| Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has evolved as an important analytical tool for the analysis of peptides, proteins, nucleic acids, oligosaccharides and polymers. However, signal suppression effects complicate the MALDI-TOF MS analyses of complex mixtures. Protein expression profiling of either neat lung tumor cell lysate or neat serum from lung cancer patient by the MALDI-TOF MS only generated a small fraction (∼50 protein ion signals) of the total number of proteins that are expected to exist in either cell lysate or serum.;In an attempt to understand the role of charge in defining the relative desorption/ionization efficiencies of peptides in a given mixture, several ionization tags were chemically attached to the N-termini of model peptides and their desorption/ionization behaviors during MALDI analysis were investigated. This work also describes a solid-phase sample preparation method for the MALDI-TOF MS analysis of a binary peptide mixture and a fractionation strategy that employs the liquid-phase isoelectric focusing technique. Both methods have been shown to reduce signal suppression effects during the MALDI analysis. Nearly 3-fold increase in the number of protein ions detected by the MALDI-TOF MS was observed for serum samples compared to when no pre-fractionation was employed. In total, about 148 unique protein ion signals were reproducibly detected in all 3 replicate fractionation trials.;Additionally, described here is a MALDI-TOF MS-based protein expression profiling strategy for the biomarker discovery for non-small cell lung cancer. Two proteins, MIF (macrophage migration inhibitory factor) and CypA (cyclophilin A) were identified as potential biomarkers in this work. Further investigation using a hydrogen/deuterium exchange-based technique unambiguously confirmed the protein identification for the biomarker, CypA. As part of this work, the dissociation constant for the CypA-CsA (cyclosporin A) complex was determined to be 32 +/- 20 nM. Our studies demonstrate that a MALDI-TOF MS based protein expression profiling can be used as an alternative strategy in the search for new diagnostic and therapeutic targets for lung cancer. |
