APP Is a Context-Sensitive Regulator of the Hippocampal Presynaptic Active Zone.

The hallmarks of Alzheimer's disease (AD) are characterized by cognitive decline and behavioral changes. The most prominent brain region affected by the progression of AD is the hippocampal formation. The pathogenesis involves a successive loss of hippocampal neurons accompanied by a decline in learning and memory consolidation mainly attributed to an accumulation of senile plaques. The amyloid precursor protein (APP) has been identified as precursor of Aß-peptides, the main constituents of senile plaques. Until now, little is known about the physiological function of APP within the central nervous system. The allocation of APP to the proteome of the highly dynamic presynaptic active zone (PAZ) highlights APP as a yet unknown player in neuronal communication and signaling. In this study, we analyze the impact of APP deletion on the hippocampal PAZ proteome. The native hippocampal PAZ derived from APP mouse mutants (APP-KOs and NexCreAPP/APLP2-cDKOs) was isolated by subcellular fractionation and immunopurification. Subsequently, an isobaric labeling was performed using TMT6 for protein identification and quantification by high-resolution mass spectrometry. We combine bioinformatics tools and biochemical approaches to address the proteomics dataset and to understand the role of individual proteins. The impact of APP deletion on the hippocampal PAZ proteome was visualized by creating protein-protein interaction (PPI) networks that incorporated APP into the synaptic vesicle cycle, cytoskeletal organization, and calcium-homeostasis. The combination of subcellular fractionation, immunopurification, proteomic analysis, and bioinformatics allowed us to identify APP as structural and functional regulator in a context-sensitive manner within the hippocampal active zone network.

The proteome of the presynaptic active zone from mouse brain.

Neurotransmitter release as well as the structural and functional dynamics of the presynaptic active zone is controlled by proteinaceous components. Here we describe for the first time an experimental approach for the isolation of the presynaptic active zone from individual mouse brains, a prerequisite for understanding the functional inventory of the presynaptic protein network and for the later analysis of changes occurring in mutant mice. Using a monoclonal antibody against the ubiquitous synaptic vesicle protein SV2 we immunopurified synaptic vesicles docked to the presynaptic plasma membrane. Enrichment studies by means of Western blot analysis and mass spectrometry identified 485 proteins belonging to an impressive variety of functional categories. Our data suggest that presynaptic active zones represent focal hot spots that are not only involved in the regulation of neurotransmitter release but also in multiple structural and functional alterations the adult nerve terminal undergoes during neural activity in adult CNS. They furthermore open new avenues for characterizing alterations in the active zone proteome of mutant mice and their corresponding controls, including the various mouse models of neurological diseases.

Perfusion reversed-phase high-performance liquid chromatography for protein separation from detergent-containing solutions: an alternative to gel-based approaches.

Detergents are frequently used for the solubilization of membrane proteins during and after purification steps. Unfortunately some of these detergents impair chromatographic separations and mass spectrometry (MS) analysis. Perfusion reversed-phase high-performance liquid chromatography (RP-HPLC) using POROS materials is suited for separating intact proteins solubilized by detergents due to the particles' highly diffusive pores and chemical stability. In this article, the use of perfusive reversed-phase material packed into small inner diameter capillary columns is presented as a cheap, rapid, and efficient method for the removal of different types of detergents from protein solutions. The ability to purify and separate the subunits of membrane protein complexes with self-packed capillary columns is exemplified for bovine cytochrome bc(1) complex. Even highly hydrophobic subunits can be detected in collected fractions by intact mass measurements and identified after proteolytic digestion and matrix-assisted laser desorption/ionization tandem MS (MALDI MS/MS). The comparison with a gel-based approach shows that this method is a valuable alternative for purification and separation of intact proteins with subsequent MS analysis and that hydrophobic proteins are even better represented in the LC-based approach.

Improving the precision of quantitative bottom-up proteomics based on stable isotope-labeled proteins.

Stable isotope dilution-based quantitative proteomics with intact labeled proteins as internal standards in combination with a bottom-up approach, i.e., with quantification on the peptide level, is an established method. To explore the technical precision of this approach, calmodulin-like protein 3 was prepared in non-labeled (light) and SILAC-type labeled (heavy) form by cell-free synthesis, mixed, digested with trypsin, and analyzed by UPLC-ESI-MS. In total, 16 light/heavy peptide pair ratios were determined. Pair-wise comparison of ratios of 12 peptides selected according to S/N ratios >50 revealed that the majority exhibited ratios, which were different at a high level of statistical significance (p < 0.001). HPLC-MALDI-MS ratio data confirmed this observation, thus excluding the ionization method as a source of the observed ratio differences. Variation of the digestion time from 0.25 to 4 h showed that the light/heavy ratios of most peptides decrease with time, indicating a kinetic isotope effect leading to preferred cleavage of light calmodulin-like protein 3. The subset of peptides with statistically identical ratios resulted in an average ratio with a RSD of 1.0 %. The light/heavy ratio calculated on the basis of these peptides probably provides the most accurate molar protein ratio.

Simple dual-spotting procedure enhances nLC-MALDI MS/MS analysis of digests with less specific enzymes.

