Henrik Thomas

Protein identification pipeline for the homology-driven proteomics.

Homology-driven proteomics is a major tool to characterize proteomes of organisms with unsequenced genomes. This paper addresses practical aspects of automated homology-driven protein identifications by LC-MS/MS on a hybrid LTQ Orbitrap mass spectrometer. All essential software elements supporting the presented pipeline are either hosted at the publicly accessible web server, or are available for free download.

Improving protein extraction and separation methods for investigating the metaproteome of anaerobic benzene communities within sediments

BTEX compounds such as benzene are frequent soil and groundwater contaminants that are easily biodegraded under oxic conditions by bacteria. In contrast, benzene is rather recalcitrant under anaerobic conditions. The analysis of anoxic degradation is often hampered by difficult sampling conditions, limited amounts of biomass and interference of matrix compounds with proteomic approaches. In order to improve the procedure for protein extraction we established a scheme consisting of the following steps: dissociation of cells from lava granules, cell lysis by ultrasonication and purification of proteins by phenol extraction. The 2D-gels revealed a resolution of about 240 proteins spots and the spot patterns showed strong matrix dependence, but still differences were detectable between the metaproteomes obtained after growth on benzene and benzoate. Using direct data base search as well as de novo sequencing approaches we were able to identify several proteins. An enoyl-CoA hydratase with cross species homology to Azoarcus evansii, is known to be involved in the anoxic degradation of xenobiotics. Thereby the identification confirmed that this procedure has the capacity to analyse the metaproteome of an anoxic living microbial community.

Separating the Wheat from the Chaff: Unbiased Filtering of Background Tandem Mass Spectra Improves Protein Identification

Only a small fraction of spectra acquired in LC-MS/MS runs matches peptides from target proteins upon database searches. The remaining, operationally termed background, spectra originate from a variety of poorly controlled sources and affect the throughput and confidence of database searches. Here, we report an algorithm and its software implementation that rapidly removes background spectra, regardless of their precise origin. The method estimates the dissimilarity distance between screened MS/MS spectra and unannotated spectra from a partially redundant background library compiled from several control and blank runs. Filtering MS/MS queries enhanced the protein identification capacity when searches lacked spectrum to sequence matching specificity. In sequence-similarity searches it reduced by, on average, 30-fold the number of orphan hits, which were not explicitly related to background protein contaminants and required manual validation. Removing high quality background MS/MS spectra, while preserving in the data set the genuine spectra from target proteins, decreased the false positive rate of stringent database searches and improved the identification of low-abundance proteins.

A Comparative Analysis of an Orthologous Proteomic Environment in the Yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe

The sequential application of protein tagging, affinity purification, and mass spectrometry enables highly accurate charting of proteomic environments by the characterization of stable protein assemblies and the identification of subunits that are shared between two or more protein complexes, termed here “proteomic hyperlinks.” We have charted the proteomic environments surrounding the histone methyltransferase, Set1, in both yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Although the composition of these nonessential Set1 complexes is remarkably conserved, they differ with respect to their hyperlinks to their proteomic environments. We speculate that conservation of the core components of protein assemblies and variability of hyperlinks represents a general principle in the molecular organization of eukaryotic proteomes.

Proteomics identifies the composition and manufacturing recipe of the 2500-year old sourdough bread from Subeixi cemetery in China.

UNLABELLED We report on the geLC-MS/MS proteomics analysis of cereals and cereal food excavated in Subeixi cemetery (500-300BC) in Xinjiang, China. Proteomics provided direct evidence that at the Subexi sourdough bread was made from barley and broomcorn millet by leavening with a renewable starter comprising bakers yeast and lactic acid bacteria. The baking recipe and flour composition indicated that barley and millet bread belonged to the staple food already in the first millennium BC and suggested the role of Turpan basin as a major route for cultural communication between Western and Eastern Eurasia in antiquity. This article is part of a Special Issue entitled: Proteomics of non-model organisms. BIOLOGICAL SIGNIFICANCE We demonstrate that organic residues of thousand year old foods unearthed by archeological excavations can be analyzed by geLC-MS/MS proteomics with good representation of protein source organisms and coverage of sequences of identified proteins. In-depth look into the foods proteome identifies the food type and its individual ingredients, reveals ancient food processing technologies, projects their social and economic impact and provides evidence of intercultural communication between ancient populations. Proteomics analysis of ancient organic residues is direct, quantitative and informative and therefore has the potential to develop into a valuable, generally applicable tool in archaeometry. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Simplified validation of borderline hits of database searches.

