Alain Dupuis

Isotope-labeled Protein Standards Toward Absolute Quantitative Proteomics

Diagnostic development and public health surveillance require technologies that provide specific identification and absolute quantification of protein biomarkers. Beside immunologically related techniques (e.g. enzyme-linked immunosorbent assay), MS is gaining increasing interest due to its high sensitivity and specificity. Furthermore, MS-based analyses are extremely accurate quantitatively, provided that suitable reference standards are available. Recently, the use of chemically synthesized isotope-labeled marker peptides for MS-based absolute quantification of proteins has led to major advances. However, we show here that the use of such peptides can lead to severe biases. In this work, we present an innovative strategy (Protein Standard Absolute Quantification) that uses in vitro-synthesized isotope-labeled full-length proteins as standards for absolute quantification. As those protein standards perfectly match the biochemical properties of the target proteins, they can be directly added into the samples to be analyzed, allowing a highly accurate quantification of proteins even in prefractionated complex samples. The power of our Protein Standard Absolute Quantification methodology for accurate absolute quantification of biomarkers was demonstrated both on water and urine samples contaminated with Staphylococcus aureus superantigenic toxins as typical biomarkers of public health interest.

Isotope dilution strategies for absolute quantitative proteomics.

The development of mass spectrometry (MS)-based methodologies for high-throughput protein identification has generated a concomitant need for protein quantification. Numerous MS-based relative quantification methodologies have been dedicated to the extensive comparison of multiple proteomes. On the other hand, absolute quantification methodologies, which allow the determination of protein concentrations in biological samples, are generally restricted to defined sets of proteins. Depending on the selected analytical procedure, absolute quantification approaches can provide accurate and precise estimations. These analytical performances are crucial for specific applications such as the evaluation of clinical biomarker candidates. According to bioanalytical guidelines, accurate analytical processes require internal standards and quality controls. Regarding MS-based analysis of small molecules, isotope dilution has been recognized as the reference method for internal standardization. However, protein quantification methodologies which rely on the isotope dilution principle have been implemented in the proteomic field only recently. In these approaches, the sample is spiked with defined amounts of isotope-labeled analogue(s) of specific proteolytic peptide(s) (AQUA and QconCAT strategies) or protein(s) (PSAQ strategy). In this review, we present a critical overview of these isotope dilution methodologies.

Protein Standard Absolute Quantification (PSAQ) for improved investigation of staphylococcal food poisoning outbreaks

Staphylococcal enterotoxins are major causing agents of food‐borne diseases. Their detection in food remnants for risk assessment or food poisoning outbreaks investigation suffers from a lack in comprehensive immunological tools. In this study, we demonstrate that the combination of immunocapture and Protein Standard Absolute Quantification (PSAQ) strategy, which uses isotope‐labeled enterotoxins as internal standards for MS‐based analysis, is powerful to specifically identify and quantify these contaminating agents in food matrices. This approach is believed to significantly improve the elucidation of staphylococcal food poisoning outbreaks.

The 49‐kDa subunit of NADH‐ubiquinone oxidoreductase (Complex I) is involved in the binding of piericidin and rotenone, two quinone‐related inhibitors

Piericidin is a potent inhibitor of the mitochondrial and bacterial type I NADH‐ubiquinone oxidoreductases (Complex I) and is considered to bind at or close to the ubiquinone binding site(s) of the enzyme. Piericidin‐resistant mutants of the bacterium Rhodobacter capsulatus have been isolated and the present work demonstrates that a single missense mutation at the level of the gene encoding the peripheral 49‐kDa/NUOD subunit of Complex I is definitely associated with this resistance. Based on this original observation, we propose a model locating the binding site for piericidin (and quinone) at the interface between the hydrophilic and hydrophobic domains of Complex I.

The Complex I from Rhodobacter capsulatus

The NADH-ubiquinone oxidoreductase (type I NDH) of Rhodobacter capsulatus is a multisubunit enzyme encoded by the 14 genes of the nuo operon. This bacterial enzyme constitutes a valuable model for the characterization of the mitochondrial Complex I structure and enzymatic mechanism for the following reasons. (i) The mitochondria-encoded ND subunits are not readily accessible to genetic manipulation. In contrast, the equivalents of the mitochondrial ND1, ND2, ND4, ND4L, ND5 and ND6 genes can be easily mutated in R. capsulatus by homologous recombination. (ii) As illustrated in the case of ND1 gene, point mutations associated with human cytopathies can be reproduced and studied in this model system. (iii) The R. capsulatus model also allows the recombinant manipulations of iron-sulfur (Fe-S) subunits and the assignment of Fe-S clusters as illustrated in the case of the NUOI subunit (the equivalent of the mitochondrial TYKY subunit). (iv) Finally, like mitochondrial Complex I, the NADH-ubiquinone oxidoreductase of R. capsulatus is highly sensitive to the inhibitor piericidin-A which is considered to bind to or close to the quinone binding site(s) of Complex I. Therefore, isolation of R. capsulatus mutants resistant to piericidin-A represents a straightforward way to map the inhibitor binding sites and to try and define the location of quinone binding site(s) in the enzyme. These illustrations that describe the interest in the R. capsulatus NADH-ubiquinone oxidoreductase model for the general study of Complex I will be critically developed in the present review.

