Walter P. Blackstock

Proteomic analysis of NMDA receptor–adhesion protein signaling complexes

N-methyl-D-aspartate receptors (NMDAR) mediate long-lasting changes in synapse strength via downstream signaling pathways. We report proteomic characterization with mass spectrometry and immunoblotting of NMDAR multiprotein complexes (NRC) isolated from mouse brain. The NRC comprised 77 proteins organized into receptor, adaptor, signaling, cytoskeletal and novel proteins, of which 30 are implicated from binding studies and another 19 participate in NMDAR signaling. NMDAR and metabotropic glutamate receptor subtypes were linked to cadherins and L1 cell-adhesion molecules in complexes lacking AMPA receptors. These neurotransmitter–adhesion receptor complexes were bound to kinases, phosphatases, GTPase-activating proteins and Ras with effectors including MAPK pathway components. Several proteins were encoded by activity-dependent genes. Genetic or pharmacological interference with 15 NRC proteins impairs learning and with 22 proteins alters synaptic plasticity in rodents. Mutations in three human genes (NF1, Rsk-2, L1) are associated with learning impairments, indicating the NRC also participates in human cognition.

Proteomics: quantitative and physical mapping of cellular proteins

Genome sequencing provides a wealth of information on predicted gene products (mostly proteins), but the majority of these have no known function. Two-dimensional gel electrophoresis and mass spectrometry have, coupled with searches in protein and EST databases, transformed the protein-identification process. The proteome is the expressed protein complement of a genome and proteomics is functional genomics at the protein level. Proteomics can be divided into expression proteomics, the study of global changes in protein expression, and cell-map proteomics, the systematic study of protein-protein interactions through the isolation of protein complexes.

Molecular characterization and comparison of the components and multiprotein complexes in the postsynaptic proteome.

Characterization of the composition of the postsynaptic proteome (PSP) provides a framework for understanding the overall organization and function of the synapse in normal and pathological conditions. We have identified 698 proteins from the postsynaptic terminal of mouse CNS synapses using a series of purification strategies and analysis by liquid chromatography tandem mass spectrometry and large‐scale immunoblotting. Some 620 proteins were found in purified postsynaptic densities (PSDs), nine in AMPA‐receptor immuno‐purifications, 100 in isolates using an antibody against the NMDA receptor subunit NR1, and 170 by peptide‐affinity purification of complexes with the C‐terminus of NR2B. Together, the NR1 and NR2B complexes contain 186 proteins, collectively referred to as membrane‐associated guanylate kinase‐associated signalling complexes. We extracted data from six other synapse proteome experiments and combined these with our data to provide a consensus on the composition of the PSP. In total, 1124 proteins are present in the PSP, of which 466 were validated by their detection in two or more studies, forming what we have designated the Consensus PSD. These synapse proteome data sets offer a basis for future research in synaptic biology and will provide useful information in brain disease and mental disorder studies.

Phosphorylation sites on Tau identified by nanoelectrospray mass spectrometry : Differences in vitro between the mitogen-activated protein kinases ERK2, c-Jun N-terminal kinase and p38, and glycogen synthase kinase-3β

Abstract: The stress‐activated kinases c‐Jun N‐terminal kinase (JNK) and p38 are members of the mitogen‐activated protein (MAP) kinase family and take part in signalling cascades initiated by various forms of stress. Their targets include the microtubule‐associated protein tau, which becomes hyperphosphorylated in Alzheimers disease. It is necessary, as a forerunner for in vivo studies, to identify the protein kinases and phosphatases that are responsible for phosphate turnover at individual sites. Using nanoelectrospray mass spectrometry, we have undertaken an extensive comparison of phosphorylation in vitro by several candidate tau kinases, namely, JNK, p38, ERK2, and glycogen synthase kinase 3β (GSK3β). Between 10 and 15 sites were identified for each kinase. The three MAP kinases phosphorylated Ser202 and Thr205 but not detectably Ser199, whereas conversely GSK3β phosphorylated Ser199 but not detectably Ser202 or Thr205. Phosphorylated Ser404 was found with all of these kinases except JNK. The MAP kinases may not be strictly proline specific: p38 phosphorylated the nonproline sites Ser185, Thr245, Ser305, and Ser356, whereas ERK2 was the most strict. All of the sites detected except Thr245 and Ser305 are known or suspected phosphorylation sites in paired helical filament‐tau extracted from Alzheimer brains. Thus, the three MAP kinases and GSK3β are importantly all strong candidates as tau kinases that may be involved in the pathogenic hyperphosphorylation of tau in Alzheimers disease.

New Phosphorylation Sites Identified in Hyperphosphorylated Tau (Paired Helical Filament‐Tau) from Alzheimer's Disease Brain Using Nanoelectrospray Mass Spectrometry

Abstract: Paired helical filaments (PHFs) are the structural constituents of neurofibrillary tangles in Alzheimers disease and are composed of hyperphosphorylated forms of the microtubule‐associated protein tau (PHF‐tau). Pathological hyperphosphorylation of tau is believed to be an important contributor to the destabilisation of microtubules and their subsequent disappearance from tangle‐bearing neurons in Alzheimers disease, making elucidation of the mechanisms that regulate tau phosphorylation an important research goal. Thus, it is essential to identify, preferably by direct sequencing, all of the sites in PHF‐tau that are phosphorylated, a task that is incomplete because of the difficulty to date of purifying insoluble PHF‐tau to homogeneity and in sufficient quantities for structural analysis. Here we describe the solubilisation of PHF‐tau followed by its purification by Mono Q chromatography and reversed‐phase HPLC. Phosphopeptides from proteolytically digested PHF‐tau were sequenced by nanoelectrospray mass spectrometry. We identified 22 phosphorylation sites in PHF‐tau, including five sites not previously identified. The combination of our new data with previous reports shows that PHF‐tau can be phosphorylated on at least 25 different sites.

