The present teachings relate to the fields of mass spectrometry and tagging reagents useful for mass spectrometry.
Methods of analyzing amine-containing compounds have been known, however, it is desirable to provide a method for the relative and absolute quantitation of amine-containing compounds. Previous methods have exhibited low sensitivity, a need for 2H, 15C, or 15N isotope-containing amino acid standards, and/or a need for other isotope-labeled standards. A need exists for a method of quantitating amine-containing compounds that overcomes these drawbacks.
According to various embodiments, the methods of the present teachings utilize mass differential, mass spectrometry (MS) tagging reagents to label amine functionality of amine-containing compounds. The labeled analytes can have distinct retention times on a reversed phase column, and distinct masses. Under high energy collision, reporter groups can be generated. The intensity or the peak area detected for each reporter group can be used for quantitation.
A plurality of exemplary mass differential reagents that can be provided and/or used according to various embodiments of the present teachings are shown in
In some embodiments, a package including each of the different reagents is provided and can include separate respective containers, for example, one for each of the different reagents. One or more standards can also be provided, for example, each comprising a known concentration of a known amine-containing compound.
According to various embodiments, the present teachings provide a method for the quantitation of amine-containing compounds. While the method can be used for the quantitation of a wide variety of amine-containing compounds, the present teachings will be particularly exemplified with reference to the quantitation of amino acids. In some embodiments, the reagents and methods can be used for relative and absolute quantitation in two-plex, three-plex, and other multi-plex assays.
According to various embodiments, a plurality of mass spectrometry (MS) tagging reagents is provided for tagging one or more amine-containing compounds. The plurality can be packaged together as a set, packaged separately, or packaged in various combinations. The reagents can comprise a first tagging reagent having a chemical structure and a first mass. The chemical structure can comprise a moiety that is reactive to bond to a nitrogen atom of the amine functionality of the amine-containing compound. An exemplary reactive moiety can comprise an ester linkage to a carbonyl moiety. The nitrogen atom of the amine functionality of the amino-containing compound can react with the active ester of the tag to form an amide linkage to the tag. The hydrogen atom can be a hydrogen atom of a primary or secondary amine. Binding of the linkage can result in releasing a release moiety or leaving group comprising a hydroxylated moiety, for example, a hydroxylated succinimide.
The plurality of MS tagging reagents also comprises a second tagging reagent having the same chemical structure as the first tagging reagent but a different atomic mass compared to the first tagging reagent. The mass of the second tagging reagent can differ from that of the first tagging reagent by one or more atomic mass units. In an exemplary embodiment, the first tagging reagent can comprise, for example, a carbon atom, a nitrogen atom, a hydrogen atom, and/or an oxygen atom, but in the second tagging reagent the same carbon atom, nitrogen atom, hydrogen atom, or oxygen atom can be replaced by a 2H, 13C, a 15N, or an 18O isotope. If the chemical structure includes two carbon atoms, hydrogen atoms, and/or nitrogen atoms, and/or at least one oxygen atom, then the second tagging reagent can comprise two 2H, 13C or 15N isotopes, or one 18O isotope, and would thus have a mass of two atomic units over the mass of the first tagging reagent. In some embodiments, the first tagging reagent can comprise an isotope and the second tagging reagent can be free of that isotope, such that the first tagging reagent need not have the smallest mass of the plurality of tagging reagents. In some embodiments, each tagging reagent of the plurality comprises at least one isotope.
