Alexandre Panchaud

Precursor Acquisition Independent From Ion Count: How to Dive Deeper into the Proteomics Ocean

Data-dependent precursor ion selection is widely used in shotgun proteomics to profile the protein components of complex samples. Although very popular, this bottom-up method presents major drawbacks in terms of detectable dynamic range. Here, we demonstrate the superior performance of a data-independent method we term precursor acquisition independent from ion count (PAcIFIC). Our results show that almost the entire, predicted, soluble bacterial proteome can be thoroughly analyzed by PAcIFIC without the need for any sample fractionation other than the C18-based liquid chromatograph used to introduce the peptide mixture into the mass spectrometer. Importantly, we also show that PAcIFIC provides unique performance for analysis of human plasma in terms of the number of proteins identified (746 at FDR < or = 0.5%) and achieved dynamic range (8 orders of magnitude at FDR < or = 0.5%), without any fractionation other than immuno-depletion of the seven most abundant proteins.

Mass spectrometry for nutritional peptidomics: How to analyze food bioactives and their health effects ☆

We describe nutritional peptidomics for discovery and validation of bioactive food peptide and their health effects. Understanding nature and bioactivity of nutritional peptides means comprehending an important level of environmental regulation of the human genome, because diet is the environmental factor with the most profound life-long influence on health. We approach the theme from three angles, namely the analysis, the discovery and the biology perspective. Food peptides derive from parent food proteins via in vitro hydrolysis (processing) or in vivo digestion by various unspecific and specific proteases, as opposed to the tryptic peptides typically generated in biomarker proteomics. A food bioactive peptide may be rare or unique in terms of sequence and modification, and many food genomes are less well annotated than e.g. the human genome. Bioactive peptides can be discovered either empirically or by prediction: we explain both the classical hydrolysis strategy and the bioinformatics-driven reversed genome engineering. In order to exert bioactivity, food peptides must be either ingested and then reach the intestine in their intact form or be liberated in situ from their parent proteins to act locally, that is in the gut, or even systemically, i.e. through the blood stream. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.

Chemical Cross-Linking and Mass Spectrometry As a Low-Resolution Protein Structure Determination Technique

Protein complexes are the foundation of a majority of cellular processes. Although a large number of protein complexes have been identified through biochemical experiments, the precise molecular details and three-dimensional structures are available for only a small fraction. Chemical cross-linking coupled with mass spectrometry (CXMS) has gained popularity in recent years for characterization of inter- and intraprotein interactions in protein complexes. This perspective provides a comprehensive and critical overview of CXMS strategies employed for structural elucidation of protein complexes. We evaluate the challenges associated with CXMS techniques with special emphasis on data analysis. As sensitivity, mass resolution, mass accuracy and ease of use of mass spectrometers have improved, the complexity of processing and interpreting CXMS data has become the central problem to be addressed. We review here a number of computer programs available to address these problems.

xComb: A Cross-Linked Peptide Database Approach to Protein−Protein Interaction Analysis

We developed an informatic method to identify tandem mass spectra composed of chemically cross-linked peptides from those of linear peptides and to assign sequence to each of the two unique peptide sequences. For a given set of proteins the key software tool, xComb, combs through all theoretically feasible cross-linked peptides to create a database consisting of a subset of all combinations represented as peptide FASTA files. The xComb library of select theoretical cross-linked peptides may then be used as a database that is examined by a standard proteomic search engine to match tandem mass spectral data sets to identify cross-linked peptides. The database search may be conducted against as many as 50 proteins with a number of common proteomic search engines, e.g. Phenyx, Sequest, OMSSA, Mascot and X!Tandem. By searching against a peptide library of linearized, cross-linked peptides, rather than a linearized protein library, search times are decreased and the process is decoupled from any specific search engine. A further benefit of decoupling from the search engine is that protein cross-linking studies may be conducted with readily available informatics tools for which scoring routines already exist within the proteomic community.

