Proteomic and peptidomic UHPLC-ESI MS/MS analysis of cocoa beans fermented using the Styrofoam-box method.

This work characterises the peptide and protein profiles of Theobroma cacao beans of the genotype IMC 67 at different fermentation stages, using the Styrofoam-box fermentation method and employing UHPLC-ESI MS/MS for the analysis of peptides and proteins extracted from the beans. A total of 1058 endogenous peptides were identified and quantified over four fermentation time points. The majority of these peptides were formed after 2 and 4 days of fermentation, and originated predominantly from the proteolysis of two storage proteins - vicilin and a 21 kDa albumin. The changes in the peptide profile over fermentation were subsequently evaluated, and potential markers for assessing the degree of fermentation were identified. In particular, changes of the relative abundance of the major cocoa proteins detected can be proposed as potential markers for the fermentation stage. Furthermore, PCA of both the peptidomic and proteomic data has allowed differentiation of beans at different fermentation stages.

UHPLC-MS/MS analysis of cocoa bean proteomes from four different genotypes.

In this study the proteomic profiles of cocoa beans from four genotypes with different flavour profiles were analysed by bottom-up label-free UHPLC-MS/MS. From a total of 430 identified proteins, 61 proteins were found significantly differentially expressed among the four cocoa genotypes analysed with a fold change of ≥2. PCA analysis allowed clear separation of the genotypes based on their proteomic profiles. Genotype-specific abundances were recorded for proteases involved in the degradation of storage proteins and release of flavour precursors. Different genotype-specific levels of other enzymes, which generate volatiles compounds that could potentially lead to flavour-inducing compounds, were also detected. Overall, this study shows that UHPLC-MS/MS data can differentiate cocoa bean varieties.

Characterization of the Proteome of Theobroma cacao Beans by Nano-UHPLC-ESI MS/MS.

Cocoa seed storage proteins play an important role in flavour development as aroma precursors are formed from their degradation during fermentation. Major proteins in the beans of Theobroma cacao are the storage proteins belonging to the vicilin and albumin classes. Although both these classes of proteins have been extensively characterized, there is still limited information on the expression and abundance of other proteins present in cocoa beans. This work is the first attempt to characterize the whole cocoa bean proteome by nano-UHPLC-ESI MS/MS analysis using tryptic digests of cocoa bean protein extracts. The results of this analysis show that >1000 proteins could be identified using a species-specific Theobroma cacao database. The majority of the identified proteins were involved with metabolism and energy. Additionally, a significant number of the identified proteins were linked to protein synthesis and processing. Several proteins were also involved with plant response to stress conditions and defence. Albumin and vicilin storage proteins showed the highest intensity values among all detected proteins, although only seven entries were identified as storage proteins. A comparison of MS/MS data searches carried out against larger non-specific databases confirmed that using a species-specific database can increase the number of identified proteins, and at the same time reduce the number of false positives. The results of this work will be useful in developing tools that can allow the comparison of the proteomic profile of cocoa beans from different genotypes and geographic origins. Data are available via ProteomeXchange with identifier PXD005586.

Determination of plant hormones in fertilizers by high-performance liquid chromatography with photodiode array detection: method development and single-laboratory validation.

A simple and reliable high-performance liquid chromatographic method that uses photodiode array detection was developed for the simultaneous determination of 12 native and synthetic plant hormones, i.e., plant growth regulators (PGRs), in fertilizers, such as 1-naphthol, 2,4-dichlorophenoxyacetic acid, 4-(2,4-dichlorophenoxy)butyric acid, 4-chlorophenoxyacetic acid, indole-3-acetic acid, 4-(3-indolyl)butyric acid, dichlorprop, (4-chloro-2-methylphenoxy)acetic acid, alpha-naphthaleneacetic acid, 1-naphthaleneacetamide, beta-naphthoxyacetic acid, and thidiazuron. The method was experimentally validated for routine regulatory application, and the following analytical parameters were assessed for all PGRs studied: linearity; specificity; precision (relative standard deviation) and accuracy, both measured at 3 concentration levels (0.1, 0.05, and 0.01%, w/w); ruggedness; limit of detection; and limit of quantification. Results were satisfactory for all method validation parameters tested and for all PGRs studied, demonstrating the suitability of the method for the determination of PGRs in fertilizers. The uncertainty of measurement was also estimated at 3 concentration levels for all PGRs by using the approach of the International Organization for Standardization, described in its Guide to the Expression of Uncertainty in Measurement. The method was applied to 20 samples of liquid fertilizer with declared biostimulant properties.