EP-4740010-A1 - NEGATIVE TUNE STANDARD FOR MASS SPECTROMETRY

EP4740010A1EP 4740010 A1EP4740010 A1EP 4740010A1EP-4740010-A1

Abstract

Methods and compositions are used for evaluating the performance of a mass spectrometer instrument. An oligonucleotide is introduced to the mass spectrometry instrument, the sample is analyzed in negative ionization mode, and the resulting mass spectrometry peaks are compared to known standards of the nucleotide-based sample for purposes of performing a qualification of the mass spectroscopy instrument in negative ionization mode. Compositions are provided for the methods and systems.

Inventors

GALLAGHER, Nevin
BETZ, Travis
REDLINSKA, Aleksandra
EPPS, JASON
BRITTAIN, Matt
SLONE, Dennis

Assignees

Agilent Technologies, Inc.

Dates

Publication Date: 20260513
Application Date: 20231020

Claims (1)

Docket No.20230112-02 / 027644.8474 CLAIMS We claim: 1. A method of evaluating performance of a mass spectrometer instrument comprising: providing a mass spectrometer (MS) instrument; introducing a standard to the MS instrument, wherein the standard comprises an oligonucleotide having a structure of Formula I, Formula II, Formula III, or Formula IV: X 1 3ʹ-PL-3ʹX 2 (I) X 1 5ʹ-PL-5ʹX 2 (II) X 1 3ʹ-PL-5ʹX 2 (III) X 1 5ʹ-PL-3ʹX 2 (IV) wherein PL is a phosphate linkage comprising -PO4-, -PO3-O-PO3-, -PO3S, -PO3BH2- or - PO 3 -O-PO 2 -O-PO 3 -; X 1 and X 2 are independently a nucleoside, a nucleotide, or an oligonucleotide; and evaluating one or more mass spectrometry signals corresponding to the oligonucleotide from the mass spectrometry instrument. 2. The method of claim 1, wherein the evaluating comprises identifying one or more mass peaks corresponding to the oligonucleotide, and comparing the identified one or more mass peaks to one or more expected mass peaks for the oligonucleotide. Docket No.20230112-02 / 027644.8474 3. The method of claim 1, further comprising adjusting the MS instrument based on the evaluating of the one or more mass spectrometry signals. 4. The method of claim 1, wherein the mass spectrometry instrument has a negative ionization mode, and wherein the standard is introduced for a negative mode mass spectrometry analysis. 5. The method of claim 4, further comprising performing a qualification of a negative ionization mode of the MS instrument. 6. The method of claim 5, wherein the qualification comprises: analyzing the oligonucleotide in a plurality of amounts; and determining whether the one or more mass spectrometry signals are substantially linear relative to the plurality of amounts. 7. The method of claim 6, wherein the analyzing is a non-linear regression analysis relative to the plurality of amounts used to calibrate or qualify a mass spectrometer. 8. The method of claim 1, further comprising performing a mass-axis calibration. 9. The method of claim 1, wherein the standard is a continuous reference standard introduced to the MS instrument on a continuous basis during sample analysis. Docket No.20230112-02 / 027644.8474 10. The method of claim 1, wherein the standard is an internal standard introduced to the MS instrument simultaneously with a sample for analysis. 11. The method of claim 1, wherein X 1 and X 2 are the same nucleoside. 12. The method of claim 1, wherein X 1 and X 2 are different nucleosides. 13. The method of claim 1, wherein the oligonucleotide of Formula I is deoxythymidine-3ʹ-PL-3ʹ-deoxythymidine. 14. The method of claim 1, wherein the oligonucleotide is in solution. 15. The method of claim 1, wherein the oligonucleotide is solubilized from a solid. 16. The method of claim 1, wherein the oligonucleotide is from 2 to 100 nucleobases in length. 17. The method of claim 1, wherein the oligonucleotide is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. Docket No.20230112-02 / 027644.8474 18. The method of claim 1, wherein the oligonucleotide is 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100 nucleobases in length. 19. The method of claim 1, wherein the oligonucleotide comprises MOE, 2’fluoro, 2’Ome, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, 6-O-methylguanine, LNA, UNA, C(et), 2-thiocytidine, methylated bases, intercalated bases, a nucleotide comprising a modified sugar (e.g., 2’-fluororibose, ribose, 2’- deoxyribose, arabinose, and hexose), or any combination thereof. 