My research interest concerns the functional interactions of plant cell organelles during development and differentiation, with an emphasis on chloroplast functions. We are using the tools of functional genomics, proteomics and systems biology to deal with the complexity of molecular responses and to unravel currently unknown dependencies in metabolic and regulatory networks. During our work we have established organellar protein inventories and we now use this information to analyze quantitative proteome and phosphoproteome dynamics under a variety of different conditions. Our research is currently focused around three major topics: i. Integration of plastids in cellular Ca²⁺ signaling, ii. Regulation and control of plastid protein import, and iii. Topology and dynamics of chloroplast phosphorylation networks.

Different functional proteomics tools are now available that enable the quantitative characterization of proteome dynamics and the mapping of posttranslational modifications. We report here examples how we used these tools to characterize the functional proteome of Arabidopsis and rice cell organelles, with a focus on plant-specific plastids. We analyzed the Arabidopsis proteome at genome-scale and provide quantitative information about organellar proteomes in different plant organs by “normalized spectral counting” (Baerenfaller et al., Science 320, 938-41; Baerenfaller et al., Integrative Biology 3, 225-237). For a functional characterization of plastid protein import, we analyzed the proteomes of protein import mutants and established protein N-termini to distinguish precursor from mature plastid proteins in WT, ppi1 and ppi2. These analyses revealed the accumulation of precursor proteins in the cytosol of protein import mutants. In order to assess the short-term regulation of the chloroplast proteome in response to environmental signals, we analyzed the chloroplast phosphoproteome and characterized its dynamics during a circadian cycle (Reiland et al., Plant Physiol. 150, 889-903). Phosphorylation motif utilization suggests that the phosphorylation network topology in chloroplasts is characterized by many-to-many relationships. To establish the kinase/substrate nodes in this network we performed comparative quantitative phosphoproteome profiling experiments with wildtype and kinase mutant plant material, starting out with the STN8 kinase. Differential protein phosphorylation was assessed by relative quantification with “extracted ion chromatograms” and the data were further validated by functional characterization of selected substrates (Reiland et al., Proc. Natl. Acad. Sci. USA, in press). We present here our data, comment on reliability and reproducibility and propose strategies to increase both at a reasonable cost.

Perdita Barran is a Senior Lecturer in Biophysical Chemistry at the University of Edinburgh. The Barran group has considerable experience in gas-phase ion chemistry, instrument development and the application of mass spectrometry to complex chemical and biological problems. Dr Barran has published over 50 papers with 595 citations to date, as well as being an invited/keynote speaker at 5 international conferences in the past two years. The Barran research group currently comprises of 7 graduate students, and 2 PDRA’s. Since Barran started as an independent researcher, 12 PhD students have successfully defended their PhD’s. Barran develops techniques to investigate changes in protein conformation that occur in response to chemical or physical intervention and relates them to their biological role. In 2009 in recognition of her achievements Barran was awarded the inaugural Joseph Black award by the RSC Analytical Division for her contribution to the developments of biological mass spectrometry.

The p53 protein is a transcription factor which plays a central role to tumour suppression. Loss of p53 function is correlated with the development of cancer in which the MDM2 protein binds to the N-terminal transcription activation domain shutting down function³. Mass spectrometry studies of WT p53 and mutants of the DNA binding domain following nano-electrospray from native conditions show a wide charge state range for charge states n= 8 - 18 for monomeric species of the general form [M+nH]ⁿ⁺. Mass spectrometry data from both instruments are qualitatively similar. The most abundant peaks are at n =9 and n=10 indicating the dominance of a compact structure. Multimeric species are observed for the WT and mutants, however the dimers shown in the WT are more intense. Generally, the collision cross-sections of the monomer are observed to increase with increasing charge in a stochastic fashion attributed to protein unfolding due principally to coulombic repulsion, which can be attributed to the ‘plasticity’ of these IDP’s . Multiple gas-phase conformers are resolved for a number of charge states, over a very wide charge state range. Subtle differences in unfolding transitions between the WT and mutated samples can be characterized. For instance the H115N mutant, altered at the boundary of loop I seems to be less structured – favoring the more extended conformations when compared with WT p53.,Conversely, the R249A/H115N mutant appears to be more compact. The thermally induced unfolding also shows interesting trends, and in particular reveals the resistance to unfolding of this important IDP. These results are interpreted in terms of the biological activity of these proteins and also in terms of the implications for the use of IM-MS to study this largely ignored, but critically important, class of proteins.

Guy Bouchoux was born in Paris in 1946. He studied chemistry at Orsay University (France) where he is teaching since 1970 and where he is presently a Professor of Chemistry. During the same period he was conducting his research at the Ecole Polytechnique (Palaiseau). His research interests include gas-phase ion chemistry (mechanisms of unimolecular and ion molecule reactions) and thermochemistry (protonation energetic, quantum chemical calculations). Guy Bouchoux is the (co-) author of ca 200 publications and one of the founders of the Société Française de Spectrométrie de Masse.

Structural characterization of molecular species by mass spectrometry supposes the knowledge of the type of ions generated and the mechanism by which they dissociate. In this context, a need for a rationalization of ESI(+)(-) mass spectra of small molecules has been recently expressed [1]. Similarly, at the other end of the mass scale, efforts are currently made to interpret the major fragmentation processes of protonated and deprotonated peptides and their reduced forms produced in electron capture (ECD) or electron transfer (ETD) experiments [2,3].

Most fragmentation processes of molecular and pseudo-molecular ions may be described by a combination of several key mechanistic steps: simple bond dissociation, hydrogen atom, hydride ion or proton migrations, formation of ion-neutral complex intermediates… Selected crucial aspects of these elementary reactions, occurring inside positively charged ions, will be recalled and illustrated by examples taken in the recent mass spectrometry literature. Emphasis will be given on the protonation process and its consequences in term of structure and energetic [4].

