholarship of ISCIII FI08/00721. C.L.N. is supported by the Cancer Prevention and Research Institute of Texas and the University of Texas Medical Branch. [11-0624]; National Cancer Institute [R33 CA 127768-03, R01CA160816, and P50-CA92629]; Sidney Kimmel Center for Prostate and Urologic Cancers; and David H. Koch through the Prostate Cancer Foundation.

The SwePep database is designed for endogenous peptides and mass spectrometry. It contains information about the peptides such as mass, pl, precursor protein and potential post-translational modifications. Here, we have improved and extended the SwePep database with tandem mass spectra, by adding a locally curated version of the global proteome machine database (GPMDB). In peptidomic experiment practice, many peptide sequences contain multiple tandem mass spectra with different quality. The new tandem mass spectra database in SwePep enables validation of low quality spectra using high quality tandem mass spectra. The validation is performed by comparing the fragmentation patterns of the two spectra using algorithms for calculating the correlation coefficient between the spectra. The present study is the first step in developing a tandem spectrum database for endogenous peptides that can be used for spectrum-to-spectrum identifications instead of peptide identifications using traditional protein sequence database searches.