The plurality of MS tagging reagents can further comprise one or more additional tagging reagents, each having the same chemical structure as the first and second tagging reagents but each having a mass that differs from the mass of the first tagging reagent and the mass of the second tagging reagent, by one or more atomic mass units. An exemplary plurality of MS tagging reagents is shown in
According to various embodiments, a kit is provided for the quantitation of one or more amine-containing compounds. The kit can comprise one or more mass differential tagging reagents as described herein, for example, each stored in a separate respective container. In some embodiments, the kit can comprise a box, envelope, bag, or other outer container, inside of which can be the stored individual respective containers for the different tagging reagents. In some embodiments, the kit can comprise buffers and various reagents, useful to early out the methods. In some embodiments, the kit can comprise a plurality of MS tagging reagents wherein each of the tagging reagents have an atomic mass that differs from the atomic masses of the other tagging reagents by two or more atomic mass units. As an example, a kit can be provided that comprises the reagent shown in
As shown in
Other exemplary tagging reagents, tagging moieties, and release moieties that can be used in accordance with various embodiments of the present invention include those described, for example, in U.S. Pat. No. 7,195,751 B2 which is incorporated herein in its entirety by reference.
In use, a first tagging reagent of the plurality can be made to contact a standard that may, or may not, be included with the tagging reagents in a kit. The standard can comprise a known amine-containing compound, for example, a previously tagged amine-containing compound at a known concentration. The contact can be made under conditions that favor a reaction between the first tagging reagent and the standard. For example, the reaction can comprise a chemical reaction that binds the standard to the carbonyl N-alkyl piperizene moiety of the ester described above. The reaction can result in the release of the N-hydroxy succinimide moiety of the ester described above.
Also, when in use, a second tagging reagent of the plurality can be made to contact a sample comprising an unknown concentration of the same amine-containing compound. As described below with reference to
An exemplary method of quantitation is shown with reference to
The next step of the method depicted in
According to various embodiments, a method is provided that can be used for the absolute quantitation of one or more amino acids, wherein standards having known concentrations of a plurality of known amino acids are used. In some embodiments, a kit or package is provided having a plurality of standards, one for each of a plurality of different amino acids sought to be tested in a sample.
Another exemplary method of relative and absolute quantitation is shown with reference to
The tagging chemistry and the methodology of the present teachings provide increased sensitivity relative to known methods, and eliminate the need for 2H-containing, 15N-containing, N-containing, or 18O-containing amino acid standards. Each analyte can have its own internal standard. The reporter signals can be specific to the standard sample and to the test sample. By adding labeled calibration standard directly to the sample, the need to obtain a matrix that is free of endogenous analyte is eliminated. In some embodiments, using PDITM increases specificity and reduces the risk of error. The reagent design makes it a good tool for FlashQuant™ System application.
In some embodiments, the tagging chemistry and the method can be run on any triple quadrupole instruments or on any instrument with a MALDI source, for example, those including, but not limited to, an AB Sciex TripleTOF™ 5600 System, 5800 MALDI TOF/TOF™ System, 4800 MALDI TOF/TOF™ System, 4700 MALDI TOF/TOF™ System, or a FlashQuant™ System with a MALDI source. Reagent kits, data analysis software, and the MS platform can together be used as an analyzer system for amino acid analysis. The method can similarly be employed for other amine-containing compounds.
Different liquid chromatography and mass spectrometry methods, systems, and software that can be used in accordance with various embodiments of the present teachings include those described in U.S. Provisional Patent Application No. 61/182,748 filed May 31, 2009, and in U.S. Patent Application No. US 2006/0183238 A1 which published on Aug. 17, 2006. Both of these references are incorporated herein in their entireties by reference.
The present teachings will be more fully understood with reference to the following Examples that are intended to illustrate, not limit, the present teachings.
40 μL of a physiological sample was transferred to a tube. 10 μL of Sulfosalicylic Acid containing 4000 pmol norleucine, was added. The tube was vortexed to mix, then spun at 10,000×g for 2 minutes. 10 μL of the supernatant was transferred to a clean tube.
Diluting with Labeling Buffer
40 μL of Labeling Buffer containing 800 pmol norvaline was added to the 10-μL aliquot of supernatant from above. The tube was vortexed to mix, then spun. 10 μL of the supernatant was transferred to a clean tube. This sample was labeled with a First Tagging Reagent (see Labeling Samples section below). The remaining supernatant was refrigerated.