The Fibrinogen- and Fibronectin-Binding Domains of Staphylococcus aureus Fibronectin-Binding Protein A Synergistically Promote Endothelial Invasion and Experimental Endocarditis†

ABSTRACT Staphylococcus aureus experimental endocarditis relies on sequential fibrinogen binding (for valve colonization) and fibronectin binding (for endothelial invasion) conferred by peptidoglycan-attached adhesins. Fibronectin-binding protein A (FnBPA) reconciles these two properties—as well as elastin binding—and promotes experimental endocarditis by itself. Here we attempted to delineate the minimal subdomain of FnBPA responsible for fibrinogen and fibronectin binding, cell invasion, and in vivo endocarditis. A large library of truncated constructs of FnBPA was expressed in Lactococcus lactis and tested in vitro and in animals. A 127-amino-acid subdomain spanning the hinge of the FnBPA fibrinogen-binding and fibronectin-binding regions appeared necessary and sufficient to confer the sum of these properties. Competition with synthetic peptides could not delineate specific fibrinogen- and fibronectin-binding sites, suggesting that dual binding arose from protein folding, irrespective of clearly defined binding domains. Moreover, coexpressing the 127-amino-acid subdomain with remote domains of FnBPA further increased fibrinogen binding by ≥10 times, confirming the importance of domain interactions for binding efficacy. In animals, fibrinogen binding (but not fibronectin binding) was significantly associated with endocarditis induction, whereas both fibrinogen binding and fibronectin binding were associated with disease severity. Moreover, fibrinogen binding also combined with fibronectin binding to synergize the invasion of cultured cell lines significantly, a feature correlating with endocarditis severity. Thus, while fibrinogen binding and fibronectin binding were believed to act sequentially in colonization and invasion, they appeared unexpectedly intertwined in terms of both functional anatomy and pathogenicity (in endocarditis). This unforeseen FnBPA subtlety might bear importance for the development of antiadhesin strategies.

Faster, Quantitative, and Accurate Precursor Acquisition Independent From Ion Count

Data-dependent precursor ion selection is widely used in shotgun proteomics to profile the protein components of complex samples. Although very popular, this bottom-up method presents major drawbacks in terms of detectable dynamic range. Recently, we demonstrated the superior performance of a data-independent method we termed precursor acquisition independent from ion count (PAcIFIC). Here, we report a faster, accurate, multiplexed, and quantitative PAcIFIC method. Our results show that the time needed to perform such analysis can be decreased by 33% to 66% using modern ion trap instruments and that high mass accuracy can be applied to such a strategy. Quantification capability is demonstrated on protein standards and a whole bacterial cell lysate using isobaric tagging. Finally, we confirm in yeast the dynamic range capabilities of such a method where proteins down to less than 50 copies per cell can be monitored without sample prefractionation.

Proteomics in Nutrition: Status Quo and Outlook for Biomarkers and Bioactives

Food and beverages are the only physical matter we take into our body, if we disregard the air we inhale and the drugs we may have to apply. While traditional nutrition research has aimed at providing nutrients to nourish populations and preventing specific nutrient deficiencies, it more recently explores health-related aspects of individual bioactive components as well as entire diets and this at group rather than population level. The new era of nutrition research translates empirical knowledge to evidence-based molecular science. Modern nutrition research focuses on promoting health, preventing or delaying the onset of disease, optimizing performance, and assessing risk. Personalized nutrition is a conceptual analogue to personalized medicine and means adapting food to individual needs. Nutrigenomics and nutrigenetics build the science foundation for understanding human variability in preferences, requirements, and responses to diet and may become the future tools for consumer assessment motivated by personalized nutritional counseling for health maintenance and disease prevention. The scope of this paper is to review the current and future aspects of nutritional proteomics, focusing on the two main outputs: identification of health biomarkers and analysis of food bioactives.