Proteomics Characterization of Abundant Golgi Membrane Proteins

A mass spectrometric analysis of proteins partitioning into Triton X-114 from purified hepatic Golgi apparatus (84% purity by morphometry, 122-fold enrichment over the homogenate for the Golgi marker galactosyl transferase) led to the unambiguous identification of 81 proteins including a novel Golgi-associated protein of 34 kDa (GPP34). The membrane protein complement was resolved by SDS-polyacrylamide gel electrophoresis and subjected to a hierarchical approach using delayed extraction matrix-assisted laser desorption ionization mass spectrometry characterization by peptide mass fingerprinting, tandem mass spectrometry to generate sequence tags, and Edman sequencing of proteins. Major membrane proteins corresponded to known Golgi residents, a Golgi lectin, anterograde cargo, and an abundance of trafficking proteins including KDEL receptors, p24 family members, SNAREs, Rabs, a single ARF-guanine nucleotide exchange factor, and two SCAMPs. Analytical fractionation and gold immunolabeling of proteins in the purified Golgi fraction were used to assess the intra-Golgi and total cellular distribution of GPP34, two SNAREs, SCAMPs, and the trafficking proteins GBF1, BAP31, and α2P24 identified by the proteomics approach as well as the endoplasmic reticulum contaminant calnexin. Although GPP34 has never previously been identified as a protein, the localization of GPP34 to the Golgi complex, the conservation of GPP34 from yeast to humans, and the cytosolically exposed location of GPP34 predict a role for a novel coat protein in Golgi trafficking.

Interrogating the human genome using uninterpreted mass spectrometry data.

The public availability of a draft assembly of the human genome has enabled us to demonstrate, for the first time, the feasibility of searching a complete, unmasked eukaryotic genome using uninterpreted mass spectrometry data. A complex LC‐MS/MS data set, containing peptides from at least 22 human proteins, was searched against a comprehensive, nonidentical protein database, an expressed sequence tag (EST) database, and the International Human Genome Project draft assembly of the human genome. The results from the three searches are compared in detail, and the merits of the different databases for this application are discussed. In the case of the EST database, the UniGene index provided a method of simplifying and summarising the search results. In the case of the genomic DNA, the presence of introns prevented matching of roughly one quarter of the spectra, but the technique can provide primary experimental verification of predicted coding sequences, and has the potential to identify novel coding sequences.

A proteomic approach for the study of Saccharomyces cerevisiae cell wall biogenesis

In fungi, cell shape is determined by the presence of a rigid cell wall which separates the cell from the extracellular medium. This highly dynamic structure is essential for the maintenance of cell integrity and is involved in several phenomena such as flocculation, adherence and pathogenicity. The composition of the fungal cell wall is well known, but issues such as the assembly and remodeling of its components remain poorly understood. In an attempt to study the de novo construction of the yeast cell wall, we have undertaken a large‐scale proteomic approach to analyze the proteins secreted by regenerating protoplasts. Upon incubation of protoplasts in regenerating conditions, numerous proteins are secreted into the culture medium. These presumably include proteins destined for the cell wall, comprising both structural proteins as well as enzymes involved in cell wall biogenesis. This work reports the establishment of a reference map of proteins secreted by regenerating protoplasts by means of two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) and their identification by mass spectrometry. Thirty‐two different proteins have been identified, including known cell wall proteins, glycolytic enzymes, heat shock proteins, and proteins involved in several other processes. Using this approach, novel proteins possibly involved in cell wall construction have also been identified. This reference map will allow comparative analyses to be carried out on a selected collection of mutants affected in the cell wall.

PROTEOME ANALYSIS: GENOMICS VIA THE OUTPUT RATHER THAN THE INPUT CODE

A knowledge of the ‘proteome,’ total protein output encoded by a genome, provides information on (1) if and when predicted gene products are translated, (2) the relative concentrations of gene products, and (3) the extent of posttranslational modification, none of which can be accurately predicted from the nucleic acid sequence alone. The current status of proteome analysis is reviewed with respect to some of the techniques employed, automation, relevance to genomic studies, mass spectrometry and bioinformatics, limitations, and recent improvements in resolution and sensitivity for the detection of protein expression in whole cells, tissues, or organisms. The concept of ‘proteomic contigs’ is introduced for the first time. Traditional approaches to genomic analysis call upon a number of strategies to produce contiguous DNA sequence information, while ‘proteomic contigs’ are derived from multiple molecular mass and isoelectric point windows in order to construct a picture of the total protein expression within living cells. In higher eukaryotes, the latter may require several dozen image subsets of protein spots to be stitched together using advanced image analysis. The utility of both experimental and theoretical peptide-mass fingerprinting (PMF) and associated bioinformatics is outlined. A previously unknown motif within the peptide sequence of Elongation Factor Tu from Thermus aquaticus was discovered using PMF. This motif was shown to possess potential significance in maintaining structural integrity of the entire molecule.

Matching peptide mass spectra to EST and genomic DNA databases.

The use of mass spectrometry data to search molecular sequence databases is a well-established method for protein identification. The technique can be extended to searching raw genomic sequences, providing experimental confirmation or correction of predicted coding sequences, and has the potential to identify novel genes and elucidate splicing patterns.