- N,N-Dimethyl leucines as novel Isobaric tandem mass tags for quantitative proteomics and peptidomics
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Herein, we describe the development and application of a set of novel N,N-dimethyl leucine (DiLeu) 4-plex isobaric tandem mass (MS2) tagging reagents with high quantitation efficacy and greatly reduced cost for neuropeptide and protein analysis. DiLeu reagents serve as attractive alternatives for isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass tags (TMTs) due to their synthetic simplicity, labeling efficiency, and improved fragmentation efficiency. DiLeu reagent resembles the general structure of a tandem mass tag in that it contains an amine reactive group (triazine ester) targeting the N-terminus and ε-amino group of the lysine side chain of a peptide, a balance group, and a reporter group. A mass shift of 145.1 Da is observed for each incorporated label. Intense a1 reporter ions at m/z 115.1, 116.1, 117.1, and 118.1 are observed for all pooled samples upon MS2. All labeling reagents are readily synthesized from commercially available chemicals with greatly reduced cost Labels 117 and 118 can be synthesized in one step and labels 115 and 116 can be synthesized in two steps. Both DiLeu and iTRAQ reagents show comparable protein sequence coverage (~43%) and quantitation accuracy ( a marine model organism, Callinectes sapidus.
- Xiang, Feng,Ye, Hui,Chen, Ruibing,Fu, Qiang,Li, Ngjun
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- Novel isotopic N,N-Dimethyl Leucine (iDiLeu) reagents enable absolute quantification of peptides and proteins using a standard curve approach
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Absolute quantification of protein targets using liquid chromatography-mass spectrometry (LC-MS) is a key component of candidate biomarker validation. One popular method combines multiple reaction monitoring (MRM) using a triple quadrupole instrument with stable isotope-labeled standards (SIS) for absolute quantification (AQUA). LC-MRM AQUA assays are sensitive and specific, but they are also expensive because of the cost of synthesizing stable isotope peptide standards. While the chemical modification approach using mass differential tags for relative and absolute quantification (mTRAQ) represents a more economical approach when quantifying large numbers of peptides, these reagents are costly and still suffer from lower throughput because only two concentration values per peptide can be obtained in a single LC-MS run. Here, we have developed and applied a set of five novel mass difference reagents, isotopic N,N-dimethyl leucine (iDiLeu). These labels contain an amine reactive group, triazine ester, are cost effective because of their synthetic simplicity, and have increased throughput compared with previous LC-MS quantification methods by allowing construction of a four-point standard curve in one run. iDiLeu-labeled peptides show remarkably similar retention time shifts, slightly lower energy thresholds for higher-energy collisional dissociation (HCD) fragmentation, and high quantification accuracy for trypsin-digested protein samples (median errors 15%). By spiking in an iDiLeu-labeled neuropeptide, allatostatin, into mouse urine matrix, two quantification methods are validated. The first uses one labeled peptide as an internal standard to normalize labeled peptide peak areas across runs (19% error), whereas the second enables standard curve creation and analyte quantification in one run (8% error).
- Greer, Tyler,Lietz, Christopher B.,Xiang, Feng,Li, Lingjun
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- Differential isotope-labeling for Leu and Val residues in a protein by E. coli cellular expression using stereo-specifically methyl labeled amino acids
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The 1H-13C HMQC signals of the 13CH 3 moieties of Ile, Leu, and Val residues, in an otherwise deuterated background, exhibit narrow line-widths, and thus are useful for investigating the structures and dynamics of larger proteins. This approach, named methyl TROSY, is economical as compared to laborious methods using chemically synthesized site- and stereo-specifically isotope-labeled amino acids, such as stereo-array isotope labeling amino acids, since moderately priced, commercially available isotope-labeled α-keto acid precursors can be used to prepare the necessary protein samples. The Ile δ1-methyls can be selectively labeled, using isotope-labeled α-ketobutyrates as precursors. However, it is still difficult to prepare a residue-selectively Leu and Val labeled protein, since these residues share a common biosynthetic intermediate, α-ketoisovalerate. Another hindering drawback in using the α-ketoisovalerate precursor is the lack of stereo-selectivity for Leu and Val methyls. Here we present a differential labeling method for Leu and Val residues, using four kinds of stereo-specifically 13CH 3-labeled [U-2H;15N]-leucine and -valine, which can be efficiently incorporated into a protein using Escherichia coli cellular expression. The method allows the differential labeling of Leu and Val residues with any combination of stereo-specifically isotope-labeled prochiral methyls. Since relatively small amounts of labeled leucine and valine are required to prepare the NMR samples; i.e., 2 and 10 mg/100 mL of culture for leucine and valine, respectively, with sufficient isotope incorporation efficiency, this approach will be a good alternative to the precursor methods. The feasibility of the method is demonstrated for 82 kDa malate synthase G.
