Cancer Proteomics Core
Cancer Proteomics Core
The Cancer Proteomics Core (CPC) provides high sensitivity, high resolution, and high-throughput proteomics methods, with particular emphasis on clinical sample analysis and in-depth proteomic consultation. The Core utilizes blood, urine, cerebrospinal fluid, cell, and tissue extracts to gain insight into cancer processes, discover new biomarkers for early detection, diagnosis, prognosis, and outcome prediction, study population effects, manage clinical trials, evaluate drug efficacy and toxicity, stratify patients, identify tumor antigens, and accelerate drug development.
Learn about the CPC’s involvement in two projects: reproducibility studies of quantitative proteomics for identification of biomarkers for early detection of ovarian cancer, and protein interaction partners in signaling networks linked to tumor development.
Reproducibility studies of quantitative proteomics for identification of biomarkers for early detection of ovarian cancer.
Shelly Tworoger, PhD (HMS), Dimitrios Spentzos, MD (BIDMC) and Susan Hankinson, MPH, ScD (HSPH).
Protein profiling by SELDI-TOF and quantitative protein identification by MALDI-TOF/TOF are being used to identify early markers of ovarian cancer in plasma samples from the Nurses Health Study. Since ovarian cancer patients are most frequently diagnosed at later stages, when the disease is incurable, there is an urgent need to identify biomarkers in the blood that can predict the development of ovarian cancer early on.
The Nurses Health Study has collected plasma from more than 50,000 nurses and has been able to continue to follow-up collecting data. Since the study’s inception, more than 250 nurses participating in the study have developed ovarian cancer.
Most proteomics studies examine one blood specimen per participant; however, it is unknown how well measures at one point in time reflect an individual’s long-term proteome pattern. Therefore, in order to compare plasma samples of nurses that developed ovarian cancer to plasma from healthy nurses, the CPC examined the reproducibility of the proteome over three years in postmenopausal women not taking hormones. Methods included using label-free quantitative mass spectrometric analysis by surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF), as well as quantitative proteomics using the iTRAQ tagging reagents and MALDI-TOF/TOF mass spectrometry. A subset of 60 women who provided >2 blood samples over three years were randomly selected.
The CPC performed in depth quality control mass spectrometry experiments with blinded, control plasma samples from the study in order to address reproducibility of the mass spectrometry results, plasma sample quality with regard to storage times, storage temperatures, and over time reproducibility. Four different proteins chip surfaces were examined by SELDI-TOF: unfractionated plasma on a CM10 and H50 chip, pH≥9 plasma fraction on a CM10 chip, and the organic fraction on the H50 chip. In parallel, the same samples were depleted of the top 20 proteins, digested with trypsin, iTRAQ labeled, and analyzed by MALDI-TOF/TOF mass spectrometry. Participant and quality control (QC) samples were aligned to a reference sample and then peak intensity in these samples for all peaks identified in the reference sample assessed. The average coefficient of variation (CV) of the peak intensity within conditions was evaluated demonstrating a well acceptable CV for further studies using the Nurses Health Study’s plasma samples.
The results from this project suggest that protein peak reproducibility over three years was reasonable among postmenopausal women not taking hormones, such that one sample may reflect the proteome pattern over time. A manuscript has already been submitted and a second manuscript is being prepared. The CPC was involved in all aspects of this project, starting with the first consultations to discuss the project and deciding on study design to performing all proteomics experiments and together with the investigators, analyzing and interpreting the data.
Protein interaction partners in signaling networks linked to tumor development.
Brendan Manning, PhD (HSPH)
The Manning laboratory focuses on the detailed characterization of signaling networks underlying tumor development. Their studies require extensive identification of specific protein-binding partners and novel phosphorylation sites on components of these networks. Highly sensitive and precise mass spectrometry technologies are essential for these studies. Furthermore, as the major constituents and important regulatory phosphorylation events within these pathways are defined, a real need exists for technology allowing quantitative measurements of the stoichiometry of post-translational modifications under different physiological conditions.
To accomplish this, the CPC has developed both a qualitative and quantitative approach for studying differences in phosphorylation sites. Methods include using the LTQ ion trap mass spectrometer, and acquiring shotgun and targeted data. The Core also developed a label-free software application that allows the lab to quantify changes in phosphorylation levels from wild type and stimulated conditions. The CPC continues to work closely with Manning and colleagues to approach challenges and to develop new methods for quantitative analysis of protein expression and phosphorylation.