1 – WMA. Niessen. Mass Spectrom Rev 2011, 30, 626-663.

2 – AG Harrison. Mass Spectrom Rev 2009, 28, 640-654.

3 – SA McLuckey, M Mentinova. J Am Soc Mass Spectrom 2011, 22, 3-12.

4 – G Bouchoux. Mass Spectrom Rev 2007, 26, 775-835.

Dr. Jennifer S. Brodbelt is a professor of chemistry at the University of Texas at Austin. She earned her B.S. degree in chemistry at the University of Virginia and her doctorate in chemistry at Purdue University under the supervision of Prof. Graham Cooks. After a post-doctoral position at the University of California at Santa Barbara, she began her academic career at the University of Texas in 1989. Her research interests focus on the development and application of photodissociation as an ion activation method for characterization of biological molecules, including peptides, proteins, nucleic acids, oligosaccharides, and lipids. Her group has also developed the use of mass spectrometry for evaluation of DNA/drug interactions.

The tremendous growth in the application of mass spectrometry for detection, quantification and characterization of biological molecules has spurred the exploration of new ion activation/dissociation methods. Although collision induced dissociation (CID) remains the gold standard for structural characterization of ions, it has several shortcomings (e.g. insufficient energy deposition, limited applicability for pinpointing post-translational modifications in peptides, etc.) that have stimulated the search for other activation methods. Photodissociation offers an especially promising alternative to traditional CID in ion traps, in addition to its success with FTICR and time-of-flight instruments.

There are several compelling advantages of using lasers to activate ions in a mass spectrometer. Photoactivation is a non-resonant process, meaning that both the selected precursor ions and primary fragment ions may be activated, leading to secondary dissociation and a richer array of fragment ions without requiring deliberate sequential stages of ion manipulation. The ability to vary energy deposition without resorting to multi-stage experiments (MSⁿ) is particularly important when analyzing macromolecules in which initial ion currents may be low and the total sample quantity is limited.

The reduction of ion losses is a critical feature when analyzing small ion populations while maintaining adequate detection sensitivity. Photoactivation can be utilized to analyze both negative and positive ions, so it offers the potential for broader characterization of the proteome. UV photodissociation using a laser offers fast, high energy deposition that yields good sequence coverage for peptide analysis.

Dr. Virginie Brun completed her undergraduate studies in Veterinary Medicine at the Lyon Veterinary School, France, in 1999. She received a PhD in Physiology at the Claude Bernard University of Lyon, in 2003. Then, she joined the "Exploring the Dynamics of Proteomes" laboratory, headed by Dr Jérôme Garin, in Grenoble, France, for a 4-years post-doctoral training. During this period, she optimized and developed methodologies for absolute quantitative proteomics, in particular the "Protein Standard Absolute Quantification" (PSAQ) strategy.

She's currently working as a research scientist in the "Exploring the Dynamics of Proteomes" laboratory at the french Atomic Energy Commission and French National Institute for Health and Medical Research ( EDyP website). She's in charge of biomedical research projects with a special focus on quantitative proteomics. Dr Virginie Brun is also the co-founder of Promise Advanced Proteomics, a spin-off company located in Grenoble, France, specialized in the synthesis of isotopically labelled proteins and related MS-based analytical services (http://www.promise-proteomics.com). 

Technological developments that will provide reliable and multiplex quantification of proteins in biofluids are critical for biomarker research and medical science. Mass-spectrometry analysis is currently attracting considerable interest due to its multiplexing capacities. Combined with stable isotope-labelled quantification standards [1], MS-based assays can provide quantitative data for hundreds of peptides generated by trypsin digestion of proteins. However, to envision MS as a relevant methodology for biomarker evaluation and biomarker measurement in clinical laboratories, some analytical variabilities still need to be resolved [2].

In 2007, we developed the PSAQ^TM method (Protein Standard Absolute Quantification) which uses full-length isotope-labelled protein standards to quantify target proteins [3]. In this presentation, we’ll demonstrate the specific advantages of the PSAQ^TM method for accurate and reliable quantification of protein biomarkers. Recent technological advances related to the production and use of PSAQ^TM standards will be presented. Several applications of the PSAQ^TM method in the health domain will also be exposed.

References

1. Brun V. et al., (2009) Isotope dilution strategies for absolute quantitative proteomics. J. Proteomics 72, 740-749.

2. Hoofnagle A.N. et al., (2010)Quantitative Clinical Proteomics by Liquid Chromatography-Tandem Mass Spectrometry: Assessing the Platform. Clin. Chem. 56, 161-164.

3. Brun V. et al (2007) Isotope-labeled protein standards: toward absolute quantitative proteomics. Mol. Cell. Proteomics 6, 2139-2149.

For the past 20 years, Prof. Pierre Chaurand has contributed to the development and characterization of various aspects of matrix-assisted laser desorption ionization (MALDI) mass spectrometry. During his Ph.D. years (1991-1994, Université de Paris Sud, Orsay, France), his contributions ranged from the fundamental understanding of ion production via the MALDI process and subsequent detection by impact on various surfaces including microchannel plate detectors. During his post-doctoral training (1994-1998, University of Düsseldorf, Germany), Prof. Chaurand’s efforts were forwarded focused on the design and construction of MALDI time-of flight mass spectrometers optimized for peptide sequencing by post-source decay. Prof. Chaurand has then spent 11 years of his professional career (1998-2009) has research faculty at Vanderbilt University (Nashville TN, USA) contributing to the development of a technology named 'imaging mass spectrometry', which through the direct analysis of thin tissue sections by MALDI mass spectrometry, allows the profiling and mapping of biomolecules including proteins, lipids and other metabolites, as well as administered pharmaceuticals. In 2009, Prof. Chaurand has joined the Dept of Chemistry at the University of Montreal, where his research efforts for the development of the imaging mass spectrometry technology will be continued.

