This two-phase grant proposal seeks to develop and perfect a new mass spectrometry technique, termed Amino-terminal Solid-Phase Isotope Coding (ASPIC), for quantitation and identification of polypeptides in complex mixtures. We will focus on the application of ASPIC to proteolytic events in biological fluids and in intact cells, but anticipate that our technology will later find much broader use in proteomics and cell biology. The key feature of our technology is to selectively couple free N-termini of two parallel polypeptide mixtures individually to a solid support that contains amine-reactive moieties of identical chemical nature, but differing by 6- to 9-Da in molecular mass. The covalently coupled mixture can be washed, digested with trypsin or other proteases, quantitatively eluted, and resolved and analyzed by 2- or 3-dimensional liquid chromatography and tandem mass spectrometry (2D or 3D LC-MS/MS). Any differences in proteolysis, i.e. creation of 'new' N-termini, between two samples will be seen as changes in peak heights, while 'old' N-termini will remain equal between the two samples. Since even 2D LC-MS/MS can readily resolve at least 162,000 peptides, identify differential peak heights in an automated fashion and sequence them, we anticipate obtaining a 'snapshot' of the proteolytic state ('degradome') of the sample. This project is divided into an initial basic technology development phase (Phase I, R21) and a subsequent technology validation and application phase (Phase II, R33). Phase I will develop and optimize the ASPIC chemistry and validate it using blood coagulation as a proof of concept. The transition from Phase I to Phase II will depend on the fulfillment of defined milestones. Phase II will use the optimized technology to determine the proteases and protease substrates that constitute the signaling pathways during (i) apoptosis of HEK293 cells and (ii) T-cell activation.
| Timmer, John C; Enoksson, Mari; Wildfang, Eric et al. (2007) Profiling constitutive proteolytic events in vivo. Biochem J 407:41-8 |
| Enoksson, Mari; Li, Jingwei; Ivancic, Melanie M et al. (2007) Identification of proteolytic cleavage sites by quantitative proteomics. J Proteome Res 6:2850-8 |
