A new approach to synthesis and application of novel classes of ligands for the affinity capture and characterisation of phosphorylated peptides

Mass spectrometry has emerged as the technique of choice for the detection of posttranslational modifications of proteins due to its capability in high-throughput, and high sensitive analyses of proteolytic ally derived peptides. The use of enrichment techniques in combination with mass spectrometry has led to the identification of numerous novel phosphorylated peptides from phosphorylated proteins from whole cell lysates. Despite recent advances in the field of mass spectrometry, the analyses of phosphorylated pep tides of very low abundance still poses major analytical challenges. This dissertation will present work that I have done to overcome the limitation of existing methods and to improve detection of phosphorylated proteins or pep tides with and without the involvement of enrichment techniques, resulting in new materials and new methods for the reproducible identification of phosphorylated peptides. The experiments performed in Chapter 2 allowed the evaluation of different sample preparation strategies for the analysis of phosphorylated peptides in MALDI TOF MS. This was achieved by using proteolytic enzymes such as trypsin, proteinase K and pepsin for the proteolysis of phosphorylated proteins performed in the presence of the acid labile mass spectrometry compatible surfactant RapiGest™ in order to enhance ionization efficiency and to improve the relative signal intensity for phosphorylated bovine casein proteolytic peptides in MALDI TOF MS without using any phosphorylated peptide enrichment techniques. Finally, in this study, phosphorylated peptide originating from an elongation factor (tufa), the most abundant phosphorylated E. coli protein, was detected without using a phosphorylated peptide enrichment column by µRPLC ESIfMSn. In Chapter 3 the evaluation of novel compounds as affinity ligands developed in the laboratories of the Centre for Green Chemistry was demonstrated. The efficiency and selectivity of various ligands was tested using a mixture of proteolytic ally derived tryptic digest peptides from bovine casein proteins containing mono- and multiphosphorylated peptides. The highest affinity and selectivity was observed for ligand 1-8 or (E)-4-(pyridin-2-ylmethyleneamino) benzoic acid. The method was further developed to optimize loading capacity and elution conditions for phosphorylated peptides. In Chapter 4, the use of melamine SPE bead material with the immobilized novel ligand 1-8 in analytical high-performance liquid chromatography (HPLC) is described. A systematic optimization of the loading and washing conditions was carried out. Phosphorylated peptides were separated from nonphosphorylated peptides by loading them under acidic conditions (pH 3- 4) onto the IL-1-8 melamine column. Subsequently, the phosphorylated peptides were eluted under alkaline conditions (pH 8-9). Then the eluted phosphorylated tryptic peptides were analysed by MALDI TOF MS. Good trapping selectivity and enrichment of IL-1-8 melamine column towards phosphorylated tryptic peptides was demonstrated in comparison to a commercially available Ti02 column. In Chapter 5, the development of novel strategies for affinity materials for the use in the field of phosphoproteomic is described. This chapter has three aims: The first aim is to compare the utility of different enrichment techniques for the phosphorylated peptide identification, one in which affinity chromatography is performed at the phosphoprotein level and one in which affinity chromatography is performed at the protein level. Here I present new and improved procedures for the Qiagen affinity chromatography phosphoprotein purification kit exemplified for the purification of phosphorylated proteins derived from bovine caseins or bacterial cell lysates. Phosphorylated peptides or proteins obtained from model proteins were used to optimize and test the procedures. The second aim is to compare the solid phase extraction (SPE) with the newly developed IL-1-8 melamine material with Ti02 SPE for the enrichment of phosphorylated peptides. The third aim is to compare SPE enrichment with a µLC enrichment of phosphorylated peptides using Ti02 RP SPE and Ti02 µRPLC. In Chapter 6, new uses of the Qiagen AC phosphoprotein purification cartridge is described, which is designed for the on-line purification of phosphorylated proteins from complex mammalian cell lysates. Then enriched phosphorylated proteins can be separated by using SDS PAGE and subjected to subsequent Coomassie staining or western blot analysis. The initial investigation was started with the enrichment of phosphorylated proteins derived either from a bovine casein or E. coli protein mixture with on-line Qiagen AC phosphoprotein purification by using fast performance liquid chromatography (FPLC), then followed by two experimental procedures. In the first experimental procedure the enriched phosphorylated proteins were purified using an ultrafiltration column with a molecular weight cut-off (MWCO) of 2 kDa (E. coli protein mixture) or 10 kDa (bovine casein protein mixture) followed by an in-solution proteolytic digestion. In the second experimental procedure enriched phosphorylated proteins were separated by SDS-PAGE, followed by an in-gel proteolytic digestion. Finally, the proteolytic derived peptides mixtures which contained non- and phosphorylated peptides were subjected to an on-line enrichment of phosphorylated peptides with a commercially available Ti02 column followed by µRPLC ESI/MSn and MALDI TOF MS. In Chapter 7, a systematic investigation was performed in order to improve the elution of phosphorylated peptides especially multiphosphorylated peptides via stepwise elution from a Ti02 column with four- and three-step elution by using different buffer solutions. Eluted phosphorylated tryptic peptides were desalted by an enrichment column and separated with a RP C18 column using 2D µRPLC ESI/MSn in conjunction with an Agilent SpectrumMill database searching strategy. However, binding of some non-phosphorylated peptides to the Ti02 column under acidic conditions and elution under basic conditions was observed. The next step was performed in conjunction with a fraction collector, then the samples were spotted onto a MALDI plate and analysed by MALDI TOF MS. This approach significantly improved the detection of phosphorylated peptides in comparison to previous described procedures without fraction collector. Above described conditions improved elution, ionization of phosphorylated peptides (strong acidic pH conditions increased the degree of protonation), decreased competition and detected number of phosphorylated peptides especially after introducing a fraction collector and MALDI TOF MS analysis. In Chapter 8 a systematic investigation was performed in order to evaluate the utility of on-probe dephosphorylation of phosphorylated tryptic peptides with alkaline phosphatase for mass spectrometry based phosphopeptide analysis. The on-probe sample preparation was developed using peptides derived from in-solution or on-probe tryptic digestion of the phosphorylated protein bovine β-CN, either performed in the presence or absence of RapiGest™. Overall, it was demonstrated that on-probe dephosphorylation in conjunction with rapid MALDI TOF MS could be employed to accurately detect phosphorylated and dephosphorylated peptides, and was successfully applied to identify the five known phosphorylation sites of bovine β-CN.