MALDI-based imaging mass spectrometry is a new technology that allows to map different biocompounds and xenobiotics directly from thin tissue sections. Numerous classes of biomolecules including metabolites, phospholipids, peptides and proteins can be detected and mapped in direct correlation with the underlying histology. Molecular profiles and images depend on the types of tissues or cells studied and certain signals can be directly correlated with the health status of the tissue specimen. Indeed, the technology is sufficiently sensitive to detect variations in the molecular composition induced by the presence of disease or by drug uptake. Numerous technical advances such as automated matrix deposition and the development of in situ chemistries now allow us to study the proteomic content of fresh frozen and formalin fixed paraffin embedded tissue specimens.

After an introduction of the technology and a description of current progresses the different fields of research of imaging mass spectrometry will be presented. In particular its enormous potential in clinical settings in complement to traditional histopathology and its important role in the study of drug distribution and effects in various biological tissues will be described. Finally, a critical outlook will be made towards the developments to be made for the technology to become a mainstream analytical tool.

Graham Cooks is a pioneer in the conception and implementation of MS/MS and of desorption ionization. These interests led to the construction of miniature ion trap mass spectrometers and their application to problems of trace chemical detection. His work on ionization methods has contributed to the ambient ionization methods including desorption electrospray ionization. Some inventions and concepts introduced by Graham Cooks are Ion soft landing, neutral loss scans, the use of matrices in mass spectrometry, multiple reaction monitoring (MRM) and single reaction monitoring (SRM), hybrid mass spectrometers, handheld mass spectrometers, the kinetic method of thermochemical determinations as well as MS/MS for mixture analysis.

The recent development of ambient ionization methods is transforming the applications of mass spectrometry (MS) allowing virtually any sample to be examined in air, rather than being introduced into the vacuum system. This talk focuses on two ambient ionization methods: (i) paper spray (PS) in which in which samples are ionized in air, directly from filter paper (and similar materials including whole plant or animal tissue) and (ii) desorption electrospray ionization (DESI) in which the sample is impacted by charged microdroplets which pick up analyte by dissolution and carry it to the MS. The physical basis of each of these experiments is described.

DESI finds application in disease diagnosis by tissue imaging and examples of human bladder, liver and brain cancer diagnostics will be given. DESI imaging combines the chemical information collected for multiple analytes from the mass spectrometer with spatial information, which makes it useful for analyzing histological sections of biological tissue. Alterations in the distribution of polar lipids are associated with malignant transformations in tissue. Multivariate statistical analysis using principal component analysis (PCA) is used to analyze the imaging MS data, magnifying differences between the lipid profiles of tissue as a function of disease state. Data showing glioma diagnosis and prostate biomarker recognition will be emphasized.

Whole blood analysis for therapeutic agents is achieved in a few seconds using paper spray. Remarkably, the method is quantitatively accurate and precise and covers the therapeutic range for many oncology drugs. The promise of this method for point-of-care diagnostics will be shown. LTP applications in food safety and bacterial identification also will be described. Ultimately ambient ionization methods are best used with handheld miniature mass spectrometers, a combination that has enormous potential to transform in situ chemical analysis. Progress in interfacing ambient ionization sources to miniature mass spectrometers is described.

The support of this work by NSF, DOE and the Alfred Mann Foundation at Purdue is gratefully acknowledged as is ongoing collaboration with Prof. Zheng Ouyang.

Phagosomes are intracellular organelles formed in a variety of cells following the engulfment of large particulate materials by phagocytosis. This organelle plays key roles in both innate and adaptive immunity by promoting the establishment of the molecular properties needed to kill microorganisms and present some of their antigens on MHC molecules.

In the last 20 years, the striking advances in the field of proteomics and systems biology have largely contributed to our understanding of the molecular mechanisms regulating phagosome functions. This presentation will highlight how the application of large-scale approaches has contributed to reveal novel aspects of the functional properties of phagosomes and their emergence during evolution.

Since the 1980s Bruno Domon has applied novel techniques to characterize proteins and glycoproteins in academia and in the pharmaceutical/biotech industry. At MIT he pioneered, with Catherine E. Costello, the MS/MS fragmentation of glycoconjugates, and developed a systematic nomenclature. He worked in the industry (1988 – 2000), heading the mass spectrometry facility at Ciba in Basel and then at Biogen in Cambridge, USA. He went on to head the large-scale proteomics facility at Celera Genomics in Rockville, MD (2001-2004), then joined the group of Ruedi Aebersold at ETH Zurich (2004-2009), where he worked, as group leader, to develop alternate proteomics workflows and targeted approaches based on selected reaction monitoring, expanding this technology to encompass large-scale screening.

In 2010 Domon assumed leadership of the new Luxembourg Clinical Proteomics Center (LCP), and now heads the effort to develop high-throughput quantitative mass spectrometry methods to qualify and biomarkers in bodily fluids, with the goal of translating them into routine clinical assays. LCP is part of a wider effort in Luxembourg to push the envelope of translational biomedical research.