20. The method of claim 1, wherein the oligonucleotide further comprises TBDMS, MOM, MEM, DMT, TOM, ACE, TC, PMB, Methylthiomethyl phosphate, ether, Pivaloyl, ether, silyl ether, methyl ester, and trityl. 21. The method of claim 1, wherein the oligonucleotide further comprises an alkyl spacer. 22. The method of claim 21, wherein the alkyl spacer is selected from dodecyl, undecyl, decyl, nonyl, octyl, heptyl, hexyl, pentyl, butyl, propyl, ethyl, and methyl. Docket No.20230112-02 / 027644.8474 23. The method of claim 1, wherein the oligonucleotide further comprises a GalNAc and/or PEG residue. 24. The method of claim 1, wherein the oligonucleotide further comprises a 2’, 3’, and/or 5’ synthesis protecting group. 25. The method of claim 24, wherein the 2’, 3’ an/or 5’ synthesis protecting group is selected from TBDMS, MOM, MEM, TOM, ACE, TC, DMT, PMB, Methylthiomethyl ether, Pivaloyl, ether, silyl ether, methyl ester, and trityl. 26. The method of claim 1, wherein the oligonucleotide is composed of 3’ and/or 5’ linkages. 27. The method of claim 1, wherein the oligonucleotide comprises a synthesis protecting group. 28. The method of claim 27, wherein the synthesis protecting group is selected from the group consisting of acetyl, isobutyl, TAC, dmf, and benzoyl. 29. The method of claim 1, wherein the oligonucleotide comprises a phosphate with a CNET protecting group. Docket No.20230112-02 / 027644.8474 30. The method of claim 1, further comprising calibrating, standardizing, or qualifying an MS/MS mode of the MS instrument using the evaluated one or more mass spectrometry signals. 31. The method of claim 30, wherein the standard is used to calibrate the MS/MS mode of a mass spectrometer. 32. The method of claim 30, wherein the standard is used to standardize the MS/MS mode of a mass spectrometer. 33. The method of claim 30, wherein the standard is used to qualify the MS/MS mode of a mass spectrometer 34. The method of claim 30, wherein the standard is infused into the MS instrument with a sample, and a known mass of the standard is used to dynamically or continuously correct or adjust the MS instrument’s m/z calibration. 35. The method of claim 1, further comprising loading the standard into a standard vessel; and the standard is introduced to the MS instrument by flowing from the standard vessel. 36. The method of claim 35, wherein the standard is continuously flowed to the MS instrument for a selected or predetermined period. Docket No.20230112-02 / 027644.8474 37. The method of claim 1, further comprise fluidically connecting the MS instrument to an oligonucleotide synthesis reaction chamber, and receiving a sample from the oligonucleotide synthesis reaction chamber. 38. The method of claim 37, wherein the standard is introduced to the MS instrument on a continuous basis during analysis of the sample from the oligonucleotide synthesis reaction chamber. 39. The method of claim 37, wherein the sample from the oligonucleotide synthesis reaction chamber comprises an oligonucleotide comprising a synthesis protecting group. 40. The method of claim 39, wherein the synthesis protecting group is selected from acetyl, isobutyl, TAC, dmf, benzoyl, TBDMS, MOM, MEM, DMT, PMB, Methylthiomethyl ether, Pivaloyl, ether, silyl ether, methyl ester, and trityl. 41. The method of claim 37, wherein the sample from the oligonucleotide synthesis reaction chamber comprises an oligonucleotide comprising a phosphate with a CNET protecting group. 42. The method of claim 37, wherein the sample from the oligonucleotide synthesis reaction chamber comprises an oligonucleotide comprising a GalNAc and/or PEG residue. Docket No.20230112-02 / 027644.8474 43. The method of claim 1, wherein the oligonucleotide of the standard comprises a plurality of oligonucleotides having a structure of Formula I, II, III, or IV. 44. The method of claim 43, wherein the two or more oligonucleotides have different molecular weights. 45. The method of claim 43, wherein the two or more oligonucleotides have different lengths.