Along with unequivocal hits produced by matching multiple MS/MS spectra to database sequences, LC‐MS/MS analysis often yields a large number of hits of borderline statistical confidence. To simplify their validation, we propose to use rapid de novo interpretation of all acquired MS/MS spectra and, with the help of a simple software tool, display the candidate sequences together with each database search hit. We demonstrate that comparing hit database sequences and independent de novo interpretations of the same MS/MS spectra assists in rapid examination of ambiguous matches.

Intensity-Independent Noise Filtering in FT MS and FT MS/MS Spectra for Shotgun Lipidomics

Shotgun lipidomics relies on the direct infusion of total lipid extracts into a high resolution tandem mass spectrometer. A single shotgun analysis produces several hundred of densely populated FT MS and FT MS/MS spectra, each of which might comprise thousands of peaks although a very small percentage of those belong to lipids. Eliminating noise by adjusting a minimal peak intensity threshold is biased and inefficient since lipid species and classes vary in their natural abundance and ionization capacity. We developed a method of peak intensity-independent noise filtering in shotgun FT MS and FT MS/MS spectra that capitalizes on a stable composition of the infused analyte leading to consistent time-independent detection of its bona fide components. Repetition rate filtering relies on a single quantitative measure of peaks detection reproducibility irrespectively of their absolute intensities, masses, or assumed elemental compositions. In comparative experiments, it removed more than 95% of signals detectable in shotgun spectra without compromising the accuracy and scope of lipid identification and quantification. It also accelerated spectra processing by 15-fold and increased the number of simultaneously processed spectra by ∼500-fold hence eliminating the major bottleneck in high-throughput bottom-up shotgun lipidomics.

Thermostable β-cyclodextrin conjugates of two similar plant amine oxidases and their properties

Syntheses of conjugates of garden pea (Pisum sativum) and grass pea (Lathyrus sativus) amine oxidases (PSAO and GPAO respectively) with BCD (β‐cyclodextrin), performed to improve the thermostability of the enzymes, are described in the present study. Periodate‐oxidized BCD reacted with the enzyme proteins via free primary amino groups in a buffered solution containing cyanoborohydride as a reductant. Although the specific activities of PSAO and GPAO partially decreased after modification, Km values determined for the best diamine substrates remained almost unchanged. Both the BCD conjugates could be incubated at 65 °C for 30 min without considerable inactivation, and the residual activity remained detectable even after incubation at 75 °C. The conjugates contained approx. 30% of neutral sugars. Molecular masses of BCD–PSAO and BCD–GPAO (180 kDa), as estimated by gel‐permeation chromatography, were higher compared with the value of 145 kDa for the native enzymes. This was in good correlation with the number of modified lysine residues determined by a spectrophotometric method. Peptide mass fingerprints of tryptic digests of BCD–PSAO and BCD–GPAO were less specific than those of the native enzymes when compared with the database sequence of PSAO. As a consequence of the modification, many unidentified peaks were observed in the digests of the studied conjugates that were not seen in the digests of native PSAO and GPAO. Only some of these peaks overlapped between BCD–PSAO and BCD–GPAO. The BCD conjugates described in the present study represent suitable candidates for biotechnological applications, e.g. in analyses using biosensors, which might benefit from increased storage stability and amine oxidation at high temperatures.

Monitoring Membrane Lipidome Turnover by Metabolic 15N Labeling and Shotgun Ultra-High-Resolution Orbitrap Fourier Transform Mass Spectrometry

Lipidomes undergo permanent extensive remodeling, but how the turnover rate differs between lipid classes and molecular species is poorly understood. We employed metabolic 15N labeling and shotgun ultra-high-resolution mass spectrometry (sUHR) to quantify the absolute (molar) abundance and determine the turnover rate of glycerophospholipids and sphingolipids by direct analysis of total lipid extracts. sUHR performed on a commercial Orbitrap Elite instrument at the mass resolution of 1.35 × 106 (m/z 200) baseline resolved peaks of 13C isotopes of unlabeled and monoisotopic peaks of 15N labeled lipids (Δm = 0.0063 Da). Therefore, the rate of metabolic 15N labeling of individual lipid species could be determined without compromising the scope, accuracy, and dynamic range of full-lipidome quantitative shotgun profiling. As a proof of concept, we employed sUHR to determine the lipidome composition and fluxes of 62 nitrogen-containing membrane lipids in human hepatoma HepG2 cells.