Analyses of Intact Proteins Using Novel Mass Spectrometric Techniques 493 views

The regulation of nearly every cellular process is directly linked to the sequence and structure of the proteins involved. The study of proteins requires comprehensive knowledge of protein primary structure. This information cannot be derived from the genome alone, as the translation of mRNA into protein does not fully dictate the composition of the final protein complement. Alternative splicing, single nucleotide polymorphisms and the vast number of ways in which a protein can be post-translationally modified expand the number of possible gene products to over 1,000,000. Each has the potential to affect different cellular outcomes. Mass spectrometry (MS) has proven to be an invaluable tool in the study of protein structure and function. The wealth of information that MS is capable of generating, including amino acid sequence and post-translational modification (PTM) identification, makes it a premier method for analysis of complex biological samples. The ultimate MS-based proteomics platform would be capable of unequivocally distinguishing closely related species from one another while concurrently characterizing any PTMs. To do this, the protein must be analyzed intact, as it exists in the system from which it is derived. However, this has proven an elusive endeavor as slow spectral acquisition rates, low signal-to-noise ratios and the complexity of the data generated in tandem mass spectrometry (MS/MS) experiments of whole proteins complicate the analysis. Herein, we describe new MS technologies and methodologies that facilitate the acquisition of high-quality MS/MS spectra capable of providing near-complete amino-acid sequence coverage of intact proteins on a chromatographic time-scale. The utility of our new approaches is demonstrated via analysis of intact histones derived from HeLa cells. We hope that the methods described will help guide the design of future instrumentation and data acquisition software for similar studies, as the knowledge potentially gained from such studies could revolutionize our understanding of numerous biological processes.

PHD (Doctor of Philosophy)

Mass Spectrometry; Intact Proteins; Electron Transfer Dissociation

English

All rights reserved (no additional license for public reuse)

2014-06-17