The generation of accurate and comprehensive data sets has become an essential element of systems biology and biomarker studies. While large scale discovery experiments one hand and targeted quantitative analyses on the other are nowadays widely used in proteomics, we are proposing an alternative approach, which is based on a novel quadrupole/orbitrap instrument. The high acquisition speed and the exquisite sensitivity of such a hybrid mass spectrometer allow performing reliable qualitative and quantitative experiments. The additional selectivity intrinsic to the high resolution orbitrap mass analyzer enables the development of novel quantification methods. Quantitative experiments can be performed either in full scan mode (using the high-resolution / accurate mass capability) or in MS/MS mode by analyzing specific fragment ions (i.e. SRM-like mode). The different modes of operation, their advantages and limitations will be presented in details. This technique has been applied to precisely quantify biomarker candidates in bodily fluids, and more specifically in urine samples. The quantitative analyses were performed in conjunction with stable isotope dilution, using second generation synthetic polypeptides, composed of one universal reporter fragment facilitating the systematic, precise quantification of multiple analytes in complex biological samples.

Dr Gavin is group leader in the Structural and Computational Biology Unit at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany. Before moving to the EMBL in 2005, she was Director of the Molecular and Cell Biology division of the biotech company Cellzome Inc. Previously she spent four years as a postdoctoral fellow at the Department for Physiology, University of Basel, Switzerland and at the EMBL Heidelberg, Germany. She received her PhD from the University of Geneva, Switzerland. Gavin’s work on the proteome organization of the yeast Saccharomyces cerevisiae represented a technology breakthrough and had a wide impact in biochemistry, quantifiable by more than 1800 citations. She received several honors and awards for her research, including the Genome Technology All-Stars award “Most Prolific in Proteomics” and the Heidelberg Molecular Life Science award. She is editorial board member of Molecular and Cellular Proteomics and BMC genomics. Gavin’s research programs integrate biochemical, mass spectrometry, structural and computational methods to characterize cellular networks and circuitry at molecular levels, both spatially and temporally. Her research aims at understanding how cellular components work collectively and achieve biological function.

Biological function emerges from the concerted action of numerous interacting biomolecules. Deciphering the molecular mechanisms behind cellular processes requires the systematic charting of the multitude of interactions between all cellular components. While protein–protein and protein– DNA networks have been the subject of many systematic surveys, others critically important cellular components, such as lipids, have to date rarely been studied in large-scale interaction screens.

The importance of protein–lipid interactions is evident from the variety of protein domains that have evolved to bind particular lipids and from the large list of disorders, such as cancer and bipolar disorder, arising from altered protein–lipid interactions. The importance of lipids in biological processes and their under-representation in current biological networks suggest the need for systematic, unbiased biochemical screens. Here, we report a screen to catalog protein–lipid interactions in yeast using a lipid arrays.

To illustrate the data set’s biological value, we studied further several novel interactions with sphingolipids, a class of conserved bioactive lipids with an elusive mode of action. Integration of live-cell imaging suggests new cellular targets for these molecules, including several with pleckstrin homology (PH) domains. The dataset presented here represents an excellent resource to enhance the understanding of lipids function in eukaryotic systems.

Seth Grant is best known for his work using mouse genetics and synapse proteomics to study synaptic function, plasticity, behavior and disease. He directs the Genes to Cognition Programme that studies the role of synaptic proteins in diseases and with collaborators is studying the involvement of these proteins in psychiatric diseases. He received degrees in physiology, medicine and surgery from the University of Sydney and postdoctoral training at Cold Spring Harbor Laboratory and with Eric Kandel at Columbia University. He is a principal scientist at the Wellcome Trust Sanger Institute in Cambridge UK and Professor of Molecular Neuroscience at Edinburgh and Cambridge Universities, Professorial Fellow at the Florey Institute for Neuroscience in Melbourne Australia and Fellow of the Royal Society of Edinburgh.

Proteomic mass spectrometry has been a groundbreaking technology for studying the composition of complex subcellular organelles. In the nervous system, proteomics has discovered more synaptic proteins than any other method and uncovered a remarkable complexity in the signaling machinery involved in the communication between nerve cells. These data sets have opened up new insights into the evolution of the brain, the organisation and architecture of complex circuits and in the diversity of behavior. The presentation will show how synapse proteomics has introduced molecular complexity into neuroscience and how from this complexity simple organizational features and novel mechanisms have emerged. Synapse proteomics has allowed a unique convergence with human genetics leading to the identification of subsets of proteins that control phenotypes that are involved with many brain diseases. The study of the human synapse proteome has revealed that postsynaptic proteome is disrupted by over 200 mutations involved with over 130 brain diseases.

Martin F. Jarrold was born and raised in England. He obtained his undergraduate and graduate degrees from the University of Warwick, and then went to the University of California, Santa Barbara as a NATO Postdoctoral Fellow. After several years at UCSB, he joined the Physics Research Division of AT&T Bell Laboratories in Murray Hill, NJ. His work at Bell Labs focused on determining the properties of metal and semiconductor clusters, particularly silicon clusters. He moved to Northwestern University in 1992 to become a Professor in the Chemistry Department. While at Northwestern he performed pioneering work on ion mobility mass spectrometry, developing methods to extract structural information from mobility measurements. In 2002 he moved to Indiana University as Professor and Robert and Marjorie Mann Chair in the Chemistry Department. His work at IU has focused on investigating phase transitions in metal clusters and in developing charge detection mass spectrometry.

This presentation will provide an overview of recent work pushing the boundaries of sensitivity and accuracy with charge detection mass spectrometry. In conventional mass spectrometry the m/z distribution is obtained for an ensemble of ions, z is then deduced from the m/z distribution of the ensemble, and the ion mass is obtained from the corresponding m/z and z. This approach only works when the charge states are resolved in the m/z distribution, which is usually not the case for large and heterogeneous ions. In charge detection mass spectrometry, m/z and z are directly measured for each individual ion, so that the mass can be determined for each ion. This approach allows true mass spectra to be determined for very large and very heterogeneous ions. The two main challenges with charge detection mass spectrometry are the sensitivity and accuracy of the charge measurements. Efforts to attain an accuracy of a single elementary charge (1e) and a sensitivity of 10e will be described. Applications to ions with masses from kilodaltons to teradaltons will be demonstrated.