Accurate quantification of cardiovascular biomarkers in serum using protein standard absolute quantification (PSAQ™) and selected reaction monitoring

Development of new biomarkers needs to be significantly accelerated to improve diagnostic, prognostic, and toxicity monitoring as well as therapeutic follow-up. Biomarker evaluation is the main bottleneck in this development process. Selected Reaction Monitoring (SRM) combined with stable isotope dilution has emerged as a promising option to speed this step, particularly because of its multiplexing capacities. However, analytical variabilities because of upstream sample handling or incomplete trypsin digestion still need to be resolved. In 2007, we developed the PSAQ™ method (Protein Standard Absolute Quantification), which uses full-length isotope-labeled protein standards to quantify target proteins. In the present study we used clinically validated cardiovascular biomarkers (LDH-B, CKMB, myoglobin, and troponin I) to demonstrate that the combination of PSAQ and SRM (PSAQ-SRM) allows highly accurate biomarker quantification in serum samples. A multiplex PSAQ-SRM assay was used to quantify these biomarkers in clinical samples from myocardial infarction patients. Good correlation between PSAQ-SRM and ELISA assay results was found and demonstrated the consistency between these analytical approaches. Thus, PSAQ-SRM has the capacity to improve both accuracy and reproducibility in protein analysis. This will be a major contribution to efficient biomarker development strategies.

Evidence for a quinone binding site close to the interface between NUOD and NUOB subunits of Complex I

Piericidin, rotenone and pyridaben are specific inhibitors of the NADH-ubiquinone oxidoreductase (Complex I) that bind to its ubiquinone binding site(s). Using site directed mutagenesis, we demonstrate that residues G409, D412, R413 and V407 of the C-terminus of Complex I NUOD subunit are directly involved in the binding of these inhibitors. We propose that the corresponding inhibitor/quinone binding site would be located close to NUOD-NUOB interface.

Peptide storage: are you getting the best return on your investment? Defining optimal storage conditions for proteomics samples.

To comply with current proteomics guidelines, it is often necessary to analyze the same peptide samples several times. Between analyses, the sample must be stored in such a way as to conserve its intrinsic properties, without losing either peptides or signal intensity. This article describes two studies designed to define the optimal storage conditions for peptide samples between analyses. With the use of a label-free strategy, peptide conservation was compared over a 28-day period in three different recipients: standard plastic tubes, glass tubes, and low-adsorption plastic tubes. The results of this study showed that standard plastic tubes are unsuitable for peptide storage over the period studied. Glass tubes were found to perform better than standard plastic, but optimal peptide recovery was achieved using low-adsorption plastic tubes. The peptides showing poor recovery following storage were mainly hydrophobic in nature. The differences in peptide recovery between glass and low-adsorption plastic tubes were further studied using isotopically labeled proteins. This study allowed accurate comparison of peptide recovery between the two tube types within the same LC-MS run. The results of the label-free study were confirmed. Further, it was possible to demonstrate that peptide recovery in low-adsorption plastic tubes was optimal whatever the peptide concentration stored.

Toward a characterization of the connecting module of complex I.

Complex I [NADH–ubiquinone oxidoreductase (complex I, EC 1.6.5.3)] couples electron transfer between NADH and ubiquinone to proton transport across the bacterial cytoplasmic membrane and the mitochondrial inner membrane. This sophisticated enzyme consists of three specialized modules: (1) a hydrophilic NADH-oxidizing module that constitutes the input machinery of the enzyme; (2) a hydrophobic module that anchors the enzyme in the membrane and must take part in proton transport; and (3) a connecting domain that links the two previous modules. Using the complex I of Rhodobacter capsulatus, we developed a genetic study of the structure and function of the connecting module. In the present review, we put together the salient results of these studies, with recent reports of the literature, to try and elucidate the structure of the connecting module and its potential role in the coupling process between electron and proton flux within complex I. From this overview, we conclude that the NUOB–NUOD dimer of the connecting module and a hydrophobic subunit such as NUOH must share a quinone-reduction site. The function of this site in the mechanism of complex I is discussed.

Innovative Application of Mass Spectrometry for the Characterization of Staphylococcal Enterotoxins Involved in Food Poisoning Outbreaks

ABSTRACT Staphylococcal poisoning is a common food-borne disease for which immunoassays to detect enterotoxins were developed, but these assays often lead to false diagnoses due to interferences or lack of specificity. Absolute quantitative mass spectrometry was for the first time successfully applied to an investigation of a staphylococcal outbreak due to coconut pearls.