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1.  An integrated approach for genome annotation of the eukaryotic thermophile Chaetomium thermophilum 
Nucleic Acids Research  2014;42(22):13525-13533.
The thermophilic fungus Chaetomium thermophilum holds great promise for structural biology. To increase the efficiency of its biochemical and structural characterization and to explore its thermophilic properties beyond those of individual proteins, we obtained transcriptomics and proteomics data, and integrated them with computational annotation methods and a multitude of biochemical experiments conducted by the structural biology community. We considerably improved the genome annotation of Chaetomium thermophilum and characterized the transcripts and expression of thousands of genes. We furthermore show that the composition and structure of the expressed proteome of Chaetomium thermophilum is similar to its mesophilic relatives. Data were deposited in a publicly available repository and provide a rich source to the structural biology community.
PMCID: PMC4267624  PMID: 25398899
3.  Hip Capsule Dimensions in Patients With Femoroacetabular Impingement: A Pilot Study 
Joint-preserving hip surgery, either arthroscopic or open, increasingly is used for the treatment of symptomatic femoroacetabular impingement (FAI). As a consequence of surgery, thickening of the joint capsule and intraarticular adhesions between the labrum and joint capsule and between the femoral neck and the joint capsule have been observed. These alterations are believed to cause persistent pain and reduced range of motion. Because the diagnosis is made with MR arthrography, knowledge of the normal capsular anatomy and thickness on MRI in patients is important. To date there is no such information available.
The purpose of this study was to establish thickness, length of the hip capsule, and the size of the perilabral recess in patients with FAI.
We reviewed the preoperative MR arthrography of 30 patients (15 men) with clinical symptoms of FAI. We measured capsular thickness and made observations on the perilabral recess.
The joint capsule was thickest (6 mm) anterosuperiorly between 1 and 2 o’clock. The average length from the femoral head-neck junction to the femoral insertion of the capsule ranged from 19 to 33 mm. A perilabral recess was present circumferentially, even across the acetabular notch, where the labrum is supported by the transverse acetabular ligament. The shortest recess occurred superiorly.
Knowledge of the capsular anatomy in patients with FAI before surgery is important to judge the postoperative changes and to plan potential further therapy including arthroscopic treatment of intraarticular adhesions.
PMCID: PMC3492636  PMID: 22810156
4.  High-throughput subtomogram alignment and classification by Fourier space constrained fast volumetric matching 
Journal of structural biology  2012;178(2):10.1016/j.jsb.2012.02.014.
Cryo-electron tomography allows the visualization of macromolecular complexes in their cellular environments in close-to-live conditions. The nominal resolution of subtomograms can be significantly increased when individual subtomograms of the same kind are aligned and averaged. A vital step for such a procedure are algorithms that speedup subtomogram alignment and improve accuracy for reference-free subtomogram classification, which will facilitate automation of tomography analysis and overall high throughput in the data processing. In this paper, we propose a fast rotational alignment method that uses the Fourier equivalent form of a popular constrained correlation measure that considers missing wedge corrections and density variances in the subtomograms. The fast rotational search is based on 3D volumetric matching, which significantly improves the rotational alignment accuracy in particular for highly distorted subtomograms with low SNR and tilt angle ranges in comparison to a fast rotational alignment based on matching of projected 2D spherical images. We further integrate our fast rotational alignment method in a reference free iterative subtomogram classification scheme, and propose a local feature enhancement strategy in the classification process. We can demonstrate that the automatic method can be used to successfully classify a large number of experimental subtomograms without the need of a reference structure.
PMCID: PMC3821800  PMID: 22420977
Cryo-electron tomography; subtomogram alignment and classification; fast rotational matching
5.  Does Bone Wax Induce a Chronic Inflammatory Articular Reaction? 
Bone wax is used to control femoral neck bleeding during open femoroacetabular impingement (FAI) surgery. Despite its widespread use, only a few case reports and small case series describe side effects after extraarticular use. It is unclear whether intraarticular use of bone wax leads to such complications. However, during revision FAI surgery, we have observed various degrees of articular inflammatory reactions.
We therefore investigated whether the bone wax used intraarticularly to control femoral neck bleeding during FAI surgery could be associated with the inflammatory reactions observed at revision surgery.
