Dominic Baeumlisberger

Lipidome and proteome of lipid droplets from the methylotrophic yeast Pichia pastoris

Lipid droplets (LD) are the main depot of non-polar lipids in all eukaryotic cells. In the present study we describe isolation and characterization of LD from the industrial yeast Pichia pastoris. We designed and adapted an isolation procedure which allowed us to obtain this subcellular fraction at high purity as judged by quality control using appropriate marker proteins. Components of P. pastoris LD were characterized by conventional biochemical methods of lipid and protein analysis, but also by a lipidome and proteome approach. Our results show several distinct features of LD from P. pastoris especially in comparison to Saccharomyces cerevisiae. P. pastoris LD are characterized by their high preponderance of triacylglycerols over steryl esters in the core of the organelle, the high degree of fatty acid (poly)unsaturation and the high amount of ergosterol precursors. The high phosphatidylinositol to phosphatidylserine of ~ 7.5 ratio on the surface membrane of LD is noteworthy. Proteome analysis revealed equipment of the organelle with a small but typical set of proteins which includes enzymes of sterol biosynthesis, fatty acid activation, phosphatidic acid synthesis and non-polar lipid hydrolysis. These results are the basis for a better understanding of P. pastoris lipid metabolism and lipid storage and may be helpful for manipulating cell biological and/or biotechnological processes in this yeast.

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.

Membrane protein analysis using an improved peptic in-solution digestion protocol.

In the proteomic analysis of membrane proteins, less‐specific proteases have become a promising tool to overcome fundamental limitations of trypsin with its unique specificity for basic residues. Pepsin is well‐known to be utilized for specific applications that require acidic conditions, but in terms of membrane protein identification and characterization, it has been disregarded for the most part. This work presents an optimization of an existing peptic digest protocol for the analysis of membrane proteins using bacteriorhodopsin from purple membranes as reference.

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 Aβ-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.

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.

Approaching the complexity of elastase-digested membrane proteomes using off-gel IEF/nLC-MALDI-MS/MS.

Liquid chromatography, coupled with tandem mass spectrometry, is an established method for the identification of proteins from a complex sample. Despite its wide application, the analysis of whole proteomes still represents a challenge to researchers, because of the complexity and dynamic range of protein concentrations in biological samples. The analysis of such samples can be improved by adding a prefractionation step or a combination of orthogonal separation techniques. Off-gel isoelectric focusing (OGE) has successfully been used for prefractionation of a tryptic digest prior to nLC separation. In contrast to previous published results, we present a complete glycerol-free OGE for the analysis of purple membranes and Corynebacterium glutamicum membranes using the less-specific enzyme elastase. More than 85% of the identified unique peptides were found in solely one fraction, with very little carryover. These results are in accordance with those published for tryptic peptides. Therefore, OGE can be used as an effective prefractionation method in a multidimensional separation experiment of nontryptic membrane peptides.

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.

Peptide mass fingerprinting after less specific in-gel proteolysis using MALDI-LTQ-Orbitrap and 4-chloro-alpha-cyanocinnamic acid.

Peptide Mass Fingerprinting (PMF) of tryptically in-gel digested samples is a well-established protein identification technique for MALDI mass spectrometry but an in-depth PMF evaluation for in-gel digestions of less specific enzymes is still missing. This study demonstrates that the MALDI-LTQ-Orbitrap provides the mass accuracy to gain significant database search results via PMF for the less specific enzymes chymotrypsin and elastase. Additionally, the highly sensitive MALDI matrix ClCCA was compared to the most widely used matrix CHCA by means of the detected peptide number, peptide composition, pI and S/N distribution, sequence coverage, and Mascot score. Therefore, several proteins were in-gel digested by chymotrypsin and elastase. Trypsin and proteinase K were included as references for specific and nonspecific proteases, respectively. Compared to CHCA, ClCCA resulted in a better mapping in all cases of the more complex peptide mixtures generated by less specific enzymes. In summary, the MALDI-LTQ-Orbitrap combined with the matrix ClCCA makes PMF of less specific digests possible in an easy and fast way. Moreover, it opens more possibilities for PMF in the analysis of difficult tasks such as membrane proteins.

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.

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.