Hans Dalebout

Fibronectin is a serum biomarker for Duchenne muscular dystrophy

To identify and validate serum biomarkers for the progression of Duchenne muscular dystrophy (DMD) using a MS‐based bottom‐up pipeline.

Automated imaging MS: Toward high throughput imaging mass spectrometry

The term molecular histology has been used to convey the potential of imaging mass spectrometry to describe tissue by its constituent peptides and proteins, and to link this with established histological features. The low throughput of imaging mass spectrometry has been one of the factors inhibiting a full investigation of the clinical potential of molecular histology. Here we report the development of an automated set-up, consisting of a controlled environment sample storage chamber, a sample loading robot, and a MALDI-TOF/TOF mass spectrometer, all controlled by a single user interface. The automated set-up is demonstrated to have the positional stability and experimental reproducibility necessary for its clinical application.

Quality control based on isotopic distributions for high-throughput MALDI-TOF and MALDI-FTICR serum peptide profiling

In this study, we have implemented a new quality control (QC) parameter for peptide profiling based on isotopic distributions. This QC parameter is an objective measure and facilitates automatic sorting of large numbers of peptide spectra. Peptides in human serum samples were enriched using reversed-phase C18-functionalized magnetic beads using a high-throughput robotic platform. High-resolution MALDI-TOF and ultrahigh resolution MALDI-FTICR mass spectra were obtained and a workflow was developed for automated analysis and evaluation of these profiles. To this end, the isotopic distributions of multiple peptides were quantified from both MALDI-TOF and MALDI-FTICR spectra. Odd peptide isotope distributions in TOF spectra could be rationalized from ultrahigh resolution FTICR spectra that showed overlap of different peptides. The comparison of isotope patterns with estimated polyaveragine distributions was used to calculate a QC value for each single mass spectrum. Sorting these QC values enabled the best MALDI spectrum to be selected from replicate spots. Moreover, using this approach spectra containing high intensities of polymers or other contaminants and lacking peptides of interest can be efficiently removed from a clinical dataset. In general, this method simplifies the exclusion of low quality spectra from further statistical analysis.

Electron capture dissociation of peptide hormone changes upon opening of the tocin ring and complexation with transition metal cations.

Electron capture dissociation (ECD) is an analytical technique in mass spectrometry (MS) that allows detailed structural study of biomolecules to gain insight in their function. In this work the ECD behavior of two peptide hormones oxytocin (OT1) and vasopressin (VP1) was studied. The results of OT1 and VP1 were compared to structural analogues OT2 and VP2, which have similar amino acid sequences but lack the tocin ring. The ECD results showed that both the fragment type (c/z versus b/y) and the cleavage sites (ring versus tail) changed upon opening of the tocin ring. All four peptides were complexed with three different transition metal cations (Zn(2+), Ni(2+) and Cu(2+)) and the ECD results were compared to those obtained from the doubly protonated species. The use of various metal ions yielded different cleavages sites within the same peptide. This can be an effect of the metal ion itself, or a consequence of a change in conformation as was suggested earlier. In addition, the type of fragment ion varied for each metal-complexed peptide, which is in agreement with previous observations.

Improving mass measurement accuracy in mass spectrometry based proteomics by combining open source tools for chromatographic alignment and internal calibration

Accurate mass determination enhances peptide identification in mass spectrometry based proteomics. We here describe the combination of two previously published open source software tools to improve mass measurement accuracy in Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). The first program, msalign, aligns one MS/MS dataset with one FTICRMS dataset. The second software, recal2, uses peptides identified from the MS/MS data for automated internal calibration of the FTICR spectra, resulting in sub-ppm mass measurement errors.

Comparative mass spectrometric and immunoassay-based proteome analysis in serum of Duchenne muscular dystrophy patients.

Duchenne muscular dystrophy (DMD) is a severe and fatal neuromuscular disease. With the current developments on novel therapeutic strategies for DMD, the need to carefully monitor disease progression or regression upon treatment using molecular markers has become urgent.

A Novel Mass Spectrometry Cluster for High-Throughput Quantitative Proteomics

We have developed and implemented a novel mass spectrometry (MS) platform combining the advantages of high mass accuracy and resolving power of Fourier transform ion cyclotron resonance (FTICR) with the economy and speed of multiple ion traps for tandem mass spectrometry. The instruments are integrated using novel algorithms and software and work in concert as one system. Using chromatographic time compression, a single expensive FTICR mass spectrometer can match the throughput of multiple relatively inexpensive ion trap instruments. Liquid chromatography (LC)-mass spectrometry data from the two types of spectrometers are aligned and combined to hybrid datasets, from which peptides are identified using accurate mass from the FTICR data and tandem mass spectra from the ion trap data. In addition, the high resolving power and dynamic range of a 12 tesla FTICR also allows precise label-free quantitation. Using two ion traps in parallel with one LC allows simultaneous MS/MS experiments and optimal application of collision induced dissociation and electrontransfer dissociation throughout the chromatographic separation for increased proteome coverage, characterization of post-translational modifications and/or simultaneous measurement in positive and negative ionization mode. An FTICR-ion trap cluster can achieve similar performance and sample throughput as multiple hybrid ion trap-FTICR instruments, but at a lower cost. We here describe the first such FTICR-ion trap cluster, its performance and the idea of chromatographic compression.

Identification of meat products by shotgun spectral matching

A new method, based on shotgun spectral matching of peptide tandem mass spectra, was successfully applied to the identification of different food species. The method was demonstrated to work on raw as well as processed samples from 16 mammalian and 10 bird species by counting spectral matches to spectral libraries in a reference database with one spectral library per species. A phylogenetic tree could also be constructed directly from the spectra. Nearly all samples could be correctly identified at the species level, and 100% at the genus level. The method does not use any genomic information and unlike targeted methods, no prior knowledge of genetic variation within a genus or species is necessary.

Authentication of Closely Related Fish and Derived Fish Products Using Tandem Mass Spectrometry and Spectral Library Matching

Proteomics methodology has seen increased application in food authentication, including tandem mass spectrometry of targeted species-specific peptides in raw, processed, or mixed food products. We have previously described an alternative principle that uses untargeted data acquisition and spectral library matching, essentially spectral counting, to compare and identify samples without the need for genomic sequence information in food species populations. Here, we present an interlaboratory comparison demonstrating how a method based on this principle performs in a realistic context. We also increasingly challenge the method by using data from different types of mass spectrometers, by trying to distinguish closely related and commercially important flatfish, and by analyzing heavily contaminated samples. The method was found to be robust in different laboratories, and 94-97% of the analyzed samples were correctly identified, including all processed and contaminated samples.

Case-control breast cancer study of MALDI-TOF proteomic mass spectrometry data on serum samples.

We introduce mass spectrometry proteomic research for diagnosis from a clinical perspective, with special reference to early-stage breast cancer detection. The nature of SELDI and MALDI mass spectrometric measurement is discussed. We explain how the mass spectral data arising from this technology may be viewed as a new data type. Some of the properties of the data are discussed and we show how such spectra may be interpreted. Sample preprocessing for mass spectrometry is introduced and a literature review of research in clinical proteomics is presented. Finally, we provide a detailed description of the study design on the breast cancer case-control study which is investigated in this special issue.