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Dysfunctional or over and under expressed enzymes play a crucial role in a variety of diseases. A tool that can identify dis-regulated enzymes in individual patients would be beneficial and would allow personalised treatment. For this purpose, a 10,000 membered ‘spit-and-mix’ PNA-encoded peptide library with a cell penetrating peptide was synthesised and interrogated with K562 cell lysate and intact K562 cells. This allowed the specific enzyme activity pattern for ABL tyrosine kinase from both inside a cell and a lysate to be obtained. Hybridisation of this library with a DNA-microarray resulted in bio-fouling by the cell lysate, thereby preventing analysis of the phosphorylation pattern. To allow extraction and purification of the peptide library from the cell lysates, a His-tag was incorporated into the library, and enabled successful library analysis. In addition to this 10,000 member library, a focused 100 PNA-encoded peptide library was synthesised. The library included peptide sequences known to be phosphorylated by specific tyrosine kinases deregulated in acute lymphoblastic leukaemia (ALL) with a PNA-tag complementary to a DNA microarray. Different ligation methods to conjugate the peptides to PNA-tags were screened – this included amide coupling, copper catalysed azide–alkyne cycloaddition, strain promoted azide–alkyne cycloaddition and Diels–Alder cycloaddition. The inverse electron demand Diels–Alder cycloaddition between a tetrazine and norbornene was chosen as the preferred ligation method, and the reaction conditions optimised. To purify the library from cell lysate, a His-tag was again coupled to each member using the strain promoted azide–alkyne cycloaddition. To test the tetrazine ligation, fluorescence in situ hybridisation (FISH) was used in cells, whereby a fluorophore was ligated onto a tetrazine–conjugated PNA probe. This was hybridised onto an mRNA in fixed cells. Results indicated that the ligation needed further optimisation.

<p>Protein kinases are enzymes that catalyse the phosphoryl transfer from the g-phosphate of ATP to acceptor amino acids in proteins. The specificity of selected model protein kinases was studied at three different levels using a) novel bi-substrate-analogue inhibitors, b) synthetic peptide substrates and c) mutated protein substrate analogues. </p><p>A new class of protein kinase bi-substrate-analogue inhibitors was designed on the basis of adenosine-5’-carboxylic acid derivatives, where a short arginine containing peptide was attached to the 5'-carbon atom of the adenosine sugar moiety via a linker chain. These compounds showed high inhibitory potential against two basophilic protein kinases, the protein kinase A (PKA) and protein kinase C (PKC), with IC50 values in the nanomolar range, but no inhibitory activity towards the acidophilic kinases CK1 and CK2. The inhibitors were efficiently applied for affinity purification of PKA using MgATP as well as L-arginine as eluting agents. </p><p>Ca2+-dependent protein kinase (CDPK-1) was purified from maize seedlings and its substrate specificity was studied using a set of synthetic peptides. These were derived from the phosphorylatable sequence RVLSRLHS(15)VRER of maize sucrose synthase 2 (SuSy2), and a consensus sequence motif A/LXRXXSXRZR (where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine) was defined from a study using arrays of systematically varied peptides attached to cellulose membrane (SPOTs^TM membranes). The SuSy2 derived peptides were also found to be efficient substrates for mammalian PKC, but showed low reactivity in the case of PKA. On the basis of this peptide motif, a positionally oriented peptide library approach based on ESI-MS detection of phosphopeptides in initial velocity conditions was designed for quantitative kinetic characterization of protein kinase specificity profiles. On the basis of the obtained data an optimal peptide substrate for PKC, FRRRRSFRRR, was designed. </p><p>The specificity of protein kinase A was studied using site-directed mutagenesis in the phosphorylation site of L-type pyruvate kinase (L-PK), and comparison of the obtained data with the data from previous studies on structurally altered peptide substrates revealed that amino acid alterations in short peptide substrates cause stronger effects on the phosphorylation rate than the corresponding alterations in the protein substrate L-PK.</p>