Dr. Pascal Kintz has a degree in Pharmacy (1985), a Diplôme d'Etudes Approfondies in Molecular Pharmacology and a PhD in Toxicology (1989) of the Université Louis Pasteur in Strasbourg. He was Associate Director of the Institute of Legal Medicine of Strasbourg and Associate Professor of Legal Medicine until the end of 2004. Then he was Head of the Scientific Affairs at ChemTox Laboratory, a private structure in Strasbourg, France (2005-2010). Currently, he is consultant in Toxicology, President of his own company, X-Pertise Consulting.

His main topics of interest include: alternative specimens with a special focus on hair and oral fluid, pharmacology of drugs of abuse, postmortem toxicology and doping control.

He is active in several national and international scientific societies, such as Société Française de Toxicologie Analytique, SFTA (President 1997-2003), The International Association of Forensic Toxicologists, TIAFT (President 2005-2008) and the Society of Hair Testing (Founding Member in 1995, President since 2008).

He received the TIAFT Award for Excellence in 2001

He is an Expert for Justice for the Court of Appeal of Colmar since 1992, appointed by the Court of Cassation since 2007, for Pharmacology / Toxicology and blood alcohol determination and an Expert certified by the Gesellschaft für Toxicologische und Forensische Chemie (Germany) and Eurotox.

Dr Kintz has published more than 300 papers in peer-reviewed journals. He is associate editor of Journal of Analytical Toxicology and regular reviewer for Journal of Chromatography, Forensic Science International, Clinical Chemistry, Journal of Pharmaceutical Sciences, and Annales de Toxicologie Analytique.

The use of a drug to modify a person’s behaviour for criminal gain is not a recent phenomenon. However, the recent increase in reports of drug-facilitated crimes (sexual assault, robbery) has caused alarm in the general public. Drugs involved can be pharmaceuticals, such as benzodiazepines (flunitrazepam, lorazepam ...), hypnotics (zopiclone, zolpidem), sedatives (scopolamine, neuroleptics, some anti-H1) or anaesthetics (GHB, ketamine), drugs of abuse, such as cannabis, ecstasy or LSD, or more often ethanol.

To perform successful toxicological examinations, the analyst must follow some important rules : 1. obtain as soon as possible the corresponding biological specimens (blood, urine and hair), 2. use sophisticated analytical techniques (LC/MS, HS/GC/MS, tandem mass spectrometry); and 3. take care on the interpretation of the findings.

Even after the publication of these guidelines for the clinicians, in most cases specimens are collected at best 24 hours after the crime has occurred.

Drugs used to facilitate sexual assaults can be difficult to detect (active products at low dosages, chemical instability), possess amnesic properties and can be rapidly cleared from the body (short half-life). Prohibiting immunoassays and using only hyphenated techniques, substances can be found in blood for 6 hours to 2 days and in urine for 12 hours to 5 days. In these situations, blood or even urine can be of poor interest. This is the reason why this laboratory developed an original approach based on hair testing.

Hair was suggested as a valuable specimen in situations where, as a result of a delay in reporting the crime, natural processes have eliminated the drug from typical biological specimens. While there is a lot of papers focused on the identification of drugs in hair following chronic drug use, those dealing with a single dose are very scarce.

This laboratory recommends to wait for 3-4 weeks after the offense and then collect 4 strands of about 100 hair. One strand will be used to test for drugs of abuse (mostly for cannabis, but also for ecstasy related compounds and cocaine that are sometimes observed), one for GHB and the other one for a screening of 30 various sedatives. The last strand can be used for a potential counter-analysis. After decontamination, hair is then segmented as follows : 0 to 2 cm (segment corresponding to the period of crime), 2 to 4 and 4 to 6 cm (which should be drug-free). For GHB, segments are of 3 mm (n=8).

Conventional GC/MS can be used to test for drugs of abuse, but given the expected concentrations to measure in low weight segments (in order to avoid the shave the victim), GHB and sedatives are tested by GC-MS/MS and UPLC-MS/MS, respectively.

The experience of the authors will be documented in cases involving GHB, zolpidem, bromazepam, alprazolam, scopolamine, alimemazine, diphenhydramine ...

Hair analysis may be a useful adjunct to conventional drug testing in sexual assault. It should not be considered as an alternative to blood and urine analyses, but as a complement. This approach may find useful applications, but appears very expensive, given the number of analyses to achieve with sophisticated equipment.

Prof. Oliver Kohlbacher holds a Diplom in chemistry from Saarland University in Saarbrücken, where he also obtained a PhD in computer science. In 2000 he became a junior group leader in Saarbrücken working on protein-protein docking. After a postdoc at Celera Genomics (Rockville, MD, USA) he became full professor for bioinformatics at the University of Tübingen, where he is also a director of the center for bioinformatics and speaker of the department of computer science. He has been working on computational methods for the analysis of high-throughput data (proteomics, metabolomics, next-generation sequencing). He is one of the initiators of the OpenMS software package for computational mass spectrometry. Additional research interests are in the area of computer-aided drug design, computational immunology and systems biology.

Over the last decade HPLC-MS has become the workhorse in proteomics and metabolomics. Increasing sensitivity and resolution of the instruments give us unprecedented coverage of the proteomes and metabolomes under investigation. The flip side of this development is the amount and complexity of data produced. Similar to what we observe in next-generation sequencing, bioinformatics analysis of a high-throughput experiment is rapidly becoming the bottleneck.