ANIBAL, Stable Isotope-based Quantitative Proteomics by Aniline and Benzoic Acid Labeling of Amino and Carboxylic Groups

Identification and relative quantification of hundreds to thousands of proteins within complex biological samples have become realistic with the emergence of stable isotope labeling in combination with high throughput mass spectrometry. However, all current chemical approaches target a single amino acid functionality (most often lysine or cysteine) despite the fact that addressing two or more amino acid side chains would drastically increase quantifiable information as shown by in silico analysis in this study. Although the combination of existing approaches, e.g. ICAT with isotope-coded protein labeling, is analytically feasible, it implies high costs, and the combined application of two different chemistries (kits) may not be straightforward. Therefore, we describe here the development and validation of a new stable isotope-based quantitative proteomics approach, termed aniline benzoic acid labeling (ANIBAL), using a twin chemistry approach targeting two frequent amino acid functionalities, the carboxylic and amino groups. Two simple and inexpensive reagents, aniline and benzoic acid, in their 12C and 13C form with convenient mass peak spacing (6 Da) and without chromatographic discrimination or modification in fragmentation behavior, are used to modify carboxylic and amino groups at the protein level, resulting in an identical peptide bond-linked benzoyl modification for both reactions. The ANIBAL chemistry is simple and straightforward and is the first method that uses a 13C-reagent for a general stable isotope labeling approach of carboxylic groups. In silico as well as in vitro analyses clearly revealed the increase in available quantifiable information using such a twin approach. ANIBAL was validated by means of model peptides and proteins with regard to the quality of the chemistry as well as the ionization behavior of the derivatized peptides. A milk fraction was used for dynamic range assessment of protein quantification, and a bacterial lysate was used for the evaluation of relative protein quantification in a complex sample in two different biological states.

Time-resolved Quantitative Proteome Analysis of In Vivo Intestinal Development

Postnatal intestinal development is a very dynamic process characterized by substantial morphological changes that coincide with functional adaption to the nutritional change from a diet rich in fat (milk) to a diet rich in carbohydrates on from weaning. Time-resolved studies of intestinal development have so far been limited to investigation at the transcription level or to single or few proteins at a time. In the present study, we elucidate proteomic changes of primary intestinal epithelial cells from jejunum during early suckling (1–7 days of age), middle suckling (7–14 days), and weaning period (14–35 days) in mice, using a label-free proteomics approach. We show differential expression of 520 proteins during intestinal development and a pronounced change of the proteome during the middle suckling period and weaning. Proteins involved in several metabolic processes were found differentially expressed along the development. The temporal expression profiles of enzymes of the glycolysis were found to correlate with the increase in carbohydrate uptake at weaning, whereas the abundance changes of proteins involved in fatty acid metabolism as well as lactose metabolism indicated a nondiet driven preparation for the nutritional change at weaning. Further, we report the developmental abundance changes of proteins playing a vital role in the neonatal acquisition of passive immunity. In addition, different isoforms of several proteins were quantified, which may contribute to a better understanding of the roles of the specific isoforms in the small intestine. In summary, we provide a first, time-resolved proteome profile of intestinal epithelial cells along postnatal intestinal development.

Data-independent proteomic screen identifies novel tamoxifen agonist that mediates drug resistance.

A label-free quantitative variation of the recently developed data-independent shotgun proteomic method precursor acquisition independent from ion count (PAcIFIC) was used to identify novel proteins implicated in cancer progression and resistance. Specifically, this screen identified the pro-metastatic protein anterior gradient 2 (AGR2) as significantly up-regulated in tamoxifen-treated cells. Highlighting the need for direct proteome profiling methods like PAcIFIC, neither data-dependent gas-phase fractionation nor a transcriptomic screen detected AGR2 protein/transcript at significantly up-regulated levels. Further cell-based experiments using human cancer cell lines and in vivo xenografts confirmed the PAcIFIC hypothesis that AGR2 is up-regulated in MCF-7 cells post tamoxifen treatment and that it is implicated in drug resistance mediation.