A synthesis of new, bi-labeled peptides for quantitative proteomics

Modzel, Maciej; Płóciennik, Halina; Kielmas, Martyna; Szewczuk, Zbigniew; Stefanowicz, Piotr

doi:10.1016/j.jprot.2014.12.001

Cited by 8 publications

(11 citation statements)

References 21 publications

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“…While peptides quantified by method A are used since 2012, [ 5 ] in 2015 a similar approach using Dabsyl labeled peptides was reported. [ 6 ] However, Dabsyl is more expensive than Nitrotyrosine, no correlation to the current gold standard AAA has been reported, and effective Dabsyl tag removal has been shown only for a handful of peptides using harsh tryptic digestion conditions (37 °C, 24 h). Several recent studies describe the use of peptides quantified by method A in targeted proteomics.…”

Section: Figurementioning

confidence: 99%

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

et al. 2020

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Targeted proteomics depends on the availability of stable isotope labeled (SIL) peptide standards, which for absolute protein quantification need to be absolutely quantified. In the present study, three new approaches for absolute quantification of SIL peptides are developed. All approaches rely on a quantification tag (Qtag) with a specific UV absorption. The Qtag is attached to the peptide during synthesis and is removed by tryptic digestion under standard proteomics workflow conditions. While one quantification method (method A) is designed to allow the fast and economic production of absolutely quantified SIL peptides, two other methods (methods B and C) are developed to enable the straightforward re-quantification of SIL peptides after reconstitution to control and monitor known problems related to peptide solubility, precipitation, and adhesion to vials. All methods yield consistent results when compared to each other and when compared to quantification by amino acid analysis. The precise quantitation methods are used to characterize the in vivo specificity of the H3 specific histone methyltransferase EZH2.

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Section: Figurementioning

confidence: 99%

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

et al. 2020

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“…24 In the rst step, the Fmoc-[ 13 C 6 15 N 2 ]KK(dab)K-ChemMatrix construct was synthesized (Fig. 2).…”

Section: Peptide Synthesismentioning

confidence: 99%

“…approximately 1000 times lower compared to the conventional micromole-scale set-up. 24 As we incorporated glycated lysine as a building block, 27 our peptide preparations were not contaminated with corresponding unglycated counterparts. Generally, due to a low scale of the synthesis, the resulting products can be quantied only by amino acid analysis (but not weighing of the crude product).…”

Section: Figmentioning

confidence: 99%

“…Due to the introduction of the dabsyl residue in the peptide sequence, this step can be replaced by the fast and efficient RPC separation with the detection of glycated standards by absorption at 473 nm. 24 Obviously, the procedure described here, provides the cheapest and fastest way to obtain a large number of different glycated sequences. Indeed, omitting a peptide-bound dye would ultimately require either a larger scale of synthesis, or introduction of the amino acid analysis step.…”

Section: Figmentioning

confidence: 99%

“…23 Recently, Modzel and co-workers proposed a quantication approach relying on bi-labeled stable isotope-containing peptide standards, comprising a dabsyl moiety for facilitating quantication and a cleavable linker for enzymatic hydrolysis. 24 This construct has several advantages over other standards used in quantitative proteomics: (i) variable and large m/z increments can be introduced, (ii) multiple peptides can be easily synthesized and puried in small quantities, and (iii) peptide standards are added before tryptic digestion (this is always desired, but typically not possible). 25 Here we show the applicability of the bi-labeled glycated peptides for the absolute quantication of individual glycation sites in plasma proteins of T2DM patients and non-diabetic individuals.…”

Section: Introductionmentioning

confidence: 99%

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Quantification of prospective type 2 diabetes mellitus biomarkers by stable isotope dilution with bi-labeled standard glycated peptides

et al. 2017

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Type 2 diabetes mellitus (T2DM) is a complex group of disorders, characterized by hyperglycemia, insulin resistance and insulin deficiency. In human blood, hyperglycemia ultimately results in the enhancement of glycation -a posttranslational modification formed by the interaction of protein amino groups with glucose. The resulting fructosamines (Amadori compounds) readily undergo further degradation resulting in advanced glycation end products (AGEs), known to be pro-inflammatory in humans. These compounds are highly heterogeneous and characteristic of advanced stages of the disease, whereas fructosamines are recognized markers of early diabetes stages (HbA 1C , glycated albumin). Recently, individual plasma protein glycation sites were proposed as promising T2DM biomarkers sensitive to short-term fluctuations of plasma glucose. However, corresponding absolute quantification strategies, applicable in regular clinical practice, are still not established. Therefore, here we propose a new analytical approach aiming at reproducible and precise quantification of multiple glycated peptides in human plasma tryptic digests. Thereby, the standard peptides comprised a 13 C, 15 N-labeled lysyl residue, a dabsyl moiety for determination of standard amounts, and a cleavable linker. Known amounts of these peptides were spiked to plasma samples prior to tryptic digestion, quantification relying on stable isotope dilution. The method was demonstrated to be applicable for quantification of individual glycated sites in T2DM patients and non-diabetic controls.

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Derivatization of the amino acids glycine and valine causes peptide formation—relevance for the analysis of prebiotic oligomerization

Bayer

Savelsbergh

Kaufmann

et al. 2020

Rapid Comm Mass Spectrometry

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A synthesis of new, bi-labeled peptides for quantitative proteomics

Cited by 8 publications

References 21 publications

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

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

Quantification of prospective type 2 diabetes mellitus biomarkers by stable isotope dilution with bi-labeled standard glycated peptides

Derivatization of the amino acids glycine and valine causes peptide formation—relevance for the analysis of prebiotic oligomerization

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