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Signal Transduction is mediated by protein complexes whose spatial- and temporal-distribution, composition and function within cells are often regulated by different post-translational modifications (PTM). As PTMs add or subtract a specific mass difference to a protein, mass spectrometry becomes very amenable for modification analysis. These modifications have conventionally been monitored by fragmenting the modified protein or peptide by collision induced dissociation (CID) within the mass spectrometer, and then screening for the characteristic neutral fragment or fragment ion (marker ion), which is particular to the modification in question. Unfortunately, there are two major issues with respect to the traditional mass spectrometric analysis of PTMs: (1) as there are over 300 known types of modifications, the characteristic fragmentation of only a fraction of these modifications has been studied and (2) the traditional mass spectrometric approaches can only monitor these modifications sequentially, and thus comprehensive modification analysis would be unfeasible considering the breadth of PTMs. The following work aims to address these issues by (1) analyzing PTMs that have never been characterized mass spectrometrically and (2) developing a multiplexed technique for comprehensive PTM monitoring by simultaneously screening for all known characteristic fragments. With respect to the first issue, the characteristic fragmentation of lipid modifications and HNO-induced modifications was investigated. The most prevalent indicator(s) of the modification within the mass spectra are as follows: fragmentation of N-terminal myristoylated peptides produced marker ions at 240 and 268 Th, fragmentation of cysteine farnesylated peptides produced a marker ion at 205 Th and a neutral fragment of 204 Da, and fragmentation of cysteine palmitoylated peptides produced a neutral fragment of 272 Th. For HNO-induced modifications, fragmentation of the sulfinamide- and sulfinic acid-modified peptides produced a neutral fragment of 65 Da and 66 Da, respectively. With respect to the second issue, a multiplexed technique for monitoring modifications that fragment as neutral losses, termed Multiple Neutral Loss Monitoring (MNM), has been developed, successfully validated, and then shown to be the most sensitive approach for PTM analysis. MNM, combined with a second multiplexed approach, targeted Multiple Precursor Ion Monitoring, has been used to provide a comprehensive PTM analysis.

A library of 2-nitrobenzenesulfonyl (Ns) derivatives incorporating isotopic labels and a range of structural variations was prepared and characterized to investigate mass spectrometric fragmentation processes. Deprotonated Ns amino acids were formed readily by negative mode electrospray ionization. Collision induced dissociation experiments established precursor-product ion relationships and indicated a novel loss of an aryl ortho substituent (NO2, F, Cl or Br). In total, four distinct fragmentation pathways of Ns-alpha- and Ns-beta-amino acid anions were identified using isotopic labeling, structural variations of the Ns derivatives, and collision induced dissociation of ions generated in source. Overall, the observation of specific fragmentation pathways correlated with anion structure and ionization site. However, the observation of only one of four possible fragmentation processes in the mass spectrum of the Ns derivative of an amino dicarboxylic acid indicated that functional group interactions must also be considered in the interpretation and prediction of fragmentation processes.

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The Hippo signaling pathway offers an intrinsic mechanism to control organ sizes, and dysfunction of this pathway can often lead to cancer. Great advancement has been made in recent years into understanding this pathway. Despite all this invaluable knowledge, much remains to be explored. Mass spectrometry offers an unbiased approach to characterize the interactome of any protein of interest and is particularly powerful for identifying potential novel regulators of signalling pathways. I therefore set out to characterize the interactome of all the Hippo pathway main components using mass spectrometry, with the goal of uncovering novel regulatory mechanism(s) of the Hippo pathway. In the end, I was able to identify over 250 novel interactors of the Hippo pathway in total. This study demonstrates the utility of mass spectrometry to identify novel regulators of the Hippo pathway and characterization of one such interactor.