Sensitive and Accurate Quantitation of Phosphopeptides Using TMT Isobaric Labeling Technique.

Phosphorylation is an essential post-translational modification for regulating protein function and cellular signal transduction. Mass spectrometry (MS) combined with isobaric tandem mass tags (TMTs) has become a powerful platform for simultaneous, large-scale phospho-proteome site identification and quantitation. To improve the accuracy of isobaric tag-based quantitation in complex proteomic samples, MS3-based acquisition methods such as Synchronous Precursor Selection (SPS) have been used. However, the method suffers from lower peptide identification rates when applied to enriched phosphopeptide samples compared with unmodified samples due to differences in phosphopeptide fragmentation patterns during tandem MS. We developed and optimized two new acquisition methods for analysis of TMT-labeled multiplexed phosphoproteome samples, which resulted in more phosphopeptide identifications with less ratio distortion when compared with previous methods. We also applied these improved methods to a large-scale study of phosphorylation levels in A549 cell lines treated with insulin or insulin growth factor 1 (IGF-1). Overall, 3378 protein groups and 12 465 phosphopeptides were identified, of which 10 436 were quantified across 10 samples without prefractionation. The accurate measurement enabled us to map to numerous signaling pathways including mechanistic target of rapamycin (mTOR), epidermal growth factor receptor (EGFR, ErbB), and insulin signaling pathways.

Inhibitor kappaB kinase beta binding by inhibitor kappaB kinase gamma.

Activation of a large multisubunit protein kinase, called the inhibitor kappaB kinase (IKK) complex, is central to the induction of the family of transcription factors nuclear factor kappaB. IKK is comprised of two catalytic subunits, IKKalpha and IKKbeta, and a regulatory IKKgamma subunit. It is known that the catalytic IKKbeta and regulatory IKKgamma subunits associate through interactions mediated by the N-terminal region of IKKgamma and an 11-mer peptide located near the C-terminus of IKKbeta. In this study, we have defined the minimal IKKgamma segment that binds IKKbeta and determined the binding affinity of the IKKbeta/IKKgamma complex. We identified that the N-terminal segment spanning residues 40-130 of IKKgamma binds the IKKbeta C-terminal domain (residues 665-756) with Kd approximately 25 nM. Several smaller N-terminal IKKgamma deletion mutants within the N-terminal 130 residues, although in some cases retained IKKbeta binding activity, showed a tendency to aggregate and formed covalently linked complexes. However, expansion of the C-terminus of these fragments to residue 210 completely changed the solution behavior of the IKKgamma N-terminus without affecting the IKKbeta binding affinity. We also found that the IKKbeta C-terminal domain formed a dimer in solution and the basic unit of the IKKbeta/IKKgamma complex was a dimer/dimer.

The cofactor-induced pre-active conformation in PhoB.

PhoB is an Escherichia coli transcription factor from a two-component signal transduction system that is sensitive to limiting environmental phosphate conditions. It consists of an N-terminal receiver domain (RD) and a C-terminal DNA-binding domain. The protein is activated upon phosphorylation at the RD, an event that depends on Mg(2+) binding. The structure of PhoB RD in complex with Mg(2+) is presented, which shows three protomers in the asymmetric unit that interact across two different surfaces. One association is symmetric and has been described as a non-active dimerization contact; the other involves the alpha4-beta5-alpha5 interface and recalls the contact found in activated PhoB. However, here this last interaction is not perfectly symmetric and helix alpha4, which in the activated molecule undergoes a helical shift, becomes strongly destabilized in one of the interacting monomers. All protomers bind the cation in a similar manner but, interestingly, at the prospective binding site for the phosphate moiety the side chains of either Glu88 (in helix alpha4) or Trp54 alternate and interact with active-site atoms. When Glu88 is inside the cavity, helix alpha4 is arranged similarly to the unliganded wild-type structure. However, if Trp54 is present, the helix loses its contacts with the active-site cavity and vanishes. Accordingly, the presence of Trp54 in the active site induces a flexible state in helix alpha4, potentially allowing a helical shift that phosphorylation would eventually stabilize.