Absolute quantification of dengue virus serotype 4 chimera vaccine candidate in Vero cell culture by targeted mass spectrometry.

Infection by dengue flavivirus is transmitted by mosquitoes and affects tens to hundreds of millions people around the world each year. Four serotypes have been described, all of which cause similar disease. Currently, there no approved vaccines or specific therapeutics for dengue, although several vaccine prototypes are in different stages of clinical development. Among them, a chimeric vaccine, built from the replication machinery of the yellow fever 17D virus, has shown promising results in phase III trials. Accurate quantitation of expressed viral particles in alive attenuated viral antigen vaccine is essential and determination of infectious titer is usually the method of choice. The current paper describes an alternative or orthogonal strategy, namely, a multiplexed and absolute assay of four proteins of the chimera yellow fever/dengue serotype 4 virus using targeted MS in SRM mode. Over 1 month, variability of the assay using a partially purified Vero cell extract was between 8 and 17%, and accuracy was between 80 and 120%. In addition, the assay was linear between 6.25 and 200 nmol/L and could therefore be used in the near future to quantify dengue virus type 4 during production and purification from Vero cells.

Overcoming biofluid protein complexity during targeted mass spectrometry detection and quantification of protein biomarkers by MRM cubed (MRM3).

Targeted mass spectrometry in the so-called multiple reaction monitoring mode (MRM) is certainly a promising way for the precise, accurate, and multiplexed measurement of proteins and their genetic or posttranslationally modified isoforms. MRM carried out on a low-resolution triple quadrupole instrument faces a lack of specificity when addressing the quantification of weakly concentrated proteins. In this case, extensive sample fractionation or immunoenrichment alleviates signal contamination by interferences, but in turn decreases assay performance and throughput. Recently, MRM(3) was introduced as an alternative to MRM to improve the limit of quantification of weakly concentrated protein biomarkers. In the present work, we compare MRM and MRM(3) modes for the detection of biomarkers in plasma and urine. Calibration curves drawn with MRM and MRM(3) showed a similar range of linearity (R(2) > 0.99 for both methods) with protein concentrations above 1 µg/mL in plasma and a few nanogram per milliliter in urine. In contrast, optimized MRM(3) methods improve the limits of quantification by a factor of 2 to 4 depending on the targeted peptide. This gain arises from the additional MS(3) fragmentation step, which significantly removes or decreases interfering signals within the targeted transition channels.

Total ApoE and ApoE4 isoform assays in an Alzheimer's disease case-control study by targeted mass spectrometry (n=669): a pilot assay for methionine-containing proteotypic peptides.

Allelic polymorphism of the apolipoprotein E (ApoE) gene (ApoE ε2, ApoE ε3 and ApoE ε4 alleles) gives rise to three protein isoforms (ApoE2, ApoE3 and ApoE4) that differ by 1 or 2 amino acids. Inheritance of the ApoE ε4 allele is a risk factor for developing Alzheimer's disease (AD). The potential diagnostic value of ApoE protein levels in biological fluids (i.e. cerebrospinal fluid, plasma and serum) for distinguishing between AD patients and healthy elderly subjects is subject to great controversy. Although a recent study reported subnormal total ApoE and ApoE4 levels in the plasma of AD patients, other studies have found normal or even elevated protein levels (versus controls). Because all previously reported assays were based on immunoenzymatic techniques, we decided to develop an orthogonal assay based on targeted mass spectrometry by tracking (i) a proteotypic peptide common to all ApoE isoforms and (ii) a peptide that is specific for the ε4 allele. After trypsin digestion, the ApoE4-specific peptide contains an oxidation-prone methionine residue. The endogenous methionine oxidation level was evaluated in a small cohort (n=68) of heterozygous ε3ε4 carriers containing both healthy controls and AD patients. As expected, the proportion of oxidized residues varied from 0 to 10%, with an average of 5%. We therefore developed a standardized strategy for the unbiased, absolute quantification of ApoE4, based on performic acid oxidization of methionine. Once the sample workflow had been thoroughly validated, it was applied to the concomitant quantification of total ApoE and ApoE4 isoform in a large case-control study (n=669). The final measurements were consistent with most previously reported ApoE concentration values and confirm the influence of the different alleles on the protein expression level. Our results illustrate (i) the reliability of selected reaction monitoring-based assays and (ii) the value of the oxidization step for unbiased monitoring of methionine-containing proteotypic peptides. Furthermore, a statistical analysis indicated that neither total ApoE and ApoE4 levels nor the ApoE/ApoE4 ratio correlated with the diagnosis of AD. These findings reinforce the conclusions of previous studies in which plasma ApoE levels had no obvious clinical significance.

Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging.

The O-linked beta-N-acetylglucosamine (O-GlcNAc) modification is an abundant post-translational modification in eukaryotic cells. This dynamic glycosylation plays a fundamental role in the activity of many nuclear and cytoplasmic proteins and is associated with pathologies like type II diabetes, Alzheimer's disease or some cancers. However the exact link between O-GlcNAc-modified proteins and their function in cells is largely undefined for most cases. Here we report a strategy based on the 1,3-dipolar cycloaddition, called click chemistry, between unnatural N-acetylglucosamine (GlcNAc) analogues (substituted with an azido or alkyne group) and the corresponding biotinylated probe to specifically detect, enrich and identify O-GlcNAc-modified proteins. This bio-orthogonal conjugation confirms that only azido analogue of GlcNAc is metabolized by the cell. Thanks to the biotin probe, affinity purification on streptavidin beads allowed us to identify 32 O-GlcNAc-azido-tagged proteins by LC-MS/MS analysis in an MCF-7 cellular model, 14 of which were previously unreported. This work illustrates the use of the click-chemistry-based strategy combined with a proteomic approach to get further insight into the pattern of O-GlcNAc-modified proteins and the biological significance of this post-translational modification. [figure: see text]

Absolute quantification of podocalyxin, a potential biomarker of glomerular injury in human urine, by liquid chromatography-mass spectrometry.

Podocalyxin is a protein present in specialized glomerulus cells called podocytes and may be released in the urine in case of kidney injury. In this context, its quantification could be of great interest in order to monitor glomerular injury. Liquid chromatography tandem mass spectrometry (LC-MS/MS), in selected reaction monitoring (SRM) mode, has been demonstrated as a powerful technique that can be applied to protein quantification. This paper describes the development of a quantification method of human podocalyxin in urine by LC-MS/MS in SRM mode by monitoring one proteotypic peptide with an isotope-dilution standardization strategy employing (13)C/(15)N labelled peptides. Inter/intra assay precisions and accuracies of the assay were below 10% and between 90% and 106.1%, respectively. In addition, the method was linear between 0.78 and 100 ng/mL and could therefore be used to quantify endogenous level of podocalyxin that was estimated between 15.2 and 44.2 ng/mL in urine samples from healthy donor.

Absolute quantification of podocin, a potential biomarker of glomerular injury in human urine, by liquid chromatography-multiple reaction monitoring cubed mass spectrometry.

Glomeruli play a major role in the kidney function since they are involved in primary urine formation. It is then crucial to dispose of methods to monitor glomerular injury, especially in drug development. In this context, quantification of podocin could be of great interest since it is a protein exclusively present in highly specialized glomerulus cells called podocytes. Immunoassays are the most commonly used approach for protein assays. However, they rely on the availability of specific antibodies. When such antibodies are not available, liquid chromatography tandem mass spectrometry (LC-MS/MS), in selected reaction monitoring (SRM) or in multiple reaction monitoring cubed (MRM(3)) mode, has been demonstrated as a powerful alternative technique, and can be applied to multiple protein quantification. This paper describes the development of a quantification method of human podocin in urine by LC-MS/MS in MRM(3) mode. Inter assay precision and accuracy ranged from 7 to 20% and from 105 to 112% respectively and the lower limit of quantification (LLOQ) was 0.39ng/mL from only 1mL of urine which is compatible for endogenous level of podocin determination.