We visually inspected the area and analyzed biopsy specimens from all 14 patients undergoing revision surgery from March 2005 to March 2006, 11 of whom had bone wax used at the time of original surgery. The three patients who did not have bone wax were used as controls.
Bone wax was identified macroscopically on the femoral neck at the time of the revision surgery in all 11 patients. In all 11 patients, biopsy results indicated a foreign body-type chronic synovial inflammation. Five patients also had an associated synovial lymphoplasmacytic inflammatory reaction. No inflammatory reaction was observed in the biopsy specimens obtained from the three patients in whom bone wax was not originally used.
Our findings suggest a synovial foreign body reaction, with or without an associated lymphoplasmacytic chronic inflammatory reaction, may be associated with intraarticular use of bone wax.
PMCID: PMC3462874  PMID: 22760602
6.  Exploring the Spatial and Temporal Organization of a Cell’s Proteome 
Journal of structural biology  2010;173(3):483-496.
To increase our current understanding of cellular processes, such as cell signaling and division, knowledge is needed about the spatial and temporal organization of the proteome at different organizational levels. These levels cover a wide range of length and time scales: from the atomic structures of macromolecules for inferring their molecular function, to the quantitative description of their abundance, and distribution in the cell. Emerging new experimental technologies are greatly increasing the availability of such spatial information on the molecular organization in living cells. This review addresses three fields that have significantly contributed to our understanding of the proteome’s spatial and temporal organization: first, methods for the structure determination of individual macromolecular assemblies, specifically the fitting of atomic structures into density maps generated from electron microscopy techniques; second, research that visualizes the spatial distributions of these complexes within the cellular context using cryo electron tomography techniques combined with computational image processing; and third, methods for the spatial modeling of the dynamic organization of the proteome, specifically those methods for simulating reaction and diffusion of proteins and complexes in crowded intracellular fluids. The long-term goal is to integrate the varied data about a proteome’s organization into a spatially explicit, predictive model of cellular processes.
PMCID: PMC3784337  PMID: 21094684
Macromolecular assemblies; Visual proteomics; Cryo electron tomography; Cryo electron microscopy; Density fitting; Brownian dynamics simulations; Reaction-diffusion modeling
7.  Mentoring in complex surgery: minimising the learning curve complications from peri-acetabular osteotomy 
International Orthopaedics  2011;36(5):921-925.
The aim of this study was to determine whether a complex surgical procedure such as peri-acetabular osteotomy could be safely learnt by using a programme involving mentoring by a distant expert. To determine this, we examined the incidence of intra-operative complications, the acetabulum correction achieved, the late incidence of re-operation and progressive degenerative arthritis.
Between 1992 and 2004, peri-acetabular osteotomy was performed in 26 hips in 23 patients. The median follow-up was ten (5–17) years. The median age of the patients at operation was 28 (14–41) years. Clinical outcomes were reported and radiographic results were determined by an independent expert.
There were no intra-articular osteotomies, sciatic nerve injuries, hingeing deformities or vascular injuries. There was one ischial nonunion. The lateral centre-edge angle improved from a median 4° pre-operatively to 25°. One revision osteotomy, one osteectomy and three total hip replacements were required, two for progression of osteoarthritis.
The programme of mentoring was successful in that there was a low incidence of the major intra-operative complications that are often reported during the learning curve period and the acetabular corrections achieved were similar to the originators.
PMCID: PMC3337117  PMID: 21898037
Medicine & Public Health; Orthopedics
8.  Cell type-specific nuclear pores: a case in point for context-dependent stoichiometry of molecular machines 
The stoichiometry of the human nuclear pore complex is revealed by targeted mass spectrometry and super-resolution microscopy. The analysis reveals that the composition of the nuclear pore and other nuclear protein complexes is remodeled as a function of the cell type.
The human NPC has a previously unanticipated stoichiometry that varies across cell types.Primarily functional Nups are dynamic, while the NPC scaffold is static.Stoichiometries of many complexes are fine-tuned toward cell type-specific needs.