In this talk, I will illustrate how wet-lab workflows have to be matched by appropriate data analysis workflows in order to enable more complex experiments. Based on our software package OpenMS/TOPP, I will illustrate how analysis workflows can be set up, adapted to specific experimental designs for different quantification strategies. The resulting analyses can then be run automatically. Automated workflows also enable more compute-intensive methods, as computer power scales much better than manual labor done by PhD students and postdocs. I will give a few examples, how these more complex analyses can result in a drastic increase in the coverage and accuracy of the experiment.

Dr. Overall is a Professor and Tier 1 Canada Research Chair in Metalloproteinase Proteomics and Systems Biology at the University of British Columbia, Centre for Blood Research. He completed his undergraduate, honours Science and Masters degrees at the University of Adelaide, South Australia; his Ph.D. in Biochemistry at the University of Toronto; and was a MRC Centennial Fellow in his post-doctoral work with Dr Michael Smith Nobel Laureate, Biotechnology Laboratory. He won the Institute of Musculoskeletal Health and Arthritis CIHR Award as 2002 CIHR Scientist of the Year, the University of British Columbia Killam Senior Researcher Award (Science) 2005, and was the Chair of the 2003 MMP and the 2010 Protease Gordon Research Conferences. On Sabbatical in 1997-1998 he was a Visiting Scientist at British Biotech Pharmaceuticals, Oxford, UK and in 2004/2008 he was a Visiting Scientist at the Expert Protease Platform, Novartis Pharma, Basel, Switzerland. He is currently an External Senior Fellow, Freiburg Institute for Advanced Studies - FRIAS, Albert-Ludwigs-Universität Freiburg. With over 8000 citations for his 173 papers and with an h factor of 52, he is a highly influential scientist in the field. He is the pioneer of Degradomics, with 6 Nature Review Papers on this, protease genomics, drug target validation, MMP therapeutics, and substrate discovery and has developed numerous approaches to decipher the roles of proteases in vivo by elucidating the protease and substrate degradomes through quantitative proteomics and N- and C-terminome analysis in cell based systems and in animal models.

Protein termini are truncated by proteolysis, but the extent to which this molds the proteome in vivo is unknown. In addition to constitutive proteolysis during protein synthesis and maturation, the processing of a mature protein often irreversibly changes its activity. Specific degradomics techniques are needed to rapidly identify and quantify the N- and C-terminomes in order to reveal the extent of post-translational modifications of protein termini and therefore the functional state of key molecules, the extent of proteolysis in a system, and to identify new protease substrates. We have devised new approaches to enrich for the N and C termini of proteins for high throughput terminome analyses of human tissues and cells for the Human Proteome project, in particular for chromosome 21 that is Canada’s focus for the HPP. Broad coverage N-terminome analysis necessitates a negative selection procedure as the variety of original mature protein N-terminal blocked peptides each present individual chemical hurdles for their enrichment by positive selection strategies.

We developed a combined N-terminomics and C-terminomics and protease substrate discovery degradomics platforms for the simultaneous quantitative analysis of the N-terminome and proteolysis on a proteome-wide scale called Terminal Amino Isotopic Labelling of Substrates (TAILS, Kleifeld et al Nature Biotech 28, 281-288; Prudova et al 2010 Mol Cell Proteomics; auf dem Keller et al 2010 Mol Cell Proteomics) and C-TAILS (Schilling et al Nature Methods 2010). By using novel polymers to deplete the internal tryptic peptides, TAILS suffers little from sample loss and low yields, so requiring only 100 microgram of sample and one MS/MS analysis per sample. By a three-day procedure with flexible labelling options, TAILS can be adapted to a variety of experimental situations including cell culture and complex biological sample analysis. Incorporating iTRAQ labelling iTRAQ-TAILS also provides wide coverage of all forms of naturally blocked N-terminal peptides and allows for their quantification through labelling of lysine side-chains in up to 8 samples. In addition to providing valuable proteome annotation this has several unique advantages. TAILS permits exploitation of the acetylated and other blocked mature protein N-terminal peptides as a statistical classifier that is then used to set isotope ratio cut offs that reveal protease activity.

We introduce a novel parameter evaluating ion intensity dependent quantification confidences of single peptide quantifications. Being a quantitative procedure, TAILS can analyse the substrate degradome of a broad specificity protease or one with no known specificity without manual data parsing, in the same experiment, and also do this in vivo. We have applied TAILS to a variety of proteases and compared protease knock out mice in models of arthritis, skin inflammation and models of breast cancer metastasis and pancreatic carcinoma in the RIP-Tag model. Typical analyses identify over 3000 N-terminal peptides from which we found that the removal of the N-terminal methionine is dependent upon the amino acid at position 2 with distinct preferences found for valine, glycine, alanine and serine. In one experiment, acetylation occurred on 731 original mature protein N-terminal peptides but at the initiator methionine in only 153 of these instances. In 578 cases, acetylation was at position 2 in the protein after removal of ¹Met, with alanine, serine and methionine being the preferred acetylated residues. Internal acetylation sites exhibit a distinct acetylation pattern that differs from the N-terminal acetylation. Finally N-terminal positional proteomics enables MS sample simplification with proteins identified in bronchoalvelar fluid and serum having abundances spanning a range greater than six orders of magnitude. This underscores the potential of TAILS to tackle the dynamic range analysis problem in complex proteomes and as a high throughput approach annotate the N and C termini in the HPP. 