The beneficial effect of high mass accuracy in mass spectrometry is especially pronounced when using less specific enzymes as the number of theoretically possible peptides increases dramatically without any cleavage specificity defined. Together with a preceding chromatographic separation, high-resolution mass spectrometers such as the MALDI-LTQ-Orbitrap are therefore well suited for the analysis of protein digests with less specific enzymes. A combination with fast, automated, and informative MALDI-TOF/TOF analysis has already been shown to yield increased total peptide and protein identifications. Here, a simple method for nLC separation and subsequent alternating spotting on two targets for both a MALDI-LTQ-Orbitrap and a MALDI-TOF/TOF instrument is introduced. This allows for simultaneous measurements on both instruments and subsequent combination of both data sets by an in-house written software tool. The performance of this procedure was evaluated using a mixture of four standard proteins digested with elastase. Three replicate runs were examined concerning repeatability and the total information received from both instruments. A cytosolic extract of C. glutamicum was used to demonstrate the applicability to more complex samples. Database search results showed that an additional 32.3% of identified peptides were found using combined data sets in comparison to MALDI-TOF/TOF data sets.

Labeling elastase digests with TMT: informational gain by identification of poorly detectable peptides with MALDI-TOF/TOF mass spectrometry.

The applicability of the less specific protease elastase for the identification of membrane and cytosolic proteins has already been demonstrated. MALDI as ionization technique particularly favors the detection of basic and to a lesser extent of weakly acidic peptides, whereas neutral peptides often remain undetected. Moreover, peptides below 700 Da are routinely excluded. In the following study, the advantage of additional information gained from tandem mass tag zero labeled peptides and the resultant increase in sequence coverage was evaluated. Through derivatization with tandem mass tag reagents, peptide measurement within the standard mass range of the MALDI reflector mode is achievable due to the mass increase. Compared to the unlabeled sample, peptides exhibiting relatively low molecular masses, pI values or higher hydrophobicity could be identified.

3-Aminoquinoline acting as matrix and derivatizing agent for MALDI MS analysis of oligosaccharides.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a widely used method in oligosaccharide analysis. Underivatized oligosaccharides are not well-suited for that purpose due to their low ionization efficiency; however, derivatization requires tedious sample purification steps which may lead to sample losses, thereby decreasing its benefit. On-target derivatization performed by the matrix 3-aminoquinoline does not require such purification and yields Schiff bases which can be measured in positive and negative ion mode from one single spot. In negative ion mode, spectra from anionic adducts of the derivatives can be acquired from 1 fmol of oligosaccharide. Furthermore, postsource decay (PSD) fragmentation in positive and negative ion mode is enhanced, providing information on oligosaccharide sequence, linkage, and branching. Optimization of reaction conditions and matrix solution led to a complete and reproducible derivatization for all tested standard oligosaccharides. Finally, the method was applied to trifucosyllacto-N-hexaose and trifucosyl-para-lacto-N-hexaose, two isomers occurring in human breast milk samples, which were easily identified and distinguished.

The benefit of combining nLC-MALDI-Orbitrap MS data with nLC-MALDI-TOF/TOF data for proteomic analyses employing elastase.

The recently established coupling of a MALDI-type ion source to a linear ion trap and an orbitrap mass analyzer offers high-accuracy mass measurements compared to common MALDI-TOF/TOF instruments. Contrary to MALDI-TOF/TOF, the fragmentation of peptides in the new hybrid mass spectrometer is less efficient due to the generation of predominantly singly charged ions by the MALDI process. Therefore, data from two MALDI instruments, TOF/TOF and Orbitrap, were combined into a single data set in order to obtain accurate precursor masses as well as superior MS/MS spectra. This study demonstrates that an accurate precursor mass is particularly important for the nLC-MS/MS analyses of less-specific proteolytic digests. A potential gain of approximately one-third additional peptides identifications was theoretically estimated from previously published MALDI-TOF/TOF data. These calculations were verified by the nLC-MS/MS analysis of two elastatically digested proteomes, one cytosolic (Corynebacterium glutamicum) and one membrane (Halobacterium salinarium). Thereby it was discovered that the error distribution of a MALDI-Orbitrap can be significantly improved by applying an easy recalibration strategy. In summary, this study represents an updated workflow for the analysis of less-specific digests using nLC-MALDI.

Regional Specializations of the PAZ Proteomes Derived from Mouse Hippocampus, Olfactory Bulb and Cerebellum.

Neurotransmitter release as well as structural and functional dynamics at the presynaptic active zone (PAZ) comprising synaptic vesicles attached to the presynaptic plasma membrane are mediated and controlled by its proteinaceous components. Here we describe a novel experimental design to immunopurify the native PAZ-complex from individual mouse brain regions such as olfactory bulb, hippocampus, and cerebellum with high purity that is essential for comparing their proteome composition. Interestingly, quantitative immunodetection demonstrates significant differences in the abundance of prominent calcium-dependent PAZ constituents. Furthermore, we characterized the proteomes of the immunoisolated PAZ derived from the three brain regions by mass spectrometry. The proteomes of the release sites from the respective regions exhibited remarkable differences in the abundance of a large variety of PAZ constituents involved in various functional aspects of the release sites such as calcium homeostasis, synaptic plasticity and neurogenesis. On the one hand, our data support an identical core architecture of the PAZ for all brain regions and, on the other hand, demonstrate that the proteinaceous composition of their presynaptic active zones vary, suggesting that changes in abundance of individual proteins strengthen the ability of the release sites to adapt to specific functional requirements.