Direct Identification of Intact Proteins Using a Low-Resolution Mass Spectrometer with CIDn/ETnoD.

Over the past decades, proteomics has become increasingly important and a heavily discussed topic. The identification of intact proteins remains a major focus in this field. While most intact proteins are analyzed using high-resolution mass spectrometry, identifying them through low-resolution mass spectrometry continues to pose challenges. In our study, we investigated the capability of identifying various intact proteins using collision-induced dissociation (CID) and electron transfer without dissociation (ETnoD). Using myoglobin as our test protein, stable product ions were generated with CID, and the identities of the product ions were identified with ETnoD. ETnoD uses a short activation time (AcT, 5 ms) to create sequential charge-reduced precursor ion (CRI). The charges of the fragments and their sequences were determined with corresponding CRI. The product ions can be selected for subsequent CID (termed CIDn) combined with ETnoD for further sequence identification and validation. We refer to this method as CIDn/ETnoD. The use of a multistage CID activation (CIDn) and ETnoD protocol has been applied to several intact proteins to obtain multiple sequence identifications.

Proteomic analysis of "Oriental Beauty" oolong tea leaves with different degrees of leafhopper infestation.

RATIONALE: Oriental Beauty, a type of oolong tea native to Taiwan, is highly prized by connoisseurs for its unique fruity aroma and sweet taste. Leaves of Oriental Beauty vary in appearance, aroma, and taste, depending on the degree of tea green leafhopper (Jacobiasca formosana) infestation. In this study, the aim is to investigate the differential expression of proteins in leaves with low, medium, and high degrees of leafhopper infestation. METHODS: Proteomic techniques 2DE (two-dimensional electrophoresis) and nanoscale liquid chromatography/tandem mass spectrometry (LC/MS/MS) were used to investigate the differential expression of proteins in tea leaves with different degrees of leafhopper infestation. RESULTS: A total of 89 proteins were found to exhibit significant differences in expression. In a gene ontology analysis, most of these proteins participated in biosynthesis, carbohydrate metabolism, transport, responses to stress, and amino acid metabolism. CONCLUSIONS: These results indicated that the unique aroma and taste of the leaves might be influenced by their protein expression profiles, as well as related factors such as defensive responses to tea green leafhopper saliva.

Rapid determination of isoflavones and other bioactive compounds in soybean using SWATH-MS.

Isoflavones are the major bioactive components in soybeans. Sequential window acquisition of all theoretical fragment ions (SWATH) is a kind of data-independent acquisition (DIA), such that all fragments of each precursor will be preserved in a SWATH-Mass Spectrometry (SWATH-MS) run. In this study, a high-throughput SWATH-MS method for the determination of 12 isoflavones in soybeans was established. Furthermore, amino acids, saponins can be semi-quantitated from the same SWATH-MS data. Combination of targeted quantification and untargeted profiling with SWATH, all bioactive compounds were analyzed within 5 min in 10 min run time, and the method had good linear regression with r2 > 0.99. The precisions (RSD %) of the intra-day and inter-day analyses ranged from 2.11% to 18.7%, and the accuracies (RE%) ranged from -14.39% to 17.48%. The matrix effect ranged from 88.66% to 114.82%. Moreover, 7 varieties of soybeans were analyzed and compared with this robust screening method.