PubMed · 10895103
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J Wernerman, O Rooyackers. 2000. Methods, who cares?. https://doi.org/10.1054/clnu.1999.0131
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Carboxylic acids (CAs) are important contributors to the flavor quality of sauce-flavor Chinese Baijiu, yet their comprehensive analysis remains challenging due to poor ionization efficiency, weak chromatographic retention, and limited annotation capability. Herein, we developed a workflow for the high-coverage discovery and annotation of CAs in Baijiu by coupling chemical isotope labeling-liquid chromatography-mass spectrometry with a structure-guided molecular network strategy (SGMNS). A bromine-containing derivatization reagent, 1-(3-aminopropyl)-3-bromoquinolin-1-ium bromide (APBQ), was designed and synthesized to exploit the natural isotope distribution of bromine and characteristic MS/MS fragmentation behavior. Following APBQ derivatization, the target CAs showed superior chromatographic retention and favorable analytical performance. Based on isotopic peak pairing in MS1 and diagnostic fragment validation in MS2, 372 potential CA derivatives were discovered from pooled Baijiu samples and 355 of them were validated by diagnostic fragments in MS2 spectra. To address the scarcity of derivatized spectral libraries, SGMNS was employed for annotation using a background network constructed from APBQ-labeled candidates derived from the Expanded Chinese Baijiu Compound Database. The developed method was further applied to profile Baijiu samples, revealing pronounced differences in CA composition across the seven fermentation rounds. Notably, rounds 3 to 5 exhibited the largest numbers of differential CAs. This study provided an effective analytical strategy for large-scale CA profiling, offering new insight into the chemical basis of flavor formation during multi-round fermentation of sauce-flavor Baijiu.
Data-independent acquisition (DIA) is increasingly preferred over data-dependent acquisition due to its higher throughput and fewer missing values. Whereas data-dependent acquisition often uses stable isotope labeling to improve quantification, DIA mostly relies on label-free approaches. Efforts to integrate DIA with isotope labeling include chemical methods like mass differential tags for relative and absolute quantification and dimethyl labeling, which, while effective, complicate sample preparation. Stable isotope labeling by amino acids in cell culture (SILAC) achieves high labeling efficiency through the metabolic incorporation of heavy labels into proteins in vivo. However, the need for metabolic incorporation limits the direct use in clinical scenarios and certain high-throughput experiments. Spike-in SILAC (SiS) methods use an externally generated heavy sample as an internal reference, enabling SILAC-based quantification even for samples that cannot be directly labeled. Here, we combine DIA-SiS, leveraging the robust quantification of SILAC without the complexities associated with chemical labeling. We developed DIA-SiS and rigorously assessed its performance with mixed-species benchmark samples on bulk and single cell-like amount level. We demonstrate that DIA-SiS substantially improves proteome coverage and quantification compared to label-free approaches and reduces incorrectly quantified proteins. Additionally, DIA-SiS proves effective in analyzing proteins in low-input formalin-fixed paraffin-embedded tissue sections. DIA-SiS combines the precision of stable isotope-based quantification with the simplicity of label-free sample preparation, facilitating simple, accurate, and comprehensive proteome profiling.
We report on a rubidium-xenon (Rb-Xe) polarization unit for the continuous production of large quantities of hyperpolarized (129)Xe. The unit includes two diagnostic systems which enable the absolute measurement of both the (85)Rb and (129)Xe polarization in situ and at high temperatures. The Rb diagnostic system allows the measurement of one- or two-dimensional images of the absolute Rb polarization and thus enables the experimental study of light penetration into the optical pumping cell. The equilibrium Xe polarization measured in the optical pumping cell and in the freezing unit is typically approximately 20%, under optimal flowing conditions, and this is much lower than the volume-averaged rubidium polarization.