Paola Picotti obtained her PhD from the University of Padova, Italy. In 2007 she joined Ruedi Aebersold’s group at ETH Zuerich, where she worked as post-doctoral fellow. Her research focused on the development of targeted proteomic techniques, including selected reaction monitoring (SRM), and their application to the analysis of biological systems. She was also involved in the development of approaches and resources to promote the collection and dissemination of SRM assays and their extension to whole proteomes. In 2011 she started an independent research group at the Institute of Biochemistry of ETH Zuerich.

Selected Reaction Monitoring (SRM) is a targeted mass spectrometry technique which emerged in the field of proteomics as a complement to the untargeted shotgun methods. The main advantages of SRM become apparent when predetermined sets of proteins need to be measured across multiple samples in a consistent and accurate manner. The technology is however still in its infancy for protein analysis and several challenges need to be addressed to demonstrate the power of the technique in biological research and increase its widespread application in non-specialized laboratories.

To evaluate the applicability of SRM to studying biological networks, we applied it to the analysis of a metabolic network constituted by proteins covering a broad range of abundances and including a large number of families of isoenzymes, sharing high sequence overlap. Proteins in the network were quantified by SRM in yeast cells grown under a series of conditions inducing radically different metabolic setups and in a growth time-course of cells transiting through a series of metabolic phases. The quantitative dataset generated highlighted how yeast metabolism adapts to changing conditions of supply and demand of nutrients and suggested differential functionality for several isoenzymes. The application showed the potential of the technique to elucidate the dynamics of cellular networks through large number of perturbing conditions.

In order to expand the capabilities of the technique, we used an approach based on libraries of unpurified synthetic peptides to develop at high-throughput SRM assays for entire proteomes. We used the method to develop SRM assays for the ~6,000 proteins that constitute the proteome of S. cerevisiae and then expanded it to the generation of SRM assays for >90% of the human proteome. The synthetic peptide libraries were also used to generate gold-standard ion trap spectral libraries to be used for spectral matching of shotgun proteomic datasets in discovery-driven experiments. The described SRM assays and spectral libraries are currently being made publicly available via the SRMAtlas user interface, which supports their dissemination across different laboratoreis. The potential of such proteome maps, for targeted and discovery proteomics, as well as their limitations will be discussed.

Christian Rolando is a senior scientist (Directeur de Recherche) at the CNRS (French National Center for Scientific Research) and works at the University of Lille. Christian Rolando is the head of the proteomics platform he founded in the early 2000. Christian Rolando has worked in mass spectrometry since his PhD, during which he managed the mass spectrometry facility at the Department of Chemistry of the Ecole Normale Supérieure in Paris. At this time the facility was equipped with two Varian MAT magnetic instruments (CH7 and 111) fitted with UV recorders… During the 90s Christian Rolando worked with the French company Nermag and participated in the development of the R3030 triple quadripole. He used this instrument to study organometallic ion-molecule reactions and he developed several analytical protocols combining organic reactions and mass spectrometry like the so-called thioacidolysis, which became the standard in lignin analysis. Since his arrival in Lille in the early 2000s, he developed a set of original analytical methodologies for proteomics, from sample preparation (new gels for electrophoresis, recipes based on pure chemicals avoiding the use of kits of unknown formulation) to data analysis (bioinformatics). He is also involved in several collaborations on different topics such as transfusion plasma safety, biomarkers of cigarette smoking or identification of proteins in cultural heritage artifacts. A part of Christian Rolando's research is also focused on the analytical applications of microfluidics and more recently on the development of two-dimensional FT-ICR MS. Christian Rolando is the co-author of 150 papers cited more than 2500 times and has directed 30 PhD theses. He was President of the Société Française de Spectrométrie de masse from 1994 to 1995 and he is now President of the Analytical Division of the French Chemical society and Deputy Director at the CNRS Institute of Chemistry in charge of Infrastructures.

The pulse sequence for 2D FT-ICR MS was first developed in 1988 [1] by MS and NMR teams respectively lead by Tino Gäumann and Geoffrey Bodenhausen [1] based on a key experiment demonstrating cyclotron excitation reversibility [2] and conveys detailed information for compounds in complex samples in one easily readable 2D mass spectrum without ion isolation. We revisited 2D FT-ICR MS [3] which, unlike 2D NMR, has no analytical applications yet. Gas-free fragmentation modes allow us to retain high resolution and sensitivity, and the advances in electronics and computer technology since 1988 enable broadband acquisition. The method, however, needs to be optimized in terms of scintillation noise, experiment time and necessary amounts of sample. In this study, we apply techniques adapted from NMR spectroscopy to optimize 2D FT-ICR MS.

Because in both dimensions the spectrum is obtained after Fourier transforming a signal that has been sampled at regular time intervals (in the vertical dimension, a delay, and in the horizontal dimension, a measurement date), the resolution of 2D mass spectra behaves in both dimensions like the resolution in one-dimensional FTMS: it increases with the number of data points and is inversely proportional to m/z ratio. In preliminary experiments, time transients were limited to 32k data points since the 32-bit-written software cannot handle files larger than 256 Mb. Using 2048 scans leads to hour-long experiments with a resolution of ~1000 in the MS and fragment ion MS/MS dimension but only a few hundred in the correlation parent ion dimension [3]. For example, we were able to distinguish the fragments of the ⁷⁹Br and ⁸¹Br isotopes and the ¹²C and ¹³C isotopes of singly charged ions of bromopride, a small brominated drug (molecular weight 344.25 g.mol^-1). The new 64-bit version of the NPK package developed by Marc-André Delsuc [4] and NMRTEC allows us to work on files larger than 16 Gb. An experiment on intact cytochrome C (molecular weight 12 kDalton) using 8192 scans of 512k points time transients shows line base separation for each multicharged ion bearing around 10 charges (ca 10 000 resolution) and quadrupole like (1 m/z unit) separation in the correlation dimension (ca 1000 resolution). Furthermore these experiments show that the resolution in both dimensions remains inversely proportional to m/z ratio, as expected.

