New Approaches for Absolute Quantification of Stable‐Isotope‐Labeled Peptide Standards for Targeted Proteomics Based on a UV Active Tag

Schnatbaum, Karsten; Solis‐Mezarino, Victor; Pokrovsky, Daniil; Schäfer, Frederike; Nagl, Dennis; Hornberger, Lars; Zerweck, Johannes; Knaute, Tobias; Avramova-Nehmer, Julia; Schutkowski, Mike; Hornung, Veit; Wenschuh, Holger; Völker-Albert, Moritz; Imhof, Axel; Reimer, Ulf

doi:10.1002/pmic.202000007

Cited by 7 publications

(5 citation statements)

References 11 publications

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“…Twelve isotopically labeled and absolute quantified peptides (ISTD*, SpikeTides TQL) were obtained from JPT Peptide Technologies GmbH (Berlin, Germany). 29 The carbons and nitrogen of the terminal lysine were substituted by 13 C and 15 N. Isotopic purity was specified as 97–99% and the purity of peptides was determined by HPLC-UV at 220 nm to 91–97%. The lyophilized peptides were mixed and aliquoted to 0.5 nmol of each peptide (absolute amount) per vial by JPT.…”

Section: Methodsmentioning

confidence: 99%

New Mass Spectrometric Approach to Quantify the Major Isoallergens of the Apple Allergen Mal d 1

Kaeswurm

Straub

Klußmann

et al. 2022

J. Agric. Food Chem.

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Patients who suffer from birch pollinosis often develop adverse reactions to the consumption of fresh apples due to the structural similarity of the allergens Bet v 1 and Mal d 1 from birch and apples, respectively. A different allergenic potential for Mal d 1 isoallergens is postulated, but approaches to quantify the Mal d 1 isoallergen-specific are missing. Therefore, a bottom-up proteomics approach was developed to quantify Mal d 1 by stable isotope dilution and microHPLC-QTOF analyses. Marker peptides for individual isoallergens (Mal d 1.01–Mal d 1.03 and Mal d 1.06), combinations thereof (Mal d 1.01 + 1.02, Mal d 1.02 + 1.06, and Mal d 1.04 + 1.05), and two global marker peptides, comprising Mal d 1.01 + 1.02 + 1.04 + 1.05 and Mal d 1.03 + 1.06 + 1.07 + 1.08 + 1.09, were identified. By the use of an extraction standard (r-Mal d 1_mut), an optimized protocol for extraction and tryptic digestion of apple proteins was developed, and the variety-specific extraction efficiency was monitored for the flesh and peel of apples. The Mal d 1 contents in flesh and peel of five commercial apple breeds and four apple varieties from orchard meadows were quantified isoallergen-specific.

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Section: Methodsmentioning

confidence: 99%

New Mass Spectrometric Approach to Quantify the Major Isoallergens of the Apple Allergen Mal d 1

Kaeswurm

Straub

Klußmann

et al. 2022

J. Agric. Food Chem.

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“…Most of the internal peptide standards were quantitated using UV absorption (at 350 and/or 428 nm) of a carboxy-terminal trypsin-cleavable “quantification tag” (Qtag) 22 . The Qtag is a tetrapeptide which includes a nitrotyrosine residue which is absorbent at 350 and 428 nm.…”

Section: Methodsmentioning

confidence: 99%

Absolute protein quantitation of the mouse macrophage Toll-like receptor and chemotaxis pathways

et al. 2022

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The Toll-like receptor (TLR) and chemotaxis pathways are key components of the innate immune system. Subtle variation in the concentration, timing, and molecular structure of the ligands are known to affect downstream signaling and the resulting immune response. Computational modeling and simulation at the molecular interaction level can be used to study complex biological pathways, but such simulations require protein concentration values as model parameters. Here we report the development and application of targeted mass spectrometry assays to measure the absolute abundance of proteins of the mouse macrophage Toll-like receptor 4 (TLR4) and chemotaxis pathways. Two peptides per protein were quantified, if possible. The protein abundance values ranged from 1,332 to 227,000,000 copies per cell. They moderately correlated with transcript abundance values from a previously published mouse macrophage RNA-seq dataset, and these two datasets were combined to make proteome-wide abundance estimates. The datasets produced during this investigation can be used for pathway modeling and simulation, as well as for other studies of the TLR and chemotaxis pathways.

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“…SIL synthetic peptides extended at C-terminus with a trypsin cleavable tag (SIL-TCT, SpikeTides™ TQL) were used as internal standards for protein quantification. The trypsin cleavable tag consists of a tetrapeptide that incorporates a nitrotyrosine residue 38 . Sequences of all proteotypic peptides used for protein quantification are in Tables 1 and 2 .…”

Section: Methodsmentioning

confidence: 99%

Simultaneous quantitative profiling of clinically relevant immune markers in neonatal stool swabs to reveal inflammation

Vidová

Benesová

Klánová

et al. 2021

Sci Rep

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An aberrant immune response developed early in life may trigger inflammatory bowel disease (IBD) and food allergies (e.g., celiac disease). Fecal levels of immune markers categorize an inflammatory response (e.g., food allergy, autoimmune) paralleled with the initial microbial colonization. The immunoaffinity assays are routinely applied to quantify circulating immune protein markers in blood/serum. However, a reliable, multiplex assay to quantify fecal levels of immune proteins is unavailable. We developed mass spectrometry assays to simultaneously quantify fecal calprotectin, myeloperoxidase, eosinophil-derived neurotoxin, eosinophil cationic protein, alpha-1-antitrypsin 1, and adaptive immunity effectors in 134 neonatal stool swabs. We optimized extraction and proteolytic protocol and validated the multiplex assay in terms of linearity of response (> 100; typically 0.04 to 14.77 µg/mg of total protein), coefficient of determination (R2; > 0.99), the limit of detection (LOD; 0.003 to 0.04 µg/mg of total protein), the limit of quantification (LOQ; 0.009 to 0.122 µg/mg of total protein) and robustness. The median CV of intra- and interday precision was 9.8% and 14.1%, respectively. We quantified breast milk-derived IGHA2 to differentiate meconium from feces samples and to detect the first food intake. An early life profiling of immune markers reflects disrupted intestinal homeostasis, and it is perhaps suitable for pre-symptomatic interception of IBD and food allergies.

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New Approaches for Absolute Quantification of Stable‐Isotope‐Labeled Peptide Standards for Targeted Proteomics Based on a UV Active Tag

Cited by 7 publications

References 11 publications

New Mass Spectrometric Approach to Quantify the Major Isoallergens of the Apple Allergen Mal d 1

New Mass Spectrometric Approach to Quantify the Major Isoallergens of the Apple Allergen Mal d 1

Absolute protein quantitation of the mouse macrophage Toll-like receptor and chemotaxis pathways

Simultaneous quantitative profiling of clinically relevant immune markers in neonatal stool swabs to reveal inflammation

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