Isotopic recoding for targeted tandem mass spectrometry
Aspects of the present disclosure include methods for detecting a low abundance protein and methods for identifying a site of N-glycosylation on a protein. In practicing methods according to certain embodiments, a eukaryotic cell is contacted with an isotopic labeling composition and isotopically labeled N-glycosylated peptides obtained from the eukaryotic cell are assessed by liquid chromatography-tandem mass spectrometry. A predetermined isotopic pattern in the mass spectrum is identified and amino acid sequences of the peptides containing the predetermined isotopic pattern are determined. Systems for identifying a predetermined isotopic pattern in mass spectra and determining amino acid sequences of peptides containing the predetermined isotopic pattern are also described.
1. A method for identifying a site of N-glycosylation on a protein, the method comprising:
incubating a eukaryotic cell with an isotopic labeling composition comprising two or more different isotopic labels to obtain an isotopically labeled sample of N-glycosylated proteins;
enzymatically producing peptides from the isotopically labeled sample of N-glycosylated proteins to obtain an enzymatically treated sample of isotopically labeled peptides;
subjecting the enzymatically treated sample of isotopically labeled peptides to liquid-chromatography-tandem mass spectrometry;
identifying a predetermined isotopic pattern of the enzymatically treated, isotopically labeled sample of peptides in a mass spectrum at one or more retention times;
determining an amino acid sequence of an isotopically labeled peptide present at the one or more retention times;
and identifying the site of N-glycosylation on the isotopically labeled protein based on the determined amino acid sequence.
2. The method according to claim 1 , wherein the isotopic labels are N-acetyl-D-glucosamine, N-[1,2- 13 C 2 ]acetyl-D-glucosamine and N-[1,2- 13 C 2 ]acetyl-D-[1- 13 C; 15 N]glucosamine.
3. The method according to claim 2 , wherein the isotopic labeling composition comprises a stoichiometric ratio of 1:2:1 of N-acetyl-D-glucosamine, N-[1,2- 13 C 2 ]acetyl-D-glucosamine and N-[1,2- 13 C 2 ]acetyl-D-[1- 13 C; 15 N]glucosamine.
4. The method according to claim 1 , wherein enzymatically producing peptides comprises contacting the N-glycosylated proteins with trypsin, Lys-C, Arg-N or chymotrypsin.
5. The method according to claim 1 , wherein identifying a predetermined isotopic pattern comprises identifying a 1:2:1 peak intensity ratio in the mass spectrum.
6. The method according to claim 1 , wherein the method further comprises generating an inclusion list of peptides having a mass spectrum that contains the predetermined isotopic pattern.
7. The method according to claim 6 , wherein the method further comprises determining an amino acid sequence for each of the peptides on the inclusion list.
8. A method for detecting a low-abundance protein in a biological sample, the method comprising:
incubating a eukaryotic cell with an isotopic labeling composition comprising two or more different isotopic labels;
subjecting an isotopically labeled biological sample obtained from the eukaryotic cell to liquid-chromatography-tandem mass spectrometry; and
identifying a predetermined isotopic pattern in a mass spectrum at one or more retention times to thereby detect the presence of the low-abundance protein in the biological sample.
9. The method according to claim 8 , wherein the isotopic labels are N-acetyl-D-glucosamine, N-[1,2- 13 C 2 ]acetyl-D-glucosamine and N-[1,2- 13 C 2 ]acetyl-D-[1- 13 C; 15 N]glucosamine.
10. The method according to claim 9 , wherein the isotopic labeling composition comprises a stoichiometric ratio of 1:2:1 of N-acetyl-D-glucosamine, N-[1,2- 13 C 2 ]acetyl-D-glucosamine and N-[1,2- 13 C 2 ]acetyl-D-[1- 13 C; 15 N]glucosamine.
11. The method according to claim 8 , wherein the isotopically labeled biological sample obtained from the eukaryotic cell comprises N-glycosylated proteins.
12. The method according to claim 11 , wherein the N-glycosylated proteins comprise the isotopic labels.
13. The method according to claim 11 , wherein the method further comprises producing peptides from the N-glycosylated proteins.
14. The method according to claim 13 , wherein producing peptides comprises contacting the N-glycosylated proteins with trypsin, Lys-C, Arg-N or chymotrypsin.
15. The method according to claim 8 , wherein identifying a predetermined isotopic pattern comprises identifying a 1:2:1 peak intensity ratio in the mass spectrum.
16. The method according to claim 8 , wherein the method further comprises determining an amino acid sequence of the low abundance protein.
17. The method according to claim 8 , wherein the method further comprises generating an inclusion list of peptides having a mass spectrum that contains the predetermined isotopic pattern.
18. The method according to claim 17 , wherein the method further comprises determining an amino acid sequence for each of the peptides on the inclusion list.