Protein Quantification Using a Cleavable Reporter Peptide

Duriez, Elodie; Trévisiol, Stéphane; Domon, Bruno

doi:10.1021/pr500764e

Cited by 7 publications

(9 citation statements)

References 45 publications

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“…The analysis resulted in values within the given range (Supporting Information S3 File). Recently, an approach based on cleavable reporter peptides to recalibrate and thereby further determine the exact concentration of peptide standards has been suggested [37]. It is worth mentioning that the initial amounts of the QPrESTs™ are all determined by a similar method, i.e., normalization towards a well-defined His-tagged standard.…”

Section: Discussionmentioning

confidence: 99%

Quantitative and Selective Analysis of Feline Growth Related Proteins Using Parallel Reaction Monitoring High Resolution Mass Spectrometry

et al. 2016

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Today immunoassays are widely used in veterinary medicine, but lack of species specific assays often necessitates the use of assays developed for human applications. Mass spectrometry (MS) is an attractive alternative due to high specificity and versatility, allowing for species-independent analysis. Targeted MS-based quantification methods are valuable complements to large scale shotgun analysis. A method referred to as parallel reaction monitoring (PRM), implemented on Orbitrap MS, has lately been presented as an excellent alternative to more traditional selected reaction monitoring/multiple reaction monitoring (SRM/MRM) methods. The insulin-like growth factor (IGF)-system is not well described in the cat but there are indications of important differences between cats and humans. In feline medicine IGF–I is mainly analyzed for diagnosis of growth hormone disorders but also for research, while the other proteins in the IGF-system are not routinely analyzed within clinical practice. Here, a PRM method for quantification of IGF–I, IGF–II, IGF binding protein (BP) –3 and IGFBP–5 in feline serum is presented. Selective quantification was supported by the use of a newly launched internal standard named QPrEST™. Homology searches demonstrated the possibility to use this standard of human origin for quantification of the targeted feline proteins. Excellent quantitative sensitivity at the attomol/μL (pM) level and selectivity were obtained. As the presented approach is very generic we show that high resolution mass spectrometry in combination with PRM and QPrEST™ internal standards is a versatile tool for protein quantitation across multispecies.

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Section: Discussionmentioning

confidence: 99%

Quantitative and Selective Analysis of Feline Growth Related Proteins Using Parallel Reaction Monitoring High Resolution Mass Spectrometry

et al. 2016

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“…The assay we describe is the HeavyPeptide IGNIS Prime Peptide Quantitation kit from Thermo Scientific which uses a cleavable reporter peptide 16 . We compare IGNIS with the conventional LC-MS approach for absolute quantitation using AQUA peptides.…”

Section: Introductionmentioning

confidence: 99%

Absolute quantitation of disease protein biomarkers in a single LC-MS acquisition using apolipoprotein F as an example

Kumar

Gangadharan

Cobbold

et al. 2017

Sci Rep

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LC-MS and immunoassay can detect protein biomarkers. Immunoassays are more commonly used but can potentially be outperformed by LC-MS. These techniques have limitations including the necessity to generate separate calibration curves for each biomarker. We present a rapid mass spectrometry-based assay utilising a universal calibration curve. For the first time we analyse clinical samples using the HeavyPeptide IGNIS kit which establishes a 6-point calibration curve and determines the biomarker concentration in a single LC-MS acquisition. IGNIS was tested using apolipoprotein F (APO-F), a potential biomarker for non-alcoholic fatty liver disease (NAFLD). Human serum and IGNIS prime peptides were digested and the IGNIS assay was used to quantify APO-F in clinical samples. Digestion of IGNIS prime peptides was optimised using trypsin and SMART Digest™. IGNIS was 9 times faster than the conventional LC-MS method for determining the concentration of APO-F in serum. APO-F decreased across NAFLD stages. Inter/intra-day variation and stability post sample preparation for one of the peptides was ≤13% coefficient of variation (CV). SMART Digest™ enabled complete digestion in 30 minutes compared to 24 hours using in-solution trypsin digestion. We have optimised the IGNIS kit to quantify APO-F as a NAFLD biomarker in serum using a single LC-MS acquisition.

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“…This strategy proved to be robust and able to generate accurate and precise data for the absolute abundances of many proteins. Strategies involving the integration of tag‐peptide sequences into internal standards have been exploited to date for calculating the amounts of standards . In many of these previous studies, non‐naturally occurring peptides were designed, prepared as synthetic peptides, and used to quantify a single standard molecule in one measurement .…”

Section: Discussionmentioning

confidence: 99%

“…Strategies involving the integration of tag‐peptide sequences into internal standards have been exploited to date for calculating the amounts of standards . In many of these previous studies, non‐naturally occurring peptides were designed, prepared as synthetic peptides, and used to quantify a single standard molecule in one measurement . Protein‐derived tag, which contains multiple peptides for monitoring the amount of a standard protein, was also used for the quantification of a single standard protein .…”

Section: Discussionmentioning

confidence: 99%

A strategy for absolute proteome quantification with mass spectrometry by hierarchical use of peptide‐concatenated standards

et al. 2016

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The accurate and precise absolute abundance of proteins can be determined using mass spectrometry by spiking the sample with stable isotope-labeled standards. In this study, we developed a strategy of hierarchical use of peptide-concatenated standards (PCSs) to quantify more proteins over a wider dynamic range. Multiple primary PCSs were used for quantification of many target proteins. Unique "ID-tag peptides" were introduced into individual primary PCSs, allowing us to monitor the exact amounts of individual PCSs using a "secondary PCS" in which all "ID-tag peptides" were concatenated. Furthermore, we varied the copy number of the "ID-tag peptide" in each PCS according to a range of expression levels of target proteins. This strategy accomplished absolute quantification over a wider range than that of the measured ratios. The quantified abundance of budding yeast proteins showed a high reproducibility for replicate analyses and similar copy numbers per cell for ribosomal proteins, demonstrating the accuracy and precision of this strategy. A comparison with the absolute abundance of transcripts clearly indicated different post-transcriptional regulation of expression for specific functional groups. Thus, the approach presented here is a faithful method for the absolute quantification of proteomes and provides insights into biological mechanisms, including the regulation of expressed protein abundance.

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Protein Quantification Using a Cleavable Reporter Peptide

Cited by 7 publications

References 45 publications

Quantitative and Selective Analysis of Feline Growth Related Proteins Using Parallel Reaction Monitoring High Resolution Mass Spectrometry

Quantitative and Selective Analysis of Feline Growth Related Proteins Using Parallel Reaction Monitoring High Resolution Mass Spectrometry

Absolute quantitation of disease protein biomarkers in a single LC-MS acquisition using apolipoprotein F as an example

A strategy for absolute proteome quantification with mass spectrometry by hierarchical use of peptide‐concatenated standards

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