17th International Mass Spectrometry Conference :: Prague, 2006
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|Session:||Proteomics: New Methods|
|Presentation date:||Tue, Aug 29, 2006|
|Presentation time:||14:30 – 16:00|
Xiao-Ming Lu1,2,3, Amy Lu1, Edward Carter1,2,3, Joseph Avruch2,3, John Burke2,3, Alan Fischman1,2,3, Ronald Tompkins1,2,31 Shriners burns hospital, Boston, United States
Correspondence address: Xiao-Ming Lu, Shriners burns hospital, Mass Spectrometry Lab, 51 Blossom street, Boston, 02114 United States.
Keywords: Electrospray Ionization (ESI); Mass Spectrometry, QTOF; MS/MS, Liquid Chromatography; Post-translational Modification.
Novel aspect: First time for systematic phosphorylation mapping of insulin receptor substrate-1 in vitro.
Insulin receptor substract-1 (IRS-1) plays a key role between the insulin action, burn stress and insulin resistance. It is essential to systematically map the phosphoprofile of IRS-1 under in vitro conditions in order to understand the critical phosphorylated residues that can be used to interact and regulate the down stream events and to treat diseases. This paper demonstrates a methodology to obtain the phosphorylation site mapping of recombinant IRS-1 with MAP, JNK, P38, GSK-3, normal and burned soleus muscle homogenates using nano-LC interfaced with tandem mass spectrometry as described before.1,2 Sensitive parent ion discovery is used to ensure the maximal phosphorylated sites recovery but minimize escaping from detection under 1 pmol injection. At the same time, false positive discoveries are excluded with MS/MS sequences and the phosphorylated sites are unambiguously confirmed with 96.97 Da (H2PO4) mass differences between phosphorylated and corresponding non-phosphorylated y or b ions. Total of 16 phosphorylated residues are identified: p-Ser194, p-Ser290, p-Ser632, p-Ser936, p-Ser1224, p-Thr769, p-Thr934, p-Thr951, p-Tyr87, p-Tyr478, p-Tyr489, p-Tyr554, p-Tyr1000, p-Tyr1010, p-Tyr1172, p-Tyr1222. P-Tyr87 is only one located in PH domain (12-115), and p-Ser194 is in PTB domain (115-259). The estimated false negative identification is about 15-20% due to small tryptic p-peptides escaping from C18 trapping column or lacking of detailed MS/MS data for phosphorylated residue verification. C-terminal long chain seems to be involved in multiple kinase actions and regulations. P-Thr951 and p-Tyr87 are found under normal and burned solues muscle homogenates. P-Ser290, p-Thr769, p-Thr934 are associated with burned soleus muscle, while p-Ser194, p-Ser632, p-Ser936 and p-Tyr489 are phosphorylated under normal soleus homogenates. These phosphopeptide mapping may reveal some insights into IRS-1 interactions with insulin receptor and downstream mediators under burn stress.
1. X. M. Lu, M. Lu, R. T. Tompkins and A. J. Fischman J. Mass Spectrom. 40, 1140 (2005).
2. X. M. Lu, M. Lu, A. J. Fischman and R. T. Tompkins J. Mass Spectrom. 40, 599 (2005).