All of our experiments were performed using nanoESI at concentrations of 1 pmol/µL, leading to sample consumption of less than 20 pmol per hour per experiment, but at the expense of signal intensity fluctuation which translates into scintillation noise in the 2D data. We adapted the NMR version of the Cadzow de-noising algorithm [5] for 2D FT-ICR mass spectra. This algorithm is based on the decomposition of the signal in singular values from which the signal at longer time may be predicted. The Cadzow noise reduction dramatically reduced fluctuations and brought out additional fragmentation peaks that were masked by the noise [6].

Results obtained using IRMPD and ECD fragmentation without any in-cell or quadrupole separation for simple compounds like individual peptides, the multicharged ion distribution of small proteins and more complex mixtures like tryptic protein digests will be presented. Finally future development of 2D FT-ICR will be briefly discussed at the sight of multidimensional NMR methodologies developed during the last twenty years. 

[1] P. Pfändler et al., J. Am. Chem. Soc. 110 (1988) 5625-5628

[2] A.G. Marshall et al., Chem. Phys. Letts. 105 (1984) 233-236

[3] M.A. van Agthoven et al., Int. J. Mass Spectrom., doi:10.1016/j.ijms.2010.10.034

[4] D. Tramesel et al., J. Magn. Res. 188 (2007) 56-67

[5] C. Brissac et al., J. Biomol. NMR 6 (1995) 361-365 

[6] M.A. van Agthoven et al., Rapid Commun. Mass Spectrom., 25 (2011) 1609–1616.

Prof. Yury O. Tsybin received his PhD degree in ion physics in 2004 from Uppsala University, Sweden performed under the supervision of Prof. Per Hakansson. For the next 2 years Tsybin was a postdoctoral research associate with Prof. Alan G. Marshall at the National High Magnetic Field Laboratory in the USA. Since 2006 Tsybin is a tenure-track assistant professor of physical and bioanalytical chemistry at the Ecole Polytechnique Fédérale de Lausanne (EPFL) in Switzerland where he heads the Biomolecular Mass Spectrometry Laboratory and serves as a Director of the MS Service Facility. Research interests of his group are around the high performance Fourier transform mass spectrometry method and technique development with subsequent applications in peptide and protein structure analysis. In 2011 Tsybin received the ERC Starting Grant to develop the super-resolution mass spectrometry for the applications in health and sustainability areas.

High magnetic field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides the unbeatable analytical performance in terms of high resolution measurements. However, the high resolution measurements require a long experimental time, e.g., 1-20 seconds per single mass spectrum acquisition, and thus are incompatible with the current and near-future requirements for the high throughput and fast data acquisition, enforced by the short peptide and protein elution time from the chromatographic column. Here, we will discuss some of the possible solutions to this problem from both, hardware and signal processing development. The FT-ICR MS hardware development follows the implementation of ultra-high, e.g., 21 T, magnetic field environments together with higher acquisition speed and harmonized ICR ion traps. The recent progress in Orbitrap FTMS development has significantly reduced the gap between Orbitrap and ICR FTMS in terms of obtained resolving power required for the mainstream MS applications, 60-200k. Finally, the state-of-the-art time-of-flight (TOF) mass analyzers have demonstrated a substantial progress in the recent years and now offer 30-60k resolving powers achieved at comparable times to the high field ICR and Orbitrap FTMS and faster. On the other hand, incredible progress in the computational power and high frequency electronics opens the doors to the advanced signal processing development, which received a particular attention in the recent years. A number of groups, including ours, are in the process of tailoring the super-resolution signal processing methods, e.g., filter diagonalization method (FDM), to the needs of the ICR and Orbitrap MS. The goal is to replace the FT-based signal processing with the methods that would require 10-100 times shorter transient time-domain signals to yield a similar level of the resolving power.

Dr. Vitek holds a bachelor’s and master’s degrees from the University of Geneva, Switzerland, and a PhD in Statistics from Purdue University. She interned at Eli Lilly and Company in Indianapolis, and held a position of post-doctoral associate in the Aebersold lab at the Institute for Systems Biology in Seattle. In Fall 2006 she came back to Purdue as an Assistant Professor in Statistics and Computer Science. Research in Dr. Vitek's lab focuses on statistical and computational methods for systems biology, in particular for functional proteomics, metabolomics, and ionomics, in order to increase the sensitivity and the accuracy of these investigations and to deepen their insight into the biological function.

The goal of many proteomic experiments is to quantify and compare the abundances of proteins in complex biological mixtures. This can be accomplished in a variety of mass spectrometry-based workflows, such as label-free or label-based LC-MS workflows, or label-free or label-based SRM workflows. Although the experimental details of these workflows vary greatly, they all output a list of identified and quantified spectral features. There is currently no consensus on how to appropriately handle the repeated quantitative measurements on a protein in a sample, and how to derive protein-level conclusions across all labels, features, samples and conditions.

We propose a general statistical modeling framework for protein quantification based on linear mixed-effects models. It is applicable to most experimental designs, LC-MS and SRM experiments, and label-based and label-free SRM workflows. We illustrate the utility of the framework in a series of investigations with fairly complex designs: a 3-way factorial label-free LC-MS, and a label-based SRM time course investigation of central carbon metabolism of S.cerevisiae. We further illustrate the sensitivity and specificity of the framework using controlled spike-in experiments, and using a series of simulated datasets. Finally, we discuss the freely available software that we developed to implement the modeling framework.