Absolute Quantification of Proteins and Metabolites by Mass Spectrometry: Fundamental and Novel Applications towards Development of a Systems-Level Assay for Lignin Biosynthesis

The combination of molecular specificity and detection sensitivity afforded by mass spectrometry is unparalleled by any other analytical technique, making it uniquely suited for systems-level analysis of complex biological networks. In particular, mass spectrometry has proven to be an invaluable stratagem for obtaining absolute and relative quantities for a variety of biochemicals due to its versatility and capacity for multiplexing. The work comprising this dissertation focuses on harnessing and augmenting quantitative assessments by mass spectrometry of both metabolites and proteins, particularly those involved in the biosynthetic pathway of the plant biopolymer, lignin.;Prior to studying lignin biosynthesis, the quantitative techniques utilized here were first refined through studies of two contrasting biological systems: aquatic insects and human plasma. The former system has seen relatively little attention, while the latter is probably the most widely studied; however, both pose unique challenges to the development and application of quantitative assays. Non-enzymatic antioxidants of aquatic insects have recently evoked attention as potential markers of environmental stress, but development of targeted assays has proven difficult due to the large biological variation within and across species. To this end, a highly robust assay was established for quantifying free cysteine and glutathione and, subsequently, applied to characterize several insect species under anaerobic stress. Human plasma, although widely studied, is perhaps the most challenging of biological specimen due to the diversity of its constituents. Proteomic analyses are particularly difficult in plasma due to the large dynamic range of protein concentrations, which makes assessment of low abundant proteins non-trivial. Two innovative sample preparation strategies, ALiPHAT and TRAP, were evaluated for augmenting detection of plasma proteins; in particular, B-type Natriuretic Peptide, a low abundant cardiac biomarker.;Albeit different systems, the experience and knowledge gained through these initial investigations were invaluable when applying similar quantitative mass spectrometric techniques to examine the biosynthetic pathway of lignin. Indeed, many of the same techniques were integrated to identify and characterize a novel multiprotein complex that plays a key role in lignin biosynthesis. Moreover, similar quantitative strategies were employed to develop a multiplexed assay for quantification of the 25 primary enzymes regulating lignin biosynthesis. This comprehensive assay constitutes the first protein-level evidence for many of these enzymes and, more importantly, the first quantitative assessments for all. As such, this assay will serve as a cornerstone for all future quantitative studies of lignin biosynthesis and will lead to a better understanding of lignin formation at a systems-level.;Beyond the direct impact for studying lignin biosynthesis, development of this assay also resulted in a finding that has profound implications for the proteomics community as a whole. More specifically, it was observed during development of this assay that the timing for introducing the internal standard peptide was critical to obtaining accurate quantification. This observation was explained mathematically and experimentally by the differential decay of the native and internal standard peptides used for quantification. Given this phenomenon was previously unappreciated, prior studies utilizing similar methods may have been biased. Nevertheless, these findings will lead to more reliable protein estimates for future assays.