Each vial containing the Tagging Reagent Δ8 was spun at room temperature to bring the solution to the bottom of the vial. Each tube was capped promptly. 70 μL of isopropanol was added to each. Each vial was dated. Each vial was vortexed to mix the solution, then spun.
To the sample diluted supernatant from the “Diluting with labeling buffer” section above, 5 μL of the Tagging Reagent Δ8 solution was added. Unused Tagging Reagent Δ8 solution was stored at −15° C. or below. The tube was vortexed to mix then spun. The tube was incubated at room temperature for at least 30 min. Then, 5 μL of Hydroxylamine was added and the tube was again vortexed and spun. For unlabeled allo-isoleucine analysis, 5 μL of the diluted supernatant from “Diluting with labeling buffer” above, was added. The unlabeled internal standard norleucine from the Sulfosalicylic Acid reagent used for the allo-isoleucine analysis was already mixed with the sample. The sample was dried completely in a centrifugal vacuum concentrator for not more than one hour. The dried labeled samples were stored at −15° C. or below.
The blood samples were prepared by spotting seventy-five microliters of whole blood onto Whatman #903 sample collection paper, as per a typical collection protocol. A ⅛ inch punch from the dried blood filter paper (3 μL of whole blood equivalent).
187.5 μL of 80% acetonitrile was added to the tube and it was shaken for 30 min. 100 μL of the supernatant was transferred to a clean tube and it was dried.
Dissolution with Labeling Buffer
8 μL of Labeling Buffer containing 160 pmol norvaline was added to the dried supernatant from above. The tube was vortexed to mix, then spun.
Each vial containing the Tagging Reagent Δ8 was spun at room temperature to bring the solution to the bottom of the vial. Each tube was capped promptly. 70 μL of isopropanol was added to each. Each vial was dated. Each vial was vortexed to mix the solution, then spun.
To the sample diluted supernatant from the “Diluting with labeling buffer” section above, 5 μL of the Tagging Reagent Δ8 solution was added. Unused Tagging Reagent Δ8 solution was stored at −15° C. or below. The tube was vortexed to mix then spun. The tube was incubated at room temperature for at least 30 min. Then, 5 μL of Hydroxylamine was added and the tube was again vortexed and spun. For unlabeled allo-isoleucine analysis, 5 μL of the diluted supernatant from “Diluting with labeling buffer” above, was added. The unlabeled internal standard norleucine from the Sulfosalicylic Acid reagent used for the allo-isoleucine analysis was already mixed with the sample. The sample was dried completely in a centrifugal vacuum concentrator for not more than one hour. The dried labeled samples were stored at −15° C. or below.
A vial of AA Internal Standard was spun to bring the lyophilized material to the bottom of the vial. The internal standard solution was prepared by reconstituting one vial of AA Internal Standard by: finding the amount of Standard Diluent that is specified on the AA Internal Standard vial label (approximately 1.8 mL); dispensing 1 mL of the Standard Diluent into the AA Internal Standard vial; vortexing the vial in 30- to 60-second increments until all material was dissolved; adding the remaining Standard Diluent (approximately 0.8 mL); and vortexing to mix.
To the dried sample from the “Labeling samples” sections above, 32 μL of AA Internal Standard solution was added. The tube was vortexed to mix and then spun. The labeled sample/internal standard mixture was transferred to an autosampler vial with a low-volume insert. To remove potential air trapped in the bottom of the vial, the vial was tapped or spun.
The samples were run using the MS system-specific acquisition method. Each 2 μL injection contained Tagging Reagent Δ8-labeled amino acids in the sample and approximately 10 pmole of each Δ0-labeled amino acid (except 5 pmole cystine) from the AA Internal Standard. The sample also had 10 pmole of norleucine and norvaline. Norleucine was introduced into the sample during the precipitation step and was monitored to follow the recovery of amino acids from the precipitate. Norvaline was introduced into the sample during the labeling step and was monitored to check the efficiency of the labeling reaction.
