Douglas Slotta

Composition of the Synaptic PSD-95 Complex

Postsynaptic density protein 95 (PSD-95), a specialized scaffold protein with multiple protein interaction domains, forms the backbone of an extensive postsynaptic protein complex that organizes receptors and signal transduction molecules at the synaptic contact zone. Large, detergent-insoluble PSD-95-based postsynaptic complexes can be affinity-purified from conventional PSD fractions using magnetic beads coated with a PSD-95 antibody. In the present study purified PSD-95 complexes were analyzed by LC/MS/MS. A semiquantitative measure of the relative abundances of proteins in the purified PSD-95 complexes and the parent PSD fraction was estimated based on the cumulative ion current intensities of corresponding peptides. The affinity-purified preparation was largely depleted of presynaptic proteins, spectrin, intermediate filaments, and other contaminants prominent in the parent PSD fraction. We identified 525 of the proteins previously reported in parent PSD fractions, but only 288 of these were detected after affinity purification. We discuss 26 proteins that are major components in the PSD-95 complex based upon abundance ranking and affinity co-purification with PSD-95. This subset represents a minimal list of constituent proteins of the PSD-95 complex and includes, in addition to the specialized scaffolds and N-methyl-d-aspartate (NMDA) receptors, an abundance of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, small G-protein regulators, cell adhesion molecules, and hypothetical proteins. The identification of two Arf regulators, BRAG1 and BRAG2b, as co-purifying components of the complex implies pivotal functions in spine plasticity such as the reorganization of the actin cytoskeleton and insertion and retrieval of proteins to and from the plasma membrane. Another co-purifying protein (Q8BZM2) with two sterile α motif domains may represent a novel structural core element of the PSD.

MassSieve: Panning MS/MS peptide data for proteins

We present MassSieve, a Java‐based platform for visualization and parsimony analysis of single and comparative LC‐MS/MS database search engine results. The success of mass spectrometric peptide sequence assignment algorithms has led to the need for a tool to merge and evaluate the increasing data set sizes that result from LC‐MS/MS‐based shotgun proteomic experiments. MassSieve supports reports from multiple search engines with differing search characteristics, which can increase peptide sequence coverage and/or identify conflicting or ambiguous spectral assignments.

NCBI Peptidome: a new repository for mass spectrometry proteomics data

Peptidome is a public repository that archives and freely distributes tandem mass spectrometry peptide and protein identification data generated by the scientific community. Data from all stages of a mass spectrometry experiment are captured, including original mass spectra files, experimental metadata and conclusion-level results. The submission process is facilitated through acceptance of data in commonly used open formats, and all submissions undergo syntactic validation and curation in an effort to uphold data integrity and quality. Peptidome is not restricted to specific organisms, instruments or experiment types; data from any tandem mass spectrometry experiment from any species are accepted. In addition to data storage, web-based interfaces are available to help users query, browse and explore individual peptides, proteins or entire Samples and Studies. Results are integrated and linked with other NCBI resources to ensure dissemination of the information beyond the mass spectroscopy proteomics community. Peptidome is freely accessible at http://www.ncbi.nlm.nih.gov/peptidome.

Recommendations for Mass Spectrometry Data Quality Metrics for Open Access Data (Corollary to the Amsterdam Principles)

Policies supporting the rapid and open sharing of proteomic data are being implemented by the leading journals in the field. The proteomics community is taking steps to ensure that data are made publicly accessible and are of high quality, a challenging task that requires the development and deployment of methods for measuring and documenting data quality metrics. On September 18, 2010, the United States National Cancer Institute convened the “International Workshop on Proteomic Data Quality Metrics” in Sydney, Australia, to identify and address issues facing the development and use of such methods for open access proteomics data. The stakeholders at the workshop enumerated the key principles underlying a framework for data quality assessment in mass spectrometry data that will meet the needs of the research community, journals, funding agencies, and data repositories. Attendees discussed and agreed up on two primary needs for the wide use of quality metrics: 1) an evolving list of comprehensive quality metrics and 2) standards accompanied by software analytics. Attendees stressed the importance of increased education and training programs to promote reliable protocols in proteomics. This workshop report explores the historic precedents, key discussions, and necessary next steps to enhance the quality of open access data. By agreement, this article is published simultaneously in the Journal of Proteome Research, Molecular and Cellular Proteomics, Proteomics, and Proteomics Clinical Applications as a public service to the research community. The peer review process was a coordinated effort conducted by a panel of referees selected by the journals.

From Peptidome to PRIDE: Public proteomics data migration at a large scale

The PRIDE database, developed and maintained at the European Bioinformatics Institute (EBI), is one of the most prominent data repositories dedicated to high throughput MS‐based proteomics data. Peptidome, developed by the National Center for Biotechnology Information (NCBI) as a sibling resource to PRIDE, was discontinued due to funding constraints in April 2011. A joint effort between the two teams was started soon after the Peptidome closure to ensure that data were not “lost” to the wider proteomics community by exporting it to PRIDE. As a result, data in the low terabyte range have been migrated from Peptidome to PRIDE and made publicly available under experiment accessions 17 900–18 271, representing 54 projects, ∼53 million mass spectra, ∼10 million peptide identifications, ∼650 000 protein identifications, ∼1.1 million biologically relevant protein modifications, and 28 species, from more than 30 different labs.