To understand the structure and function of large molecular machines, accurate knowledge of their stoichiometry is essential. In this study, we developed an integrated targeted proteomics and super-resolution microscopy approach to determine the absolute stoichiometry of the human nuclear pore complex (NPC), possibly the largest eukaryotic protein complex. We show that the human NPC has a previously unanticipated stoichiometry that varies across cancer cell types, tissues and in disease. Using large-scale proteomics, we provide evidence that more than one third of the known, well-defined nuclear protein complexes display a similar cell type-specific variation of their subunit stoichiometry. Our data point to compositional rearrangement as a widespread mechanism for adapting the functions of molecular machines toward cell type-specific constraints and context-dependent needs, and highlight the need of deeper investigation of such structural variants.
PMCID: PMC3619942  PMID: 23511206
fluorophore counting; nucleoporin; protein complex-based analysis; super-resolution microscopy; targeted proteomics
10.  The quantitative proteome of a human cell line 
The majority of all proteins expressed in the human osteosarcoma cell line U2OS were absolutely quantified by mass spectrometry. The quantified proteins span a concentration range of seven orders of magnitude up to 20 000 000 copies per cell.
The generation of mathematical models of biological processes, the simulation of these processes under different conditions, and the comparison and integration of multiple data sets are explicit goals of systems biology that require the knowledge of the absolute quantity of the system's components. To date, systematic estimates of cellular protein concentrations have been exceptionally scarce. Here, we provide a quantitative description of the proteome of a commonly used human cell line in two functional states, interphase and mitosis. We show that these human cultured cells express at least ∼10 000 proteins and that the quantified proteins span a concentration range of seven orders of magnitude up to 20 000 000 copies per cell. We discuss how protein abundance is linked to function and evolution.
PMCID: PMC3261713  PMID: 22068332
mass spectrometry; protein abundance; proteomics
11.  Absolute quantification of microbial proteomes at different states by directed mass spectrometry 
The developed, directed mass spectrometry workflow allows to generate consistent and system-wide quantitative maps of microbial proteomes in a single analysis. Application to the human pathogen L. interrogans revealed mechanistic proteome changes over time involved in pathogenic progression and antibiotic defense, and new insights about the regulation of absolute protein abundances within operons.
The developed, directed proteomic approach allowed consistent detection and absolute quantification of 1680 proteins of the human pathogen L. interrogans in a single LC–MS/MS experiment.The comparison of 25 extensive, consistent and quantitative proteome maps revealed new insights about the proteome changes involved in pathogenic progression and antibiotic defense of L. interrogans, and about the regulation of protein abundances within operons.The generated time-resolved data sets are compatible with pattern analysis algorithms developed for transcriptomics, including hierarchical clustering and functional enrichment analysis of the detected profile clusters.This is the first study that describes the absolute quantitative behavior of any proteome over multiple states and represents the most comprehensive proteome abundance pattern comparison for any organism to date.
Over the last decade, mass spectrometry (MS)-based proteomics has evolved as the method of choice for system-wide proteome studies and now allows for the characterization of several thousands of proteins in a single sample. Despite these great advances, redundant monitoring of protein levels over large sample numbers in a high-throughput manner remains a challenging task. New directed MS strategies have shown to overcome some of the current limitations, thereby enabling the acquisition of consistent and system-wide data sets of proteomes with low-to-moderate complexity at high throughput.
In this study, we applied this integrated, two-stage MS strategy to investigate global proteome changes in the human pathogen L. interrogans. In the initial discovery phase, 1680 proteins (out of around 3600 gene products) could be identified (Schmidt et al, 2008) and, by focusing precious MS-sequencing time on the most dominant, specific peptides per protein, all proteins could be accurately and consistently monitored over 25 different samples within a few days of instrument time in the following scoring phase (Figure 1). Additionally, the co-analysis of heavy reference peptides enabled us to obtain absolute protein concentration estimates for all identified proteins in each perturbation (Malmström et al, 2009). The detected proteins did not show any biases against functional groups or protein classes, including membrane proteins, and span an abundance range of more than three orders of magnitude, a range that is expected to cover most of the L. interrogans proteome (Malmström et al, 2009).