Mobile Phase A
For each liter of Mobile Phase A, 1 mL of Mobile Phase Modifier A was mixed with 100 μL of Mobile Phase Modifier B with 998.9 mL of Milli-Q water, or equivalent HPLC-grade water.
Mobile Phase B
For each 500 mL of Mobile Phase B, 0.5 mL of Mobile Phase Modifier A was mixed with 50 μL of Mobile Phase Modifier B with 499.5 mL of HPLC-grade methanol.
The following apparatus, parameters, and conditions were used:
Agilent 1100 system
Binary pump G1312A
Well-plate autosampler G1367A
Column oven G1316A
Micro vacuum degasser G1379B
Agilent 1200 system
Binary pump G1312A
Well-plate autosampler G1367B
Column oven G1316A
Micro vacuum degasser G1379B
Shimadzu Prominence system
System controller CBM-20A
2 Isocratic pumps LC-20AD (includes automatic purge kit and semi-micro gradient mixer SUS-20A)
On-line degasser DGU-20A3
Autosampler SIL-20AC
Column oven CTO-20AC
AAA C18 reversed-phase column, 5 μM, 150×4.6 min
None
Water+0.1% formic acid+0.01% heptafluorobutyric acid
Methanol+0.1% formic acid+0.01% heptafluorobutyric acid
Flow Rate: 0.8 mL/min
Column oven temperature: 50° C.
MS/MS detection was optimized for the systems API 3200™, API 4000™, 3200 QTRAP®, and 4000 QTRAP® LC/MS/MS. The following conditions were used.
TurboIonSpray® ion source
Positive polarity
Scan type: MRM
Resolution Q1: unit
Resolution Q3: unit
Dynamic Range Using the aTRAQ™ Kit (on the 3200 QTRAP® System)
The accuracy of each amino acid determination was calculated from 0.01 μM to 10,000 μM. The dynamic range was set where all the accuracies were between 80% and 120%. The dynamic range was ≦1 to ≧10,000 μM.
The Control Plasma sample was characterized using conventional ninhydrin amino acid analysis methods to determine a reference range. The aTRAQ method gave an average accuracy of 103.2% with an average % CV of 2.9%. The least accurate amino acids (Asn, Met, and Trp) are those that can sometimes present problems in conventional amino acid analysis. The resulting data is shown in
The Control Plasma was used to validate the alternate sample preparation method used for samples dried on Whatman #903 sample collection paper (i.e. pediatric blood spots). A punch out ⅛″ disc of each spotted sample (3 ul) was analyzed using the aTRAQ™ kit with an internal standard for every amino acid. The standard solution method and alternate spot method were run in parallel. The resulting data is shown in
Three identical Control Plasma samples were labeled with the 115, 117, and 121 reagents and then mixed together with the internal standard labeled with the 113 reagent. The single mixed sample was analyzed and the concentrations for each sample determined. The results are shown in
The Urine Control sample is a urine matrix into which amino acids have been spiked to known levels. The aTRAQ method gave an average accuracy of 103.3% with an average % CV of 2.7%. The resulting data is shown in
As can be seen from the table above, the theoretical values in the CHO sample for ethanolamine, putrescine, and spermine are 13.6, 0.543, and 15.6 mg/L, respectively.
A sample of salmon was stored at different temperatures for 3 days and then labeled with the aTRAQ™ reagent and the amount of biogenic amine was determined. The results are shown in
Other embodiments of the present teachings will be apparent to those skilled in the art from consideration of the present specification and practice of the present teachings disclosed herein. It is intended that the present specification and examples be considered exemplary only.
The present application claims a priority benefit from earlier filed U.S. Provisional Patent Application No. 61/286,491 filed Dec. 15, 2009, which is incorporated herein in its entirety by reference.
Number | Date | Country | |
---|---|---|---|
61286491 | Dec 2009 | US |