Mass Spectrometric Methods for Quantifying NPY 1-36 and NPY 3-36

• US 20130214151A1
• Filed: 07/22/2011
• Published: 08/22/2013
• Est. Priority Date: 07/22/2010
• Status: Active Grant

• Current Assignee: Georgetown University
• Original Assignee: Georgetown University
• Inventors: Soldin, Steven J.
• Granted Patent: US 9,269,550 B2
• Time in Patent Office: Days
• US Class Current: 250/282
• CPC Class Codes: G01N 2333/5755 Neuropeptide Y
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry
• CPC Class Codes: H01J 49/0036 Step by step routines describing the handling of the data generated during a measurement

Abstract

Provided are methods of detecting the presence or amount of NPY 1-36 or NPY 3-36 in a sample using mass spectrometry. Importantly, by methods of the invention, both NPY 1-36 and NPY 3-36 can be quantified simultaneously, separately, and independently in a sample that contains both peptides. The methods provide enhanced specificity, and excellent sensitivity with limits of quantitation (LOQ) of about 0.1 ng/mL, and are accomplished in less time and with less sample preparation than required in other assays for NPY. In certain embodiments, because the methods of the invention are specific for both NPY 1-36 and NPY 3-36, the methods provide a major advantage over existing immunoassays. Therefore, the methods may be used to obtain reliable concentration data in several important patient populations, such as patients with hypertension, heart disease, or cancer.

Claims

1. A method of assaying for NPY 1-36 or a fragment of NPY 1-36, which method comprises:

a) preparing a test sample, wherein the test sample comprises NPY 1-36 or a fragment of NPY 1-36;

b) combining the test sample with a known quantity of a reference peptide, thereby forming a test sample comprising an internal standard; and

c) determining by mass spectrometry the quantity of the NPY 1-36 or the fragment of NPY 1-36 in the test sample and the quantity of the reference peptide in the test sample, and calibrating the quantity of the NPY 1-36 or the fragment of NPY 1-36 in the test sample against the known and determined quantities of the reference peptide in the test sample.

2. The method of claim 1, wherein the reference peptide is differentially labeled with one or more mass spectrometrically distinct groups, such that the NPY 1-36 or the fragment of NPY 1-36 and the reference peptide can be distinguished by mass spectrometry.

3. The method of claim 1, wherein the reference peptide is NPY 1-36, human NPY 1-36, porcine NPY 1-36, radioisotopically labeled NPY 1-36, or radioisotopically labeled NPY 3-36.

4. The method of claim 1, wherein the reference peptide is deuterated NPY 1-36 or deuterated NPY 3-36.

5. The method of claim 1, wherein step (c) comprises:

i) in a mass spectrometer selecting and fragmenting ions of a mass to charge ratio corresponding to the reference peptide, and detecting and producing a first mass spectrum of first fragment ions;

ii) in a mass spectrometer selecting and fragmenting ions of a mass to charge ratio corresponding to the NPY 1-36 or the fragment of NPY 1-36, and detecting and producing a second mass spectrum of second fragment ions; and

iii) determining the quantity of the NPY 1-36 or the fragment of NPY 1-36 in the test sample on the basis of the quantity of the second fragment ions in the second mass spectrum relative to the quantities of the first fragment ions from the reference peptide in the second mass spectrum.

6. The method of claim 1, wherein the test sample comprises both NPY 1-36 and a fragment of NPY 1-36.

7. The method of claim 1, wherein the test sample comprises both NPY 1-36 and NPY 3-36.

8. The method of claim 1, wherein the test sample comprises a biological fluid.

9. The method of claim 1, wherein the performance of steps (a), (b), and (c) takes place in less than about six hours.

10. The method of claim 1, wherein the NPY 1-36 or the fragment of NPY 1-36 is present in the test sample at a concentration of about 1 pg/mL to about 3,000 pg/mL.

11. The method of claim 1, wherein the mass spectrometer is a Quadrupole Time-of-Flight (Q-TOF) mass spectrometer, Ion Trap Time-of-Flight (IT-TOF) mass spectrometer, Time-of-Flight (TOF) mass spectrometer or a triple QUAD mass spectrometer.

12. The method of claim 1, further comprising the step of separating the components of the test samples prior to step (a).

13. The method of claim 5, wherein at least one of the second fragment ions has a mass/charge ratio of between about 500 and about 1800.

14. The method of claim 5, wherein at least one of the first fragment ions or second fragment ions results from the loss of water from a parent ion.

15. The method of claim 1, wherein the NPY 1-36 or fragment of NPY 1-36 in the test sample is free NPY 1-36 or free fragment of NPY 1-36.

16. The method of claim 1, further comprising the step of adding an additive to the test sample.

17. The method of claim 16, wherein the additive is a protease inhibitor, a peptidase inhibitor, or an anti-oxidant.

18. The method of claim 16, wherein the additive is an anti-oxidant.

19. The method of claim 18, wherein the anti-oxidant is DTT.