Affinity Proteomics for Fast, Sensitive, Quantitative Analysis of Proteins in Plasma.

The improving efficacy of many biological therapeutics and identification of low-level biomarkers are driving the analytical proteomics community to deal with extremely high levels of sample complexity relative to their analytes. Many protein quantitation and biomarker validation procedures utilize an immunoaffinity enrichment step to purify the sample and maximize the sensitivity of the corresponding liquid chromatography tandem mass spectrometry measurements. In order to generate surrogate peptides with better mass spectrometric properties, protein enrichment is followed by a proteolytic cleavage step. This is often a time-consuming multistep process. Presented here is a workflow which enables rapid protein enrichment and proteolytic cleavage to be performed in a single, easy-to-use reactor. Using this strategy Klotho, a low-abundance biomarker found in plasma, can be accurately quantitated using a protocol that takes under 5 h from start to finish.

Isotopically nonstationary MFA (INST-MFA) of autotrophic metabolism.

Metabolic flux analysis (MFA) is a powerful approach for quantifying plant central carbon metabolism based upon a combination of extracellular flux measurements and intracellular isotope labeling measurements. In this chapter, we present the method of isotopically nonstationary (13)C MFA (INST-MFA), which is applicable to autotrophic systems that are at metabolic steady state but are sampled during the transient period prior to achieving isotopic steady state following the introduction of (13)CO2. We describe protocols for performing the necessary isotope labeling experiments, sample collection and quenching, nonaqueous fractionation and extraction of intracellular metabolites, and mass spectrometry (MS) analysis of metabolite labeling. We also outline the steps required to perform computational flux estimation using INST-MFA. By combining several recently developed experimental and computational techniques, INST-MFA provides an important new platform for mapping carbon fluxes that is especially applicable to autotrophic organisms, which are not amenable to steady-state (13)C MFA experiments.