Development of novel protein digestion and quantitation methods for mass spectrometic analysis

Proteins are the workhorses of biology, playing multifaceted roles in maintaining cellular function, signaling, and response to environmental cues. Understanding their abundance and dynamics is pivotal for unraveling the complexities of biological processes, which underpins the foundations of molecular and cellular biology. Accurate measurement of protein quantities provides insights into cellular homeostasis, facilitates the discovery of biomarkers, and sheds light on the molecular mechanisms of diseases, bridging the gap between the molecular intricacies of proteins and their functional consequences in health and disease. The evolution of protein quantitation methodologies, from classical colorimetric assays to sophisticated mass spectrometry-based approaches, has expanded the analytical precision and dynamic range, enabling the detection of proteins at low abundance levels. The development of targeted proteomic techniques has further refined the quantitation process, allowing researchers to investigate specific proteins or post-translational modifications with high sensitivity and accuracy. However, bottom-up targeted proteomic analysis and quantitation often requires overnight tryptic digestion to ensure complete protein cleavage into peptides, which may significantly hamper experimental efficiency and induce some chemical modifications during the long digestion process. In addition, isotope-labeled internal standards are needed for the absolute quantitation of the target proteins. Syntheses of those standards are time-consuming and costly, and some of standards might not be available or difficult to synthesize if the surrogate peptides contain post-translational modifications (PTMs). Therefore, developing new technologies to speed up the sample preparation process like digestion and standard-free quantitation strategies is essential and holds the promise of unlocking deeper insights into biology, driving innovation in diagnostics, and advancing the development of targeted therapies. Accordingly, the goal of this work is to develop novel methods for the ultrafast tryptic digestion of proteins and standard-free quantitation for peptides and proteins absolute quantitation without building the calibration curve. Three projects are included here with two novel technologies, i.e., ultrafast microdroplet digestion and coulometric mass spectrometry, showing the different applications. Firstly, the investigation of tryptic protein digestion in microdroplets and in bulk solution is conducted to comprehensively evaluate the microdroplet digestion versus bulk overnight digestion by examining digestion efficiency. Secondly, standard-free coulometric mass spectrometry (CMS) is applied for the absolute quantitation of tryptophan-containing peptides and amyloid beta-peptide fragments. Thirdly, coulometric mass spectrometry is further extended for the absolute quantitation of protein mixture sample, host cell proteins as well as deamidation modification. The successful application of those emerging technologies shows the potential for fast and cost-efficient quantitation of peptides and proteins in clinical and pharmaceutical settings

Investigations of barley powdery mildew effectors (CSEPs) in the non-host plant wheat and host plant barley

Blumeria graminis f. sp. hordei is a biotrophic obligate fungus that can survive and cause powdery mildew disease exclusively on living barley plants. Over 500 Candidate Secreted Effector Proteins (CSEPs), which are considered to be secreted by the fungal feeding structure haustoria, have been identified. Some of the CSEPs, such as CSEP0064 and CSEP0264, were found to contribute to the pathogen virulence. Wheat is resistant to Blumeria graminis f. sp. hordei-This is a form of non-host resistance. Recently, a Pseudomonas fluorescens based effector-to host analyser strain (EtHAn) was developed as a modified T3SS, which can deliver foreign effectors into plants. In this study, I used the EtHAn strain to deliver CSEP0064 and CSEP0264 into wheat, and screened for effector-triggered responses in a mapping population of wheat (WAGTAIL). Although the bacterium itself induced pattern-triggered immunity (PTI) in wheat, three cultivars (Rialto, Abbot and Madrigal) were capable of consistently recognizing and differentiating introduced CSEP0064 or CSEP0264, leading to an enhancement of EtHAn-triggered PTI. Subsequently, I tested whether the recognition of EtHAn-delivered effectors in these three lines can affect the susceptibility of wheat to subsequent fungal infection. The results showed that the recognition of introduced CSEPs triggered systemic resistance in Rialto and Madrigal, whereas induced systemic susceptibility in Abbot, to the adapted pathogen Blumeria graminis f. sp. tritici. Delivered CSEPs did not affect systemic wheat immunity to sebsequent infection of the non-adapted pathogen Blumeria graminis f. sp. hordei. These results suggest that either wheat host resistance and non-host resistance are controlled by different systemic signalling pathways or basal plant immunity is sufficient to restrict non-host pathogen invasion and additional up-regulation of systemic immunity does not provide any additional advantage. In parallel, the expression of CSEP0064 in host plant barley was also investigated. In this study, I observed that CSEP0064 is detectable by western blotting in heavily infected barley leaves and isolated haustoria. Unexpectedly, a protein with an apparent molecular mass around 200 kDa was recognized by the anti-CSEP0064 antibody on western blots. Here, I refer to this protein as ‘Big CSEP’. This ‘Big CSEP’ was detected in fungal structure-containing materials only. TFMS-mediated deglycosylation successfully removed this ‘Big CSEP’ signal, suggesting that the ‘Big CSEP’ may be a hyper/O-glycosylated CSEP0064, or a glycosylated complex containing CSEP0064. Large-scale shotgun proteomics have been performed on Blumeria graminis f. sp. hordei, leading to the identification of some haustoria-exclusive effectors including CSEP0064. Here, MRM-MS was used to detect and quantify CSEP0064 in different fractions of infected barley leaf. An enzymatic method was conducted to isolate haustoria from infected barley epidermis. GAPDH (Blumeria graminis f. sp. hordei) and GAPDH (H. vulgare) were used as reference for fungal and plant proteins. CSEP0064 was more abundant than GAPDH (Blumeria graminis f. sp. hordei) in isolated haustoria and the plant cytoplasm fractions. Moreover, CSEP0064 was four times more abundant than GAPDH (H. vulgare) in the plant cytoplasm fraction. Finally, the results demonstrate the secreation of CSEP0064 from haustoria and the uptake of CSEP0064 by the host cell.Open Acces