Description

Docket No.20230112-02 / 027644.8474 NEGATIVE TUNE STANDARD FOR MASS SPECTROMETRY CROSS-REFERENCE TO RELATED APPLICATIONS [001] This application claims the benefit of U.S. Provisional Patent Application No. 63/511,843 filed July 3, 2023, the contents of which are incorporated herein by reference in their entirety. FIELD OF THE INVENTION [002] The present disclosure relates to methods and compositions for assessing a mass spectrometer’s linearity response and mass axis calibration when in negative ionization mode through the use of an oligonucleotide and novel compositions related thereto. BACKGROUND OF THE INVENTION [003] Mass spectrometry (MS) is an important analytical technique utilized in many scientific disciplines. It measures the mass-to-charge ratio (m/z) of one or more molecules present in a sample following an ionization process. The results of the measurements are usually presented as a spectrum, which plots the mass-to-charge (m/z) ratio against the relative abundance of the molecule(s) of interest. [004] The MS instrument comprises a detector configured for analyzing a sample by measuring the m/z ratio of ions received by the detector. The mass spectrometry results may be presented as a mass spectrum, a plot of intensity as a function of the mass-to-charge ratio in relation to molecular abundance, or a chromatographic representation of the summation of ions observed (i.e., a total ion chromatogram (TIC)). Mass spectrometry is useful for identifying and quantifying components of complex mixtures. Docket No.20230112-02 / 027644.8474 [005] Analysis by mass spectrometry typically begins with the samples being ionized by any number of means, including but not limited to, Matrix Assisted Laser Desorption Ionization (MALDI) or Electrospray Ionization (ES). In MALDI techniques, analytes are embedded in a solid, IR or UV-absorbent matrix. The matrix embedded analyte is placed in the ion source of a mass spectrometer. The matrix is vaporized by a short laser pulse and the analyte molecule is thereby transported into the gas phase in a non-fragmented state. The analyte is ionized by colliding and reacting with the matrix ions generated at the same time. A voltage is applied which accelerates the ions into a field-free flight tube. Due to their different masses, the ions in the ion source are accelerated to different speeds with the smaller ions reaching the detector earlier than the larger ions. The varying times of flight are converted into the different masses of the ions. [006] Electrospray ionization allows for the ionization/vaporization of polar molecules. The sample is dissolved in a solvent and then pumped through a thin capillary which is raised to a high potential. Small charged droplets are sprayed from the ES capillary into a bath gas at atmospheric pressure and travel down a pressure and potential gradient towards an orifice in a mass- spectrometer high-vacuum system. As the droplets traverse this path, they break into smaller droplets until either an ion desorbs from the droplet or the solvent is removed. [007] Ionization can also produce unwanted adducts of the analytes which complicate the quality and resolution of the MS spectrum. [008] Ionization may create positive ions (cations) or negative ions (anions), which can then be introduced to the MS instrument. Mass spectrometry instruments are often utilized with a cation sample, which is commonly referred to as “positive mode.” Alternatively, mass spectrometry Docket No.20230112-02 / 027644.8474 instruments may be utilized with an anion sample, which is referred to in this disclosure as “negative ionization mode” or “negative mode.” [009] To ensure accuracy and reliability, MS instruments need to be qualified. Qualification is a scheduled process to determine whether the MS instrument elicits reproducible relative abundance results that follow a linear standard as the concentration or volume of an analyte is increased. Qualification is the formal verification of accuracy, linearity, and other system attributes performed through instrument calibration using a pre-defined standard as intended by the vendor. Installation Qualification (IQ) is generally required for new or pre-owned instruments, and it is usually based on factory specifications and follows manufacturer guidelines. Operational Qualification (OQ) verifies and records a MS instrument’s ability to meet the specified operational criteria after an installation, following repetitive use or after major service intervention. Performance Qualification (PQ) is a customer or instrument supplier defined performance protocol that is run on a system configuration that is representative of its regular operational state (ie (U)HPLC/MS). PQ is usually run after any service intervention. [0010] Linearity for an MS instrument refers to whether a linear relationship exists between the amount of an analyte and the resulting MS signal(s) from that analyte. Linearity can be demonstrated by intro