To elucidate mechanistic proteome changes over time involved in pathogenic progression and antibiotic defense of L. interrogans, we generated time-resolved proteome maps of cells perturbed with serum and three different antibiotics at sublethal concentrations that are currently used to treat Leptospirosis. This yielded an information-rich proteomic data set that describes, for the first time, the absolute quantitative behavior of any proteome over multiple states, and represents the most comprehensive proteome abundance pattern comparison for any organism to date. Using this unique property of the data set, we could quantify protein components of entire pathways across several time points and subject the data sets to cluster analysis, a tool that was previously limited to the transcript level due to incomplete sampling on protein level (Figure 4). Based on these analyses, we could demonstrate that Leptospira cells adjust the cellular abundance of a certain subset of proteins and pathways as a general response to stress while other parts of the proteome respond highly specific. The cells furthermore react to individual treatments by ‘fine tuning' the abundance of certain proteins and pathways in order to cope with the specific cause of stress. Intriguingly, the most specific and significant expression changes were observed for proteins involved in motility, tissue penetration and virulence after serum treatment where we tried to simulate the host environment. While many of the detected protein changes demonstrate good agreement with available transcriptomics data, most proteins showed a poor correlation. This includes potential virulence factors, like Loa22 or OmpL1, with confirmed expression in vivo that were significantly up-regulated on the protein level, but not on the mRNA level, strengthening the importance of proteomic studies. The high resolution and coverage of the proteome data set enabled us to further investigate protein abundance changes of co-regulated genes within operons. This suggests that although most proteins within an operon respond to regulation synchronously, bacterial cells seem to have subtle means to adjust the levels of individual proteins or protein groups outside of the general trend, a phenomena that was recently also observed on the transcript level of other bacteria (Güell et al, 2009).
The method can be implemented with standard high-resolution mass spectrometers and software tools that are readily available in the majority of proteomics laboratories. It is scalable to any proteome of low-to-medium complexity and can be extended to post-translational modifications or peptide-labeling strategies for quantification. We therefore expect the approach outlined here to become a cornerstone for microbial systems biology.
Over the past decade, liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) has evolved into the main proteome discovery technology. Up to several thousand proteins can now be reliably identified from a sample and the relative abundance of the identified proteins can be determined across samples. However, the remeasurement of substantially similar proteomes, for example those generated by perturbation experiments in systems biology, at high reproducibility and throughput remains challenging. Here, we apply a directed MS strategy to detect and quantify sets of pre-determined peptides in tryptic digests of cells of the human pathogen Leptospira interrogans at 25 different states. We show that in a single LC–MS/MS experiment around 5000 peptides, covering 1680 L. interrogans proteins, can be consistently detected and their absolute expression levels estimated, revealing new insights about the proteome changes involved in pathogenic progression and antibiotic defense of L. interrogans. This is the first study that describes the absolute quantitative behavior of any proteome over multiple states, and represents the most comprehensive proteome abundance pattern comparison for any organism to date.
PMCID: PMC3159967  PMID: 21772258
absolute quantification; directed mass spectrometry; Leptospira interrogans; microbiology; proteomics
12.  Template-free detection of macromolecular complexes in cryo electron tomograms 
Bioinformatics  2011;27(13):i69-i76.
Motivation: Cryo electron tomography (CryoET) produces 3D density maps of biological specimen in its near native states. Applied to small cells, cryoET produces 3D snapshots of the cellular distributions of large complexes. However, retrieving this information is non-trivial due to the low resolution and low signal-to-noise ratio in tomograms. Current pattern recognition methods identify complexes by matching known structures to the cryo electron tomogram. However, so far only a small fraction of all protein complexes have been structurally resolved. It is, therefore, of great importance to develop template-free methods for the discovery of previously unknown protein complexes in cryo electron tomograms.
Results: Here, we have developed an inference method for the template-free discovery of frequently occurring protein complexes in cryo electron tomograms. We provide a first proof-of-principle of the approach and assess its applicability using realistically simulated tomograms, allowing for the inclusion of noise and distortions due to missing wedge and electron optical factors. Our method is a step toward the template-free discovery of the shapes, abundance and spatial distributions of previously unknown macromolecular complexes in whole cell tomograms.
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC3117359  PMID: 21685103
13.  Visual proteomics of the human pathogen Leptospira interrogans 
Nature methods  2009;6(11):817-823.