- Miyanoiri, Yohei,Takeda, Mitsuhiro,Okuma, Kosuke,Ono, Akira M.,Terauchi, Tsutomu,Kainosho, Masatsune
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- Protein quantitative labeling reagent and preparation method and application thereof
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The invention provides a protein quantitative labeling reagent as well as a preparation method and application thereof. According to the protein quantitative labeling reagent provided by the invention, the high reaction activity of a o-phthalaldehyde grou
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Paragraph 0052; 0055; 0071-0072
(2020/04/17)
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- Stereoselective synthesis of L-[15N] amino acids with glucose dehydrogenase and galactose mutarotase as NADH regenerating system
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We have developed an efficient stereospecific enzymatic synthesis of L-[15N]-valine, L-[15N]-leucine, L-[15N]- norvaline, L-[15N]-norleucine and L-[15N]-isoleucine from the corresponding α-keto acids by coupling the reactions catalysed by leucine dehydrogenase and glucose dehydrogenase/galactose mutarotase. Giving high yields of L-amino acids, the procedure is economical and easy to perform and to monitor at a synthetically useful scale (1-10 g). Copyright
- Chiriac, Maria,Lupan, Iulia,Bucurenci,Popescu,Palibroda
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p. 171 - 174
(2008/09/19)
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- STABLE ISOTOPE-LABELED AMINO ACID, METHOD OF INTEGRATING THE SAME INTO TARGET PROTEIN, METHOD OF NMR STRUCTURAL ANALYSIS OF PROTEIN AND PROCESS FOR PRODUCING SITE-SELECTIVE STABLE ISOTOPE-LABELED FUMARIC ACID AND TARTARIC ACID
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The present invention provides a stable isotope-labeled amino acid which is at least one of amino acids constituting a protein and which has at least one of the following labeling patterns:(a) hydrogen atoms except at least one hydrogen atom in one or more methylene groups are deuterated,(b) hydrogen atoms in one of prochiral gem-methyl groups are completely deuterated,(c) hydrogen atoms in prochiral methyl groups are partially deuterated, and(d) all hydrogen atoms except one of them in methyl group are deuterated and hydrogen atoms in the aromatic ring are partially deuterated. With the stable isotope-labeled amino acid, the deuteration of protein can be attained without damaging the NMR sensitivity of remaining hydrogen nucleus and, in addition, the rapid, accurate analysis of NMR spectrum of a high-molecular protein which is beyond the limitation in the prior art and the determination of the stereo-structure can be performed at the same time.
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- Stereoselective synthesis of stable isotope-labeled L-α-amino acids: Electrophilic amination of oppolzer's acyl sultams in the synthesis of L-[15N]alanine, L-[15N]valine, L-[15N]leucine, L-[15N]phenylalanine and
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Using 1-chloro-1-[15N]nitrosocyclohexane, we have prepared five L-[α-15N]amino acids. The stereoselective electophillic hydroxyamination of (S)-acylbornane-10,2-sultams, followed by Zn(o)/H+ reduction, and alkaline cleavag
- Lodwig,Unkefer
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p. 239 - 248
(2007/10/03)
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- Chemo-Enzymatic Syntheses of Isotopically Labelled L-Amino Acids
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L-Alanine labelled with carbon-13 and nitrogen-15 has been prepared in good yield and high optical purity from achiral precursors using alanine dehydrogenase.This versatile approach may be simply adapted for the preparation of a range of isotopically labe
- Kelly, Nicholas M.,O'Neill, Bridget C.,Probert, John,Reid, Gordon,Stephen, Rosamund,et al.
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p. 6533 - 6536
(2007/10/02)
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