Towards the Development of a Proteomics Workflow for High-throughput Protein Biomarker Discovery
Two popular workflows exist for quantitative proteome analysis: two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), with staining to visualize proteins, and multidimensional solution phase separations of isotopically labelled peptides coupled to mass spectrometry (MS). However, the development of an alternative strategy, which combines easy-to-read differential profiling as seen in 2D-PAGE, with the sensitivity of MS for detection and identification, is needed. This thesis presents work towards the development of a workflow for high-throughput protein biomarker discovery. Multidimensional separations are vital to obtain sufficient fractionation of complex proteome mixtures. As a first dimension of separation, ion exchange chromatography (IEC) is a common choice, though it has yet to be thoroughly evaluated in terms of its effectiveness as a proteome prefractionation tool. This study used a defined set of protein standards to establish the resolution and proteome yield obtained through IEC. The evaluation uncovered significant bias in terms of protein recovery and separation. To improve throughput of a multidimensional separation strategy, a multiplexed (8-column) reversed phase liquid chromatography (RPLC) platform was constructed. The system design allowed for even distribution of flow across all columns with limited cross-loading during sample loading. This system was directly coupled to matrix-assisted laser desorption/ionization (MALDI) through a novel well plate device. The Teflon wells allowed for high recovery and no cross-contamination during collection/spotting, improved throughput, and greatly reduced the number of sample manipulation steps. An evaluation of MALDI MS, using the ThermoFisher vMALDI LTQ, for quantitative profiling was performed, employing the multiplexed LC-MALDI platform. The use of MALDI MS allowed for fast (< 5.5 hours) acquisition of quantitative data from isotopically differentiated samples partitioned over 640 fractions from two-dimensional LC. Proteins comprising 0.1% of the proteome were detected and quantified using this method. Finally, the effects of varying concentrations of acetonitrile (ACN) upon the products generated from tryptic digestions were explored. Poor enzymatic efficiency in 80% ACN was found to be responsible for an increased concentration of peptides containing missed cleavage sites. These peptides often contained unique amino acid sequences, which were not detected from complete digestions, resulting in improved protein sequence coverage following MS analysis.