Stable Isotope Labeling by Amino acid<i>in Vivo</i>(SILAV): a new method to explore protein metabolism

Lehmann, Sylvain; Vialaret, Jérôme; Combe, Guillaume Gras; Bauchet, Luc; Hanon, Olivier; Girard, M.; Gabelle, Audrey; Hirtz, Christophe

doi:10.1002/rcm.7289

Cited by 11 publications

(13 citation statements)

References 29 publications

(52 reference statements)

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“…Lastly, the stable isotope labeling approach recently described by Lehmann et al ( 2015 ) could be of great value for quantifying: (i) the rates of synthesis and clearance of a large range of proteins in the lumbar and ventricular CSF; and (ii) the large interindividual differences in protein concentration found in patients with CH.…”

Section: Discussionmentioning

confidence: 99%

Interactions between Flow Oscillations and Biochemical Parameters in the Cerebrospinal Fluid

Puy

Zmudka-Attier

Capel

et al. 2016

Front. Aging Neurosci.

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The equilibrium between the ventricular and lumbar cerebrospinal fluid (CSF) compartments may be disturbed (in terms of flow and biochemistry) in patients with chronic hydrocephalus (CH). Using flow magnetic resonance imaging (MRI) and CSF assays, we sought to determine whether changes in CSF were associated with biochemical alterations. Nine elderly patients with CH underwent phase-contrast MRI. An index of CSF dynamics (Idyn) was defined as the product of the lumbar and ventricular CSF flows. During surgery, samples of CSF were collected from the lumbar and ventricular compartments and assayed for chloride, glucose and total protein. The lumbar/ventricular (L/V) ratio was calculated for each analyte. The ratio between measured and expected levels (Ibioch) was calculated for each analyte and compared with Idyn. Idyn varied from 0 to 100.103μl2.s2. In contrast to the L/V ratios for chloride and glucose, the L/V ratio for total protein varied markedly from one patient to another (mean ± standard deviation (SD): 2.63 ± 1.24). The Ibioch for total protein was strongly correlated with the corresponding Idyn (Spearman’s R: 0.98; p < 5 × 10−5).We observed correlated alterations in CSF flow and biochemical parameters in patients with CH. Our findings also highlight the value of dynamic flow analysis in the interpretation of data on CSF biochemistry.

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Section: Discussionmentioning

confidence: 99%

Interactions between Flow Oscillations and Biochemical Parameters in the Cerebrospinal Fluid

Puy

Zmudka-Attier

Capel

et al. 2016

Front. Aging Neurosci.

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“…After the validation of proteomics results using enzyme‐linked immunosorbent assays, the authors of this work related the levels of fibrinogen, plasminogen, transthyretin, transferrin, and Tamm‐Horsfall proteins levels with the congenital bilateral hydronephrosis . Due to the small volume and difficulty to obtain, cerebrospinal fluid (CSF) has only been used in a few temporal studies to investigate the effect of the gamma‐secretase inhibitor semagacestat in the CSF peptidome, or to develop a stable isotope labeling by amino acids in vivo methodology . Further biofluid samples such as sperm, dental pellicle, Gingival crevicular fluid, broncho alveolar lavage, and breast milk have also been studied to a lesser extent (, Supporting Information).…”

Section: Protein Expressionmentioning

confidence: 99%

Mass Spectrometry‐Based Analysis of Time‐Resolved Proteome Quantification

Valdés

Lind

2019

Proteomics

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The aspect of time is essential in biological processes and thus it is important to be able to monitor signaling molecules through time. Proteins are key players in cellular signaling and they respond to many stimuli and change their expression in many time-dependent processes. Mass spectrometry (MS) is an important tool for studying proteins, including their posttranslational modifications and their interaction partners-both in qualitative and quantitative ways. In order to distinguish the different trends over time, proteins, modification sites, and interacting proteins must be compared between different time points, and therefore relative quantification is preferred. In this review, the progress and challenges for MS-based analysis of time-resolved proteome dynamics are discussed. Further, aspects on model systems, technologies, sampling frequencies, and presentation of the dynamic data are discussed.

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“…D3‐Leu has been frequently used to endogenously label proteins even though alternatives, such as carbon‐13 and nitrogen‐15, that provide better separation between the isotopologs of unlabeled and labeled peptides, are available . However, isotopolog separation does not guarantee reduced interference, even in MS2 scans that contain less interference than in MS1 scans .…”

Section: Introductionmentioning

confidence: 99%

“…Endogenous labeling typically uses a constant tracer infusion strategy with an initial bolus, achieving maximum enrichment at the equilibrium plateau. Even with the primed constant infusion method however, tracer incorporation is very low for most plasma proteins . Our PRM strategy, which is built on concepts derived from data‐independent acquisition (DIA) methods and neutron encoded mass signatures (NeuCode) , circumvents the technical issues above by co‐isolating and fragmenting the D0‐ and D3‐Leu peptides for PRM at the highest resolution to separate the 2HM3 ion from background ions and its M3 isotopolog .…”

Section: Introductionmentioning

confidence: 99%

Automation of PRM‐dependent D3‐Leu tracer enrichment in HDL to study the metabolism of apoA‐I, LCAT and other apolipoproteins

et al. 2017

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We developed an automated quantification workflow for PRM-enabled detection of D3-Leu labeled apoA-I in HDL isolated from humans. Subjects received a bolus injection of D3-Leu and blood was drawn at seven time points over three days. HDL was isolated and separated into six size fractions for subsequent proteolysis and PRM analysis for the detection of D3-Leu signal from ~0.03 to 0.6 % enrichment. We implemented an intensity-based quantification approach that takes advantage of high resolution/accurate mass PRM scans to identify the D3-Leu 2HM3 ion from non-specific peaks. Our workflow includes five modules for extracting the targeted PRM peak intensities (XPIs): Peak centroiding, noise removal, fragment ion matching using Δm/z windows, nine intensity quantification options, and validation and visualization outputs. We optimized the XPI workflow using in vitro synthesized and clinical samples of D0/D3-Leu labeled apoA-I. Three subjects’ apoA-I enrichment curves in six HDL size fractions, and LCAT, apoA-II and apoE from two size fractions were generated within a few hours. Our PRM strategy and automated quantification workflow will expedite the turnaround of HDL apoA-I metabolism data in clinical studies that aim to understand and treat the mechanisms behind dyslipidemia.

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Stable Isotope Labeling by Amino acidin Vivo(SILAV): a new method to explore protein metabolism

Abstract: The Stable Isotope Labeling Amino acid in Vivo (SILAV) method presented here, which yields unprecedented information about protein metabolism in humans, constitutes a promising new approach which certainly holds great potential in the field of clinical proteomics.

Cited by 11 publications

References 29 publications

Interactions between Flow Oscillations and Biochemical Parameters in the Cerebrospinal Fluid

Interactions between Flow Oscillations and Biochemical Parameters in the Cerebrospinal Fluid

Mass Spectrometry‐Based Analysis of Time‐Resolved Proteome Quantification

Automation of PRM‐dependent D3‐Leu tracer enrichment in HDL to study the metabolism of apoA‐I, LCAT and other apolipoproteins

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