Katelyn R. Ludwig

Over 2300 Phosphorylated Peptide Identifications with Single-Shot Capillary Zone Electrophoresis-Tandem Mass Spectrometry in a 100 min Separation

Ultraperformance liquid chromatography (UPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) is typically employed for phosphoproteome analysis. Alternatively, capillary zone electrophoresis (CZE)-ESI-MS/MS has great potential for phosphoproteome analysis due to the significantly different migration times of phosphorylated and unphosphorylated forms of peptides. In this work, we systematically compared UPLC-MS/MS and CZE-MS/MS for phosphorylated peptide identifications (IDs) using an enriched phosphoproteome from the MCF-10A cell line. When the sample loading amount of UPLC was 10 times higher than that of CZE (2 μg vs 200 ng), UPLC generated more phosphorylated peptide IDs than CZE (3313 vs 1783). However, when the same sample loading amounts were used for CZE and UPLC (2-200 ng), CZE-MS/MS consistently and significantly outperformed UPLC-MS/MS in terms of phosphorylated peptide and total peptide IDs. This superior performance is most likely due to the higher peptide intensity generated by CZE-MS/MS. More importantly, compared with UPLC data from a 2 μg sample, CZE-MS/MS can identify over 500 unique phosphorylated peptides from a 200 ng sample, suggesting that CZE and UPLC are complementary for phosphorylated peptide IDs. With further improved loading capacity via a dynamic pH junction method, 2313 phosphorylated peptides were identified with single-shot CZE-MS/MS in a 100 min analysis. This number of phosphorylated peptide IDs is over 1 order of magnitude higher than the number of phosphorylated peptide IDs previously reported by single-shot CZE-MS/MS.

Glucose Restriction Combined with Autophagy Inhibition and Chemotherapy in HCT 116 Spheroids Decreases Cell Clonogenicity and Viability Regulated by Tumor Suppressor Genes

Drug resistance is a prevalent phenomenon that decreases the efficacy of cancer treatments and contributes to cancer progression and metastasis. Weakening drug-resistant cancer cells prior to chemotherapy is a potential strategy to combat chemoresistance. One approach to damage resistant cancer cells is modulation of nutritional intake. The combination of nutrient restriction with targeted compound treatment results in pronounced molecular changes. This study provides valuable information about augmenting existing chemotherapeutic regimes with simultaneous glucose restriction and autophagy inhibition in colorectal cancer cells. In this study, we explore the chemical pathways that drive the cellular response to nutrient restriction, autophagy inhibition, and the chemotherapy irinotecan using global quantitative proteomics and imaging mass spectrometry. We determined that significant pathways were altered including autophagy and metabolism via glycolysis, gluconeogenesis, and sucrose degradation. We also found that period circadian clock 2 (PER2), a tumor suppressor protein, was significantly up-regulated only when glucose was restricted with autophagy inhibition and chemotherapy. The upstream regulators of these differentially regulated pathways were determined to have implications in cancer, showing an increase in tumor suppressor proteins and a decrease in nuclear protein 1 (NUPR1) an important protein in chemoresistance. We also evaluated the phenotypic response of these cells and discovered autophagy inhibition and chemotherapy treatment increased apoptosis and decreased cell clonogenicity and viability. When glucose restriction was combined with autophagy inhibition and chemotherapy, all of the phenotypic results were intensified. In sum, our results indicate that glucose metabolism is of great importance in the ability of cancer cells to survive chemotherapy. By weakening cancer cells with glucose restriction and autophagy inhibition prior to chemotherapy, cancer cells become more sensitive to therapy.

iTRAQ Quantitative Proteomic Profiling and MALDI–MSI of Colon Cancer Spheroids Treated with Combination Chemotherapies in a 3D Printed Fluidic Device

For a patient with metastatic colorectal cancer there are limited clinical options aside from chemotherapy. Unfortunately, the development of new chemotherapeutics is a long and costly process. New methods are needed to identify promising drug candidates earlier in the drug development process. Most chemotherapies are administered to patients in combinations. Here, an in vitro platform is used to assess the penetration and metabolism of combination chemotherapies in three-dimensional colon cancer cell cultures, or spheroids. Colon carcinoma HCT 116 cells were cultured and grown into three-dimensional cell culture spheroids. These spheroids were then dosed with a common combination chemotherapy, FOLFIRI (folinic acid, 5-fluorouracil, and irinotecan) in a 3D printed fluidic device. This fluidic device allows for the dynamic treatment of spheroids across a semipermeable membrane. Following dosing, the spheroids were harvested for quantitative proteomic profiling to examine the effects of the combination chemotherapy on the colon cancer cells. Spheroids were also imaged to assess the spatial distribution of administered chemotherapeutics and metabolites with MALDI-imaging mass spectrometry. Following treatment, we observed penetration of folinic acid to the core of spheroids and metabolism of the drug in the outer proliferating region of the spheroid. Proteomic changes identified included an enrichment of several cancer-associated pathways. This innovative dosing device, along with the proteomic evaluation with iTRAQ-MS/MS, provides a robust platform that could have a transformative impact on the preclinical evaluation of drug candidates. This system is a high-throughput and cost-effective approach to examine novel drugs and drug combinations prior to animal testing.

