Rapid Screening of Ellagitannins in Natural Sources via Targeted Reporter Ion Triggered Tandem Mass Spectrometry

Complex biomolecules present in their natural sources have been difficult to analyze using traditional analytical approaches. Ultrahigh-performance liquid chromatography (UHPLC-MS/MS) methods have the potential to enhance the discovery of a less well characterized and challenging class of biomolecules in plants, the ellagitannins. We present an approach that allows for the screening of ellagitannins by employing higher energy collision dissociation (HCD) to generate reporter ions for classification and collision-induced dissociation (CID) to generate unique fragmentation spectra for isomeric variants of previously unreported species. Ellagitannin anions efficiently form three characteristic reporter ions after HCD fragmentation that allows for the classification of unknown precursors that we call targeted reporter ion triggering (TRT). We demonstrate how a tandem HCD-CID experiment might be used to screen natural sources using UHPLC-MS/MS by application of 22 method conditions from which an optimized data-dependent acquisition (DDA) emerged. The method was verified not to yield false-positive results in complex plant matrices. We were able to identify 154 non-isomeric ellagitannins from strawberry leaves, which is 17 times higher than previously reported in the same matrix. The systematic inclusion of CID spectra for isomers of each species classified as an ellagitannin has never been possible before the development of this approach

Gas phase analysis of biomolecular ions in a quadrupole ion trap: Protein analysis and instrumentation

Ion/ion reactions were performed in a quadrupole mass spectrometer in order to facilitate top down protein identification, charge reduction, and charge inversion. The first portion of the dissertation is focused on original instrumental development of an electrospray interface optimized to efficiently transmit ions generated from electrospray through the ring electrode. Additionally, emitter voltage manipulation schemes to enable more robust ion/ion reaction capacity from duel emitters on a single source were also developed. The second portion of the dissertation focuses on a series of proton transfer ion/ion reactions that are used for distinctly different applications. A top-down protein identification strategy for complex protein mixtures was derived from post ion/ion mass spectra formed from a series of ion-parking, collision-induced dissociation, and proton transfer reaction steps. Simple proton hydrate clusters were utilized to facilitate proton transfer reactions that successfully charge reduced intact Arixtra molecules by partitioning some portion of the ion/ion reaction exothermicity into the desolvation of the proton hydrates. Finally, a sequential ion/ion reaction scheme was used to generate radical [M-2H]-. protein anions that were charge inverted [M]+. cations via a proton transfer reaction

Metabolomics approach for predicting response to neoadjuvant chemotherapy for breast cancer

Breast cancer is a clinically heterogeneous disease, which necessitates a variety of treatments and leads to different outcomes. As an example, only some women will benefit from chemotherapy. Identifying patients who will respond to chemotherapy and thereby improve their long‐term survival has important implications to treatment protocols and outcomes, while identifying non responders may enable these patients to avail themselves of other investigational approaches or other potentially effective treatments. In this study, serum metabolite profiling was performed to identify potential biomarker candidates that can predict response to neoadjuvant chemotherapy for breast cancer. Metabolic profiles of serum from patients with complete (n = 8), partial (n = 14) and no response (n = 6) to chemotherapy were studied using a combination of nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography–mass spectrometry (LC–MS) and statistical analysis methods. The concentrations of four metabolites, three (threonine, isoleucine, glutamine) from NMR and one (linolenic acid) from LC–MS were significantly different when comparing response to chemotherapy. A prediction model developed by combining NMR and MS derived metabolites correctly identified 80% of the patients whose tumors did not show complete response to chemotherapy. These results show promise for larger studies that could result in more personalized treatment protocols for breast cancer patients., ► Metabolomics differentiates response to neoadjuvant breast cancer chemotherapy.► Four serum metabolites are found to correlate with response to chemotherapy.► A 4‐metabolite model identifies 80% of the patients not showing complete response.► Additional studies on larger patient cohorts are needed to validate the findings

Infused Grandinin MS-MS

The MS/MS files for the grandinin ellagitannin standard

Custom Perl Script

<div>The custom perl script used to extract precursors subjected to CID or HCD from mgf files at the end of the proteome discoverer workflow.</div><div><br></div

Infused Roburin D MS-MS

The MS/MS files for the roburin D ellagitannin standard

Infused Castalagin MS-MS

The MS/MS files for the castalagin ellagitannin standard

UHPLC Dilution of Standards

Systematic dilution of a mixture of ellagitannin standards to observe the elution profile and associated data points with fixed injection volumes

Infused Vescalagin MS-MS

The MS/MS files for the vescalagin ellagitannin standard

UHPLC False Positive Controls, Amaranth

Application of different charge state and reporter ion triggering conditions on amaranth control samples, ellagitannin standards, and a mixture of the ellagitannin standards and the amaranth control