Protein kinases are enzymes that catalyse the phosphoryl transfer from the g-phosphate of ATP to acceptor amino acids in proteins. The specificity of selected model protein kinases was studied at three different levels using a) novel bi-substrate-analogue inhibitors, b) synthetic peptide substrates and c) mutated protein substrate analogues. A new class of protein kinase bi-substrate-analogue inhibitors was designed on the basis of adenosine-5’-carboxylic acid derivatives, where a short arginine containing peptide was attached to the 5'-carbon atom of the adenosine sugar moiety via a linker chain. These compounds showed high inhibitory potential against two basophilic protein kinases, the protein kinase A (PKA) and protein kinase C (PKC), with IC50 values in the nanomolar range, but no inhibitory activity towards the acidophilic kinases CK1 and CK2. The inhibitors were efficiently applied for affinity purification of PKA using MgATP as well as L-arginine as eluting agents. Ca2+-dependent protein kinase (CDPK-1) was purified from maize seedlings and its substrate specificity was studied using a set of synthetic peptides. These were derived from the phosphorylatable sequence RVLSRLHS(15)VRER of maize sucrose synthase 2 (SuSy2), and a consensus sequence motif A/LXRXXSXRZR (where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine) was defined from a study using arrays of systematically varied peptides attached to cellulose membrane (SPOTsTM membranes). The SuSy2 derived peptides were also found to be efficient substrates for mammalian PKC, but showed low reactivity in the case of PKA. On the basis of this peptide motif, a positionally oriented peptide library approach based on ESI-MS detection of phosphopeptides in initial velocity conditions was designed for quantitative kinetic characterization of protein kinase specificity profiles. On the basis of the obtained data an optimal peptide substrate for PKC, FRRRRSFRRR, was designed. The specificity of protein kinase A was studied using site-directed mutagenesis in the phosphorylation site of L-type pyruvate kinase (L-PK), and comparison of the obtained data with the data from previous studies on structurally altered peptide substrates revealed that amino acid alterations in short peptide substrates cause stronger effects on the phosphorylation rate than the corresponding alterations in the protein substrate L-PK.

This thesis deals with the analysis of natural peptide libraries using mass spectrometry. In the course of the study, both ribosomal and non-ribosomal classes of peptides have been investigated. Microheterogeneity, post-translational modifications (PTM), isobaric amino acids and disulfide crosslinks present critical challenges in routine mass spectral structure determination of natural peptides. These problems form the core of this thesis. Chapter 2 describes an approach where chemical derivatization, in unison with high resolution LC-MSn experiments, resulted in deconvolution of a microheterogenous peptide library of B. subtilis K1. Chapter 3 describes an approach for distinction between isobaric amino acids (Leu/Ile/Hyp), by the use of combined ETD-CID fragmentation, through characteristic side chain losses. Chapters 4-6 address a long standing problem in structure elucidation of peptide toxins; the determination of disulfide connectivity. Through the use of direct mass spectral CID fragmentation, a methodology has been proposed for determination of the S-S pairing schemes in polypeptides. Further, an algorithm DisConnect has been developed for a rapid and robust solution to the problem. This general approach is applicable to both peptides and proteins, irrespective of the size and the number of disulfide bonds present. The method has been successfully applied to a large number of peptide toxins from marine cone snails, conotoxins, synthetic foldamers and proteins. Chapter 7 describes an attempt to integrate next generation sequencing (NGS) data with mass spectrometric analysis of the crude venom. This approach couples rapidly generated cDNA sequences, with high-throughput LC-ESI-MS/MS analysis, which provides mass spectral fragmentation information. An algorithm has been developed that allows the construction of a putative conus peptide database from the NGS data, followed by a protocol that permits rapid annotation of tandem MS data. The approach is exemplified by an analysis of the peptide components present in the venom of Conus amadis, yielding 225 chemically unique sequences, with identification of more than 150 sites of PTMs. In summary, this thesis presents different methodologies that address the existing limitations of de novo mass spectral structure determination of natural peptides and presents new methodologies that permit for rapid and efficient analysis of complex mixtures.