New Technologies: Protein Interactions and Post Translational Modifications

Li Yang

A novel photocleavable and mass spectrometry identifiable cross-linker pcPIR (photocleavable protein interaction reporter) is developed to study cellular protein-protein interactions. pcPIR can be dissociated under ultraviolet light irradiation to enable identification of the cross-linked peptide pairs with cross-linking type specific mass relationships. The released peptides from photocleavage are further fragmented under collision induced dissociation to allow traditional database search and sequence identification. The pcPIR technology was demonstrated with the use of standard peptides, bovine serum albumin, and human hemoglobin tetramer protein complex. In vivo protein topologies and protein-protein interactions were measured by applying pcPIR technology to the E. coli cellular system. More than 1700 labeled peptides from E. coli were identified, indicating many protein sites reacted with pcPIR in vivo. From those labeled sites, 54 in vivo inter-cross-linked peptide pairs were identified and manually validated. Approximately half of the interactions have been reported using other techniques. Another PIR compound which contains mass spectrometry fragile bonds instead of the photocleavable bonds were applied to the E. coli cells as well. 65 cross-linked peptide pairs were identified, which is the largest data set produced to date. Many of these cross-links agreed excellently with protein and complex crystal structures where available. On the other hand, protein posttranslational modifications, such as lysine acetylation, can serve to modulate protein functional roles in vivo. Global scale proteomics studies of lysine acetylation indicate acetylation involves in a greater diversity of functional roles and subcellular localizations than previously recognized. However, fasted/fed organ-specific regulation of acetylome on a whole organism basis has not yet been reported. Here, the acetylome patterns of mice insulin sensitive organs (liver, white and brown adipose), insulin sensitive muscles (skeletal muscle and heart) and insulin non-sensitive tissues (brain and kidney) are identified and quantified by using immunoprecipitation coupled with LC-MSMS and label free quantification strategy. Moreover, the tissue specific acetylome patterns under different physiological scenarios of energy deprivation (fasting) and energy availability (re-feeding) are compared. 733 acetlyated peptides from 337 proteins were quantified, out of which 58 acetylated peptides were significantly changed under fasted/re-fed conditions amongst tissues.

New Technologies: Protein Interactions and Post Translational Modifications

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