Comparison of In-Solution, FASP, and S-Trap Based Digestion Methods for Bottom-Up Proteomic Studies

Bottom-up proteomic strategies rely on efficient digestion of proteins into peptides for mass spectrometry analysis. In-solution and filter-based strategies are commonly used for proteomic analysis. In recent years, filter-aided sample preparation (FASP) has become the dominant filter-based method due to its ability to remove SDS prior to mass spectrometry analysis. However, the time-consuming nature of FASP protocols have led to the development of new filter-based strategies. Suspension traps (S-Traps) were recently reported as an alternative to FASP and in-solution strategies as they allow for high concentrations of SDS in a fraction of the time of a typical FASP protocol. In this study, we compare the yields from in-solution, FASP, and S-Trap based digestions of proteins extracted in SDS and urea-based lysis buffers. We performed label-free quantification to analyze the differences in the portions of the proteome identified using each method. Overall, our results show that each digestion method had a high degree of reproducibility within the method type. However, S-Traps outperformed FASP and in-solution digestions by providing the most efficient digestion with the greatest number of unique protein identifications. This is the first work to provide a direct quantitative comparison of two filter-based digestion methods and a traditional in-solution approach to provide information regarding the most efficient proteomic preparation.

Evaluation of the mirn23a cluster through an iTRAQ-based quantitative proteomic approach

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that are implicated in a number of disease states. MiRNAs can exist as individual entities or may be clustered and transcribed as a single polycistron. The mirn23a cluster consists of three miRNAs: miR-23a, miR-24-2, and miR-27a. Although these miRNAs are transcribed together, they often exist at varying levels in the cell. Despite the fact that the mirn23a cluster is known to play a role in a number of diseases and developmental processes, few direct targets have been identified. In this study, we examined the effects of miR-23a, miR-24-2, miR-27a, or the mirn23a cluster overexpression on the proteome of 70Z/3 pre-B lymphoblast cells. Quantitative mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ) allowed for the global profiling of cell lines after miRNA overexpression. We identified a number of targets of each miRNA that contained predicted miRNA seed sequences and are likely direct targets. In addition, we discovered a cohort of shared miRNA targets and cluster targets, demonstrating the importance of studying miRNA clusters in their entirety.

Calcitriol Supplementation Causes Decreases in Tumorigenic Proteins and Different Proteomic and Metabolomic Signatures in Right versus Left-Sided Colon Cancer

Vitamin D deficiency is a common problem worldwide. In particular, it is an issue in the Northern Hemisphere where UVB radiation does not penetrate the atmosphere as readily. There is a correlation between vitamin D deficiency and colorectal cancer incidence and mortality. Furthermore, there is strong evidence that cancer of the ascending (right side) colon is different from cancer of the descending (left side) colon in terms of prognosis, tumor differentiation, and polyp type, as well as at the molecular level. Right-side tumors have elevated Wnt signaling and are more likely to relapse, whereas left-side tumors have reduced expression of tumor suppressor genes. This study seeks to understand both the proteomic and metabolomic changes resulting from treatment of the active metabolite of vitamin D, calcitriol, in right-sided and left-sided colon cancer. Our results show that left-sided colon cancer treated with calcitriol has a substantially greater number of changes in both the proteome and the metabolome than right-sided colon cancer. We found that calcitriol treatment in both right-sided and left-sided colon cancer causes a downregulation of ribosomal protein L37 and protein S100A10. Both of these proteins are heavily involved in tumorigenesis, suggesting a possible mechanism for the correlation between low vitamin D levels and colon cancer.

Combined Short-Term Glucose Starvation and Chemotherapy in 3D Colorectal Cancer Cell Culture Decreases 14-3-3 Family Protein Expression and Phenotypic Response to Therapy

AbstractShort-term glucose starvation prior to chemotherapy has the potential to preferentially weaken cancer cells, making them more likely to succumb to treatment, while protecting normal cells. In this study, we used 3D cell cultures of colorectal cancer and assessed the effects of short-term glucose starvation and chemotherapy compared to treatment of either individually. We evaluated both phenotypic changes and protein expression levels. Our findings indicate that the combined treatment results in more significant phenotypic responses, including decreased cell viability and clonogenicity. These phenotypic responses can be explained by the decreased expression of LDHA and 14-3-3 family proteins, which were found only in the combined treatment groups. This study indicates that short-term glucose starvation has the potential to increase the efficacy of current cancer treatment regimes. Graphical Abstractᅟ

Abstract IA04: Targeting the expression of EWS-FLI1

Post-translational modifications (PTMs) of transcription factors represent potential therapeutic targets for a variety of diseases, including cancer. In the majority of cases of the bone and soft tissue tumor Ewing sarcoma (ES), a chromosomal translocation, t(11:22), results in expression of the fusion transcription factor EWS-FLI1. Few PTMs of EWS-FLI1 have been identified. Using functional genetic methods and mass spectrometry analysis, we have identified a phosphorylated serine residue in the FLI1 domain of EWS-FLI1 that regulates the stability of the EWS-FLI1 oncoprotein. Loss of phosphorylation of this serine residue triggers ubiquitination and proteasomal degradation of EWS-FLI1, and apoptotic cell death. Xenograft studies suggest this post-translational modification of EWS-FLI1 can be targeted in vivo and that this inhibits ES tumor growth. Citation Format: Nirmalya Sen, Katelyn Ludwig, Guillermo O. Rangel-Rivera, Suntae Kim, Konrad Huppi, Lisa Jenkins, Jennifer E. Dwyer, Shelley Hoover, Lee Helman, Mark Simpson, Arnulfo Mendoza, Amanda B. Hummon, Natasha J. Caplen. Targeting the expression of EWS-FLI1 [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr IA04.