Kinetic isotope effects for the chymotrypsin catalyzed hydrolysis of ethoxyl-18O labeled specific ester substrates

Sawyer, Charles B.; Kirsch, Jack F.

doi:10.1021/ja00840a065

Cited by 16 publications

(8 citation statements)

References 2 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The linearity of the data and the standard deviations of the replicates indicate that the precision of the isotope ratios obtained from the product ion should be sufficient to permit heavy atom isotope effects to be determined on nucleotides. Similar results were obtained for the protonated base product ion when [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] O]uridine was analyzed (data not shown). The procedures described to obtain and analyze IDs by tandem MS for nucleotides (and peptides [22]) will permit heavy atom isotope effect studies to be conducted on these biomacromolecular substrates.…”

Section: Application To Time-of-flight Mass Analyzers and Tandem Masssupporting

confidence: 79%

“…One alternative to these methods is whole molecule mass spectrometry, a technique that was used as early as the 1970s for isotope ratio analysis of formyl-methyl ester and ethanol by gas chromatography quadrupole mass spectrometry (QMS) [11,12]. Today, the development of gentler ionization methods such as electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) allow larger molecules to be analyzed.…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

“…18 O 1 ]pGp were purified by HPLC (300 mm × 3.9 mm, 10 μm C 18 packing) eluted isocratically using 50 mM diisopropylethylamine (Aldrich), 1% acetic acid in water (pH 4.3) as the mobile phase. For [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] O]uridine, samples were resolved by a gradient of acetonitrile in 0.2 M ammonium acetate (5% -20% acetonitrile in 60 min.). Appropriate fractions containing either uridine, UMP, TMP or pGp were collected.…”

Section: ′-Uridine-[nonbridging-18 O 1 ]Phosphatementioning

confidence: 99%

“…The application of the analysis of isotope ratios by analysis of IDs was extended to tandemquadrupole time of flight mass spectrometry by analysis of [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] O]uridine and UM[ 18 O 1 ]P with an Applied Biosystems Q-STAR. All analyses were performed by direct infusion of the nucleoside/nucleotide in 50% acetonitrile 1% formic acid.…”

Section: Time Of Flight Mass Spectrometrymentioning

confidence: 99%

“…IRMS can only analyze small, non-polar gasses such as CO 2 , N 2 , and H 2 ; thus, this technique can only be used when the atom of interest can be quantitatively converted to one of these gasses. Scintillation counting requires substantial double-labeling schemes involving specific isotopic pairs (usually 3 H and 14 C, although others can be used [10]), again limiting the molecules that can be analyzed.One alternative to these methods is whole molecule mass spectrometry, a technique that was used as early as the 1970s for isotope ratio analysis of formyl-methyl ester and ethanol by gas chromatography quadrupole mass spectrometry (QMS) [11,12]. Today, the development of gentler ionization methods such as electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) allow larger molecules to be analyzed.…”

mentioning

confidence: 99%

See 4 more Smart Citations

Inaccuracies in selected ion monitoring determination of isotope ratios obviated by profile acquisition: nucleotide 18O/16O measurements

Cassano

Wang

Anderson

et al. 2007

Analytical Biochemistry

View full text Add to dashboard Cite

Precise and accurate measurements of isotopologue distributions (IDs) in biological molecules are needed for the determination of isotope effects, quantitation by isotope dilution and quantifying isotope tracers employed in both metabolic and biophysical studies. While single ion monitoring (SIM) yields significantly greater sensitivity and signal/noise than profile mode acquisitions, we show that small changes in the SIM window width and/or center can alter experimentally determined isotope ratios by up to 5%, resulting in significant inaccuracies. This inaccuracy is attributed to mass granularity, the differential distribution of digital data points across the m/z ranges sampled by SIM. Acquiring data in the profile mode and fitting the data to an equation describing a series of equally spaced and identically shaped peaks eliminates the inaccuracies associated with mass granularity with minimal loss of precision. Additionally a method of using the complete ID profile data that inherently corrects for "spillover" and for the natural abundance ID has been used to determine 18 Keywords isotope ratio; isotopologue distribution; mass isotopomer distribution; tandem mass spectrometry; heavy atom isotope effect; oxygen-18; nucleotide; mass granularity; whole molecule mass spectrometry 1 This work supported by NIH grants GM56740 (M.E.H.), R33 DK07029 (VEA & SP) and a HHMI predoctoral fellowship (A.G.C.). *Corresponding Authors: Vernon E. Anderson and Michael E. Harris, Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH 44106-4935, Phone: 216-368-2511 (VEA); 216-368-4779 (MEH), Fax: 216-368-3419, Email: vernon.anderson@case.edu; michael.harris@cwru.edu. 3 Current address: Department of Chemistry, Drew University, Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptAnal Biochem. Author manuscript; available in PMC 2008 August 1. Published in final edited form as:Anal Biochem. 2007 August 1; 367(1): 28-39. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptHeavy atom isotope effects generated by substituting 13 C, 15 N or 18 O are important tools for probing the mechanisms and transition states of chemical reactions [1], including enzymecatalyzed reactions [2][3][4][5][6][7]. Because the observed effects for isotopic substitution of heavy atoms are extremely small, typically a few percent, they cannot be determined by direct comparison of experimentally determined rate constants. Instead, heavy atom isotope effects are generally measured by internal competition methods that...

show abstract