Tuesday, March 03, 2015

US patent 8,969,251: mass spec for phenotype analysis

The abstract

A method for assaying phenotypic similarity or dissimilarity between organisms is disclosed in which a composite sample of admixed first and second samples is provided. The first, standard sample contains average concentrations of compounds of molecular mass less than about 1000 AMU present in the organism species. The second, assay sample contains compounds of having a similar molecular mass present in the organism whose phenotype is to be assayed. The constituents of both samples are (i) in a liquid medium and (ii) each compound of a sample has the same, first and second respective amounts of first and second stable isotopes of a first atom. The composite sample is mass spectroscopically analyzed for analytes, with the ratio of first to second isotope being determined for each analyte, along with a composite sample median ratio. The ratios for each analyte are compared to the median, with outlying ratios indicating dissimilarity.

--[ The assignee: Methabolic Analyses, Inc. (Chapel Hill, NC) ]

The '251 patent gives background on the use of stable isotope analysis:


The use of stable isotopes for the determination of biological information has a long and illustrious history [see, Hellerstein, Metabolic Engineering 6:85-100 (2004)]. The oldest and most frequent such usage is in studies probing metabolism wherein a stable isotope is incorporated into a specific molecule at a specific location. This isotopically-labeled molecule, or "precursor", is fed to an in vivo organism, in vitro cell system, or in vitro cell-free system for either a brief or extended period of time, after which the fate of the isotope is determined, either by use of NMR, mass spectrometry (MS), chemical degradation, or other detection technique.

In contrast to the use of radioactive isotopes, the use of stable isotopes is generally regarded as safe and free of regulation. Although in general, a study typically uses a single isotope incorporated into a specific location in order to achieve a precision in understanding the metabolic fate of a molecule, another embodiment of the use of stable isotopes utilizes wholly-labeled molecules (>99% of an atom is replaced with an isotopic equivalent), or universally-labeled (the isotope is universally distributed within the target molecule at less than saturation levels). There are many known studies in which more than one isotope is incorporated into a target molecule, and all of the isotopic fragments are examined for their differential fates. In all cases, these methods are targeted analyses; i.e., they seek the incorporation of a specific labeled atom into other specific molecules.

Yet another use of stable isotopically labeled compounds is as internal standards for their non-labeled counterparts. In such an experiment an isotopically enriched molecule is added to a sample or extract at a known concentration prior to an analysis, and the final measurement determines the exact concentration of the non-labeled material by comparison. In this type of study, it is not uncommon for a researcher to add more than one isotopically-distinct standard if more than one molecule is to be quantified. Indeed, there are extreme forms where one prepares an extremely complex mixture by growing a complex organism on an isotopically-defined feedstock such that the entire organism is heavily, if not entirely, composed of molecules consisting of only one isotope [Wu et al., Anal Biochem 336:164-171 (2005)]. In this situation, the same standard is introduced into all samples, but there is no information carried by the standard other than for purposes of relative quantitation; i.e., the standard has no relation to the experiment at hand. Historically, such standards are carefully constructed to differ from any other analyte by a specific mass difference.


One notes a technique which can involve stable isotope analysis is MIMS [from US Patent 6,320,101 [ Enhancing inorganic carbon fixation by photosynthetic organisms ] : The rates were assessed from measurements during steady state photosynthesis using a membrane inlet mass spectrometer (MIMS)]; See also Shevela, Front Plant Sci. 2013; 4: 473: Studying the oxidation of water to molecular oxygen in photosynthetic and artificial systems by time-resolved membrane-inlet mass spectrometry


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