4 research outputs found
Application of Displacement Chromatography to Online Two-Dimensional Liquid Chromatography Coupled to Tandem Mass Spectrometry Improves Peptide Separation Efficiency and Detectability for the Analysis of Complex Proteomes
Investigation of the Proteomes of the Truffles Tuber albidum pico, T. aestivum, T. indicum, T. magnatum, and T. melanosporum
Truffles of the Tuber species are known as expensive foods, mainly for their distinct aroma and taste. This high price makes them a profitable target of food fraud, e.g., the misdeclaration of cheaper truffle species as expensive ones. While many studies investigated truffles on the metabolomic level or the volatile organic compounds extruded by them, research at the proteome level as a phenotype determining basis is limited. In this study, a bottom-up proteomic approach based on LC-MS/MS measurements in data-independent acquisition mode was performed to analyze the truffle species Tuber aestivum, Tuber albidum pico, Tuber indicum, Tuber magnatum, and Tuber melanosporum, and a protein atlas of the investigated species was obtained. The yielded proteomic fingerprints are unique for each of the of the five truffle species and can now be used in case of suspected food fraud. First, a comprehensive spectral library containing 9000 proteins and 50,000 peptides was generated by two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS). Then, samples of the truffle species were analyzed in data-independent acquisition (DIA) proteomics mode yielding 2715 quantified proteins present in all truffle samples. Individual species were clearly distinguishable by principal component analysis (PCA). Quantitative proteome fingerprints were generated from 2066 ANOVA significant proteins, and side-by-side comparisons of truffles were done by T-tests. A further aim of this study was the annotation of functions for the identified proteins. For Tuber magnatum and Tuber melanosporum conclusive links to their superior aroma were found by enrichment of proteins responsible for sulfur-metabolic processes in comparison with other truffles. The obtained data in this study may serve as a reference library for food analysis laboratories in the future to tackle food fraud by misdeclaration of truffles. Further identified proteins with their corresponding abundance values in the different truffle species may serve as potential protein markers in the establishment of targeted analysis methods. Lastly, the obtained data may serve in the future as a basis for deciphering the biochemistry of truffles more deeply as well, when protein databases of the different truffle species will be more complete
Application of Displacement Chromatography to Online Two-Dimensional Liquid Chromatography Coupled to Tandem Mass Spectrometry Improves Peptide Separation Efficiency and Detectability for the Analysis of Complex Proteomes
The complexity of
mammalian proteomes is a challenge in bottom-up
proteomics. For a comprehensive proteome analysis, multidimensional
separation strategies are necessary. Online two-dimensional liquid
chromatography–tandem mass spectrometry (2D-LC-MS/MS) combining
strong cation exchange (SCX) in the first dimension with reversed-phase
(RP) chromatography in the second dimension provides a powerful approach
to analyze complex proteomes. Although the combination of SCX with
RP chromatography provides a good orthogonality, only a moderate separation
is achieved in the first dimension for peptides with two (+2) or three
(+3) positive charges. The aim of this study was to improve the performance
of online SCX-RP-MS/MS by applying displacement chromatography to
the first separation dimension. Compared to gradient chromatography
mode (GCM), displacement chromatography mode (DCM) was expected to
improve the separation of +2-peptides and +3-peptides, thus reducing
complexity and increasing ionization and detectability. The results
show that DCM provided a separation of +2-peptides and +3-peptides
in remarkably sharp zones with a low degree of coelution, thus providing
fractions with significantly higher purities compared to GCM. In particular,
+2-peptides were separated over several fractions, which was not possible
to achieve in GCM. The better separation in DCM resulted in a higher
reproducibility and significantly higher identification rates for
both peptides and proteins including a 2.6-fold increase for +2-peptides.
The higher number of identified peptides in DCM resulted in significantly
higher protein sequence coverages and a considerably higher number
of unique peptides per protein. Compared to conventionally used salt-based
GCM, DCM increased the performance of online SCX-RP-MS/MS and enabled
comprehensive proteome profiling in the low microgram range