Sulfoproteomics Workflow with Precursor Ion Accurate Mass Shift Analysis Reveals Novel Tyrosine Sulfoproteins in the Golgi.

Tyrosine sulfation in the Golgi of secreted and membrane proteins is an important post-translational modification (PTM). However, its labile nature has limited analysis by mass spectrometry (MS), a major reason why no sulfoproteome studies have been previously reported. Here, we show that a phosphoproteomics experimental workflow, which includes serial enrichment followed by high resolution, high mass accuracy MS, and tandem MS (MS/MS) analysis, enables sulfopeptide coenrichment and identification via accurate precursor ion mass shift open MSFragger database search. This approach, supported by manual validation, allows the confident identification of sulfotyrosine-containing peptides in the presence of high levels of phosphorylated peptides, thus enabling these two sterically and ionically similar isobaric PTMs to be distinguished and annotated in a single proteomic analysis. We applied this approach to isolated interphase and mitotic rat liver Golgi membranes and identified 67 tyrosine sulfopeptides, corresponding to 26 different proteins. This work discovered 23 new sulfoproteins with functions related to, for example, Ca2+-binding, glycan biosynthesis, and exocytosis. In addition, we report the first preliminary evidence for crosstalk between sulfation and phosphorylation in the Golgi, with implications for functional control.

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

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.

Quantitative Imaging Assessment for Clinical Trials in Oncology.

BACKGROUND: Objective radiographic assessment is crucial for accurately evaluating therapeutic efficacy and patient outcomes in oncology clinical trials. Imaging assessment workflow can be complex; can vary with institution; may burden medical oncologists, who are often inadequately trained in radiology and response criteria; and can lead to high interobserver variability and investigator bias. This article reviews the development of a tumor response assessment core (TRAC) at a comprehensive cancer center with the goal of providing standardized, objective, unbiased tumor imaging assessments, and highlights the web-based platform and overall workflow. In addition, quantitative response assessments by the medical oncologists, radiologist, and TRAC are compared in a retrospective cohort of patients to determine concordance. PATIENTS AND METHODS: The TRAC workflow includes an image analyst who pre-reviews scans before review with a board-certified radiologist and then manually uploads annotated data on the proprietary TRAC web portal. Patients previously enrolled in 10 lung cancer clinical trials between January 2005 and December 2015 were identified, and the prospectively collected quantitative response assessments by the medical oncologists were compared with retrospective analysis of the same dataset by a radiologist and TRAC. RESULTS: This study enlisted 49 consecutive patients (53% female) with a median age of 60 years (range, 29-78 years); 2 patients did not meet study criteria and were excluded. A linearly weighted kappa test for concordance for TRAC versus radiologist was substantial at 0.65 (95% CI, 0.46-0.85; standard error [SE], 0.10). The kappa value was moderate at 0.42 (95% CI, 0.20-0.64; SE, 0.11) for TRAC versus oncologists and only fair at 0.34 (95% CI, 0.12-0.55; SE, 0.11) for oncologists versus radiologist. CONCLUSIONS: Medical oncologists burdened with the task of tumor measurements in patients on clinical trials may introduce significant variability and investigator bias, with the potential to affect therapeutic response and clinical trial outcomes. Institutional imaging cores may help bridge the gap by providing unbiased and reproducible measurements and enable a leaner workflow.

Characterization of O-sulfopeptides by negative ion mode tandem mass spectrometry: superior performance of negative ion electron capture dissociation.

Positive ion mode collision-activated dissociation tandem mass spectrometry (CAD MS/MS) of O-sulfopeptides precludes determination of sulfonated sites due to facile proton-driven loss of the highly labile sulfonate groups. A previously proposed method for localizing peptide and protein O-sulfonation involves derivatization of nonsulfonated tyrosines followed by positive ion CAD MS/MS of the corresponding modified sulfopeptides for diagnostic sulfonate loss. This indirect method relies upon specific and complete derivatization of nonsulfonated tyrosines. Alternative MS/MS activation methods, including positive ion metastable atom-activated dissociation (MAD) and metal-assisted electron transfer dissociation (ETD) or electron capture dissociation (ECD) provide varying degrees of sulfonate retention. Sulfonate retention has also been reported following negative ion MAD and electron detachment dissociation (EDD), which also operates in negative ion mode in which sulfonate groups are less labile than in positive ion mode. However, an MS/MS activation technique that can effectively preserve sulfonate groups while providing extensive backbone fragmentation (translating to sequence information, including sulfonated sites) with little to no noninformative small molecule neutral loss has not previously been realized. Here, we report that negative ion CAD, EDD, and negative ETD (NETD) result in sulfonate retention mainly at higher charge states with varying degrees of fragmentation efficiency and sequence coverage. Similar to previous observations from CAD of sulfonated glycosaminoglycan anions, higher charge states translate to a higher probability of deprotonation at the sulfonate groups thus yielding charge-localized fragmentation without loss of the sulfonate groups. However, consequently, higher sulfonate retention comes at the price of lower sequence coverage in negative ion CAD. Fragmentation efficiency/sequence coverage averaged 19/6% and 33/20% in EDD and NETD, respectively, both of which are only applicable to multiply-charged anions. In contrast, the recently introduced negative ion ECD showed an average fragmentation efficiency of 69% and an average sequence coverage of 82% with complete sulfonate retention from singly- and doubly-deprotonated sulfopeptide anions.

Examining the proteome of Drosophila across organism lifespan.

A survey of the proteome of Drosophila melanogaster at nine time points across the adult lifespan based on several mass-spectrometry-based techniques is presented. In total, there is evidence for 5902 unique peptides corresponding to 1699 different proteins. Of hundreds of relatively abundant components, many appear to be highly dynamic as the adult fly ages. Of those proteins that we observe changing with age, a majority, associated with metabolism, reproduction, and development, are down-regulated. Other biological pathways such as defense response also show variable changes, where some proteins are down-regulated and others are up-regulated. The observed variations are compared with a report of genome-wide changes at the transcriptome level at different ages and the similarities and differences are presented.