Systems biology conceptualizes biological systems as dynamic networks of interacting elements, whereby functionally important properties are thought to emerge from the structure of such networks. Due to the ubiquitous role of complexes of interacting proteins in biological systems, their subunit composition and temporal and spatial arrangement within the cell are of particular interest. ‘Visual proteomics’ attempts to localize individual macromolecular complexes inside of intact cells by template matching reference structures into cryo electron tomograms. Here we have combined quantitative mass spectrometry and cryo electron tomography to detect, count and localize specific protein complexes within the cytoplasm of the human pathogen Leptospira interrogans. We describe a novel scoring function for visual proteomics and assess its performance and accuracy under realistic conditions. We discuss current and general limitations of the approach, as well as expected improvements in the future.
PMCID: PMC2862215  PMID: 19838170
14.  Probing Native Protein Structures by Chemical Cross-linking, Mass Spectrometry, and Bioinformatics* 
Chemical cross-linking of reactive groups in native proteins and protein complexes in combination with the identification of cross-linked sites by mass spectrometry has been in use for more than a decade. Recent advances in instrumentation, cross-linking protocols, and analysis software have led to a renewed interest in this technique, which promises to provide important information about native protein structure and the topology of protein complexes. In this article, we discuss the critical steps of chemical cross-linking and its implications for (structural) biology: reagent design and cross-linking protocols, separation and mass spectrometric analysis of cross-linked samples, dedicated software for data analysis, and the use of cross-linking data for computational modeling. Finally, the impact of protein cross-linking on various biological disciplines is highlighted.
PMCID: PMC2938055  PMID: 20360032
15.  Do Normal Radiographs Exclude Asphericity of the Femoral Head-Neck Junction? 
Asphericity of the femoral head-neck junction is one cause for femoroacetabular impingement of the hip. However, the asphericity often is underestimated on conventional radiographs. This study compares the presence of asphericity on conventional radiographs with its appearance on radial slices of magnetic resonance arthrography (MRA). We retrospectively reviewed 58 selected hips in 148 patients who underwent a surgical dislocation of the hip. To assess the circumference of the proximal femur, alpha angle and height of asphericity were measured in 14 positions using radial slices of MRA. The hips were assigned to one of four groups depending on the appearance of the head-neck junction on anteroposterior pelvic and lateral crosstable radiographs. Group I (n = 19) was circular on both planes, Group II (n = 19) was aspheric on the crosstable view, Group III (n = 4) was aspheric on the anteroposterior view, and Group IV (n = 13) was aspheric on both views. In all four groups, the highest alpha angle was found in the anterosuperior area of the head-neck junction. Even when conventional radiographs appeared normal, an increased alpha angle was present anterosuperiorly. Without the use of radial slices in MRA, the asphericity would be underestimated in these patients.
Level of Evidence: Level II, prognostic study. See the Guidelines for Authors for a complete description of levels of evidence.
PMCID: PMC2635443  PMID: 19023635
16.  Groin Pain after Open FAI Surgery: The Role of Intraarticular Adhesions 
Femoroacetabular impingement (FAI) is an established cause of osteoarthrosis of the hip. Surgery is intended to remove the cause of impingement with hip dislocation and resection of osseous prominences of the acetabular rim and of the femoral head-neck junction. Using the Merle d’Aubigné score and qualitative categories, recent studies suggest good to excellent outcomes in 75% to 80% of patients after open surgery with dislocation of the femoral head. Unsatisfactory outcome is mainly related to pain, located either in the area of the greater trochanter or in the groin. There are several reasons for persisting groin pain. Joint degeneration with joint space narrowing and/or osteophyte formation, insufficient correction of the acetabula, and femoral pathology are known factors for unsatisfactory outcome. Recently, intraarticular adhesions between the femoral neck and joint capsule have been identified as an additional cause of postoperative groin pain. The adhesions form between the joint capsule and the resected area on the femoral neck and may lead to soft tissue impingement. MR-arthrography is used for diagnosis and the adhesions can be treated successfully by arthroscopy. While arthroscopic resection improves outcome it is technically demanding. Avoiding the formation of adhesions is important and is perhaps best accomplished by passive motion exercises after the initial surgery.
Level of Evidence: Level V, expert opinion. See the Guidelines for Authors for a complete description of levels of evidence.
