Metal-Oxide Enrichment and Gas-Phase Characterization of Sulfopeptides using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Though not as well studied as phosphopeptides, sulfopeptides are important for many biological processes, including proper endocrine function and extracellular signaling. The discovery of sulfopeptides dates back to the 1920s; however, their enrichment and characterization have only recently become of broader interest. With a limited toolbox for analyzing sulfopeptides, we employ several chemistries to develop robust enrichment and characterization methods. At the heart of each method lies Fourier transform ion cyclotron resonance mass spectrometry, a gas-phase detection method with the power to differentiate even the slightest mass differences, such as phosphate vs. sulfonate. First, Lewis acid-base characteristics inherent to transition metal oxides are examined for the selective interaction and enrichment of sulfopeptides in the presence of mixtures of competing poly-oxyanions. Careful control of the binding and elution pH with an optimized amount of sulfopeptide loaded onto the metal oxide surface can enhance enrichment selectivity of sulfopeptides up to 97% relative abundance compared to as low as 4% prior to enrichment. Second, the utility of gas-phase activation methods for structural characterization of sulfopeptides is investigated. The sulfonate post-translational modification (PTM) is extremely labile at low pH, high temperature, and during gaseous collisional activation. This fragility has challenged researchers to discover new techniques for analysis of intact sulfonated biomolecules. As recently as 2011, authors have accepted that the sulfonate modification is lost during mass spectrometric analysis. We have found that a combination of different activation techniques can elucidate sulfopeptide sequence while keeping the labile sulfonate residue intact, allowing for unambiguous localization of this PTM. In particular, negative ion electron capture dissociation was found to yield >50% fragmentation efficiency with complete sulfonate retention. Finally, ideas are explored for improving fragmentation efficiency in electron detachment dissociation, which commonly leads to extensive neutral loss from carboxylic acids, precluding efficient backbone fragmentation and subsequent structural elucidation. To block carbon dioxide loss, chemical derivatization and anion adduction were employed. We found that chloride adduction to acidic peptides improves the fragmentation efficiency and provides nearly complete sequence coverage for several peptides. In addition, N-acetylation was shown to alter observed fragmentation pathways, presumably through changes in peptide gas-phase structures.Ph.D.ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/91429/1/kehersbe_1.pd

Role of pelvic CT during surveillance of patients with resected biliary tract cancer

Abstract Background The aim of the study was to identify the frequency of isolated pelvic metastasis with the goal of determining the utility of pelvic CT as a surveillance strategy in patients with resected biliary tract cancer (BTC). Methods Study eligibility criteria included patients 18 years or older with BTC who underwent R0 or R1 surgical resection at University of Michigan between 2004 and 2018, with a minimum 6-month disease-free surveillance period. CT and MRI reports were independently graded by two radiologists as positive (organ metastasis, peritoneal carcinomatosis, or enlarged lymph nodes), equivocal (borderline lymph nodes or non-nodular ascites), or negative (absence of or benign findings) in the abdomen and pelvis separately. A 3rd blinded radiologist reviewed all positive and equivocal scans. Clinic notes were reviewed to identify new or worsening signs and symptoms that would warrant an earlier pelvic surveillance scan. A 95% binomial proportion confidence interval was used to find the probability of isolated pelvic metastasis. Results BTC were anatomically classified as extra-hepatic (distal and hilar) cholangiocarcinoma (38; 25%), intra-hepatic cholangiocarcinoma (57; 38%), and gallbladder cancer (56; 37%). 151 patients met eligibility criteria, of which 123 (81%) had no pelvic metastasis, 51 (34%) had localized upper abdominal metastasis, and 23 (15%) had concomitant abdominal and pelvic metastasis. Median follow-up time was 19.2 months. One (0%) subject with resected BTC (intra-hepatic) developed isolated osseous pelvic metastasis during surveillance (95% CI 0.004–0.1; p = 0.0003). 3 (2%) subjects developed isolated simple ascites (equivocal grade) without concurrent upper abdominal metastasis. Conclusion Isolated pelvic metastasis is a rare occurrence during surveillance in patients with resected BTCs, and therefore, follow-up pelvic CT in absence of specific symptoms may be unnecessary