PMCID: PMC2635436  PMID: 19082679
17.  Impingement-free Hip Motion: The ‘Normal’ Angle Alpha after Osteochondroplasty 
Femoroacetabular impingement is considered a cause of hip osteoarthrosis. In cam impingement, an aspherical head-neck junction is squeezed into the joint and causes acetabular cartilage damage. The anterior offset angle α, observed on a lateral crosstable radiograph, reflects the location where the femoral head becomes aspheric. Previous studies reported a mean angle α of 42° in asymptomatic patients. Currently, it is believed an angle α of 50° to 55° is normal. The aim of this study was to identify that angle α which allows impingement-free motion. In 45 patients who underwent surgical treatment for femoroacetabular impingement, we measured the angle α preoperatively, immediately postoperatively, and 1 year postoperatively. All hips underwent femoral correction and, if necessary, acetabular correction. The correction was considered sufficient when, in 90° hip flexion, an internal rotation of 20° to 25° was possible. The angle α was corrected from a preoperative mean of 66° (range, 45°–79°) to 43° (range, 34°–60°) postoperatively. Because the acetabulum is corrected to normal first, the femoral correction is tested against a normal acetabulum. We therefore concluded an angle α of 43° achieved surgically and with impingement-free motion, represents the normal angle α, an angle lower than that currently considered sufficient.
PMCID: PMC2635435  PMID: 19018605
18.  Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans 
Nature  2009;460(7256):762-765.
Mass spectrometry based methods for relative proteome quantification have broadly impacted life science research. However, important research directions, particularly those involving mathematical modeling and simulation of biological processes, also critically depend on absolutely quantitative data, i.e. knowledge of the concentration of the expressed proteins as a function of cellular state. Until now, absolute protein concentration measurements of a significant fraction of the proteome (73%) have only been derived from genetically altered S. cerevisiae cells 1, a technique that is not directly portable from yeast to other species. In this study we developed and applied a mass spectrometry based strategy to determine the absolute quantity i.e. the average number of protein copies per cell in a cell population, for a significant fraction of the proteome in genetically unperturbed cells. Applying the technology to the human pathogen Leptospira interrogans, a spirochete responsible for Leptospirosis 4, we generated an absolute protein abundance scale for 83% of the mass spectrometry detectable proteome, from cells at different states. Taking advantage of the unique cellular dimensions of L. interrogans, we used cryo electron tomography (cryoET) morphological measurements to verify at the single cell level the average absolute abundance values of selected proteins determined by mass spectrometry on a population of cells. As the strategy is relatively fast and applicable to any cell type we expect that it will become a cornerstone of quantitative biology and systems biology.
PMCID: PMC2723184  PMID: 19606093
19.  Identification of cross-linked peptides from large sequence databases 
Nature methods  2008;5(4):315-318.
We describe a method to identify cross-linked peptides from complex samples and large protein sequence databases. The advance was achieved by combining isotopically tagged cross-linkers, chromatographic enrichment, targeted proteomics, and a novel search engine called xQuest. This software reduces the search space by an upstream candidatepeptide search before the recombination step; we show that xQuest can identify cross-linked peptides from a total E. coli lysate with an unrestricted database search.
PMCID: PMC2719781  PMID: 18327264
20.  Luminal particles within cellular microtubules 
The Journal of Cell Biology  2006;174(6):759-765.
The regulation of microtubule dynamics is attributed to microtubule-associated proteins that bind to the microtubule outer surface, but little is known about cellular components that may associate with the internal side of microtubules. We used cryoelectron tomography to investigate in a quantitative manner the three dimensional structure of microtubules in intact mammalian cells. We show that the lumen of microtubules in this native state is filled with discrete, globular particles with a diameter of 7 nm and spacings between 8 and 20 nm in neuronal cells. Cross-sectional views of microtubules confirm the presence of luminal material in vitreous sections of brain tissue. Most of the luminal particles had connections to the microtubule wall, as revealed in tomograms. A higher accumulation of particles was seen near the retracting plus ends of microtubules. The luminal particles were abundant in neurons, but were also observed in other cells, such as astrocytes and stem cells.
PMCID: PMC2064330  PMID: 16954350

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