Cyclic Ion Mobility for Hydrogen/Deuterium Exchange-Mass Spectrometry Applications.

Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful tool to probe protein dynamics. As a bottom-up technique, HDX-MS provides information at peptide-level resolution, allowing structural localization of dynamic changes. Consequently, the HDX-MS data quality is largely determined by the number of peptides that are identified and monitored after deuteration. Integration of ion mobility (IM) into HDX-MS workflows has been shown to increase the data quality by providing an orthogonal mode of peptide ion separation in the gas phase. This is of critical importance for challenging targets such as integral membrane proteins (IMPs), which often suffer from low sequence coverage or redundancy in HDX-MS analyses. The increasing complexity of samples being investigated by HDX-MS, such as membrane mimetic reconstituted and in vivo IMPs, has generated need for instrumentation with greater resolving power. Recently, Giles et al. developed cyclic ion mobility (cIM), an IM device with racetrack geometry that enables scalable, multipass IM separations. Using one-pass and multipass cIM routines, we use the recently commercialized SELECT SERIES Cyclic IM spectrometer for HDX-MS analyses of four detergent solubilized IMP samples and report its enhanced performance. Furthermore, we develop a novel processing strategy capable of better handling multipass cIM data. Interestingly, use of one-pass and multipass cIM routines produced unique peptide populations, with their combined peptide output being 31 to 222% higher than previous generation SYNAPT G2-Si instrumentation. Thus, we propose a novel HDX-MS workflow with integrated cIM that has the potential to enable the analysis of more complex systems with greater accuracy and speed.

The protective role of postoperative radiation therapy in low and intermediate grade major salivary gland malignancies: A study of the Canadian Head and Neck Collaborative Research Initiative.

BACKGROUND: The objective of this study was to examine the utility of postoperative radiation for low and intermediate grade cancers of the parotid and submandibular glands. METHODS: The authors conducted a retrospective, Canadian-led, international, multi-institutional analysis of a patient cohort with low or intermediate grade salivary gland cancer of the parotid or submandibular gland who were treated from 2010 until 2020 with or without postoperative radiation therapy. A multivariable, marginal Cox proportional hazards regression analysis was performed to quantify the association between locoregional recurrence (LRR) and receipt of postoperative radiation therapy while accounting for patient-level factors and the clustering of patients by institution. RESULTS: In total, 621 patients across 14 tertiary care centers were included in the study; of these, 309 patients (49.8%) received postoperative radiation therapy. Tumor histologies included 182 (29.3%) acinic cell carcinomas, 312 (50.2%) mucoepidermoid carcinomas, and 137 (20.5%) other low or intermediate grade primary salivary gland carcinomas. Kaplan-Meier LRR-free survival at 10 years was 89.0% (95% confidence interval [CI], 84.9%-93.3%). In multivariable Cox regression analysis, postoperative radiation therapy was independently associated with a lower hazard of LRR (adjusted hazard ratio, 0.53; 95% CI, 0.29-0.97). The multivariable model estimated that the marginal probability of LRR within 10 years was 15.4% without radiation and 8.8% with radiation. The number needed to treat was 16 patients (95% CI, 14-18 patients). Radiation therapy had no benefit in patients who had early stage, low-grade salivary gland cancer without evidence of nodal disease and negative margins. CONCLUSIONS: Postoperative radiation therapy may reduce LLR in some low and intermediate grade salivary gland cancers with adverse features, but it had no benefit in patients who had early stage, low-grade salivary gland cancer with negative margins.

Ultrahigh-Throughput Sample Analysis Using Liquid Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

Mass spectrometry (MS) allows for automated analysis of complex samples at high resolution without the need for labeling/derivatization. Liquid atmospheric pressure matrix-assisted laser desorption/ionization (LAP-MALDI) enables rapid sample preparation and MS analysis using microtiter-plate formats and high-performing mass spectrometers. We present a step change in high-speed, large-scale MS sample analysis of peptides at 20 samples/s and an enzymatic assay at 40 samples/s, i.e., an order of magnitude faster than current MS platforms. LAP-MALDI requires only low amounts of sample volume (<2 µL), of which only a fraction (<1%) is typically consumed, and allows for multiplexing and high-speed MS/MS analysis, demonstrated at ∼10 samples/s. Its high ion signal stability and similarity to electrospray ionization enables CVs below 10% and the analysis of multiply charged peptide ions at these extreme speeds. LAP-MALDI MS fulfills the speed requirements for large-scale population diagnostics and compound screening with the potential of analyzing >1 million samples per day.

Psychosocial outcomes of human papillomavirus (HPV)- and non-HPV-related head and neck cancers: A longitudinal study.

OBJECTIVES: Human papillomavirus (HPV) has prompted a need to further investigate how this new biomarker changes the head and neck cancer (HNC) psychosocial landscape. This study aimed to: (a) characterize the sociodemographic, psychological, and social profiles of patients with HPV-positive versus -negative squamous cell carcinoma of the head and neck; and (b) identify how HPV status contributes to anxiety and depression (primary outcome), quality of life (QoL), and sexuality needs. METHODS: We conducted a prospective longitudinal study of 146 patients newly diagnosed with oral, oropharyngeal, nasopharyngeal, and hypopharyngeal cancer. Seventy-nine patients were HPV-positive and 67 HPV-negative. Patients completed self-administered psychometric measures upon HNC and 3-month follow-up, and Structured Clinical Interviews for DSM Diagnoses. RESULTS: Patients with HPV-negative tumors generally presented with higher anxiety and depression and lower QoL immediately post-HNC diagnosis (<2 weeks) compared to HPV-positive cancers. A Major Depressive Disorder (MDD) immediately post-HNC diagnosis negatively affected patients' anxiety and depression and QoL levels upon diagnosis only when the cancer was HPV-positive. Immediately posttreatment, HPV status was not associated with outcomes. A previous history of suicidal ideation, and upon cancer diagnosis cigarette smoking, anxiety and depression, and feeling close to one's partner were instead explanatory. CONCLUSION: While patients with HPV-positive HNC generally present with initially lower psychological distress, their vulnerability immediately posttreatment indicates an equal need for support. Head and neck clinics may need to better address MDD, anxiety and depression, a prior history of suicidal ideation, health behavior change, and quality of relationships.

Mapping Isomeric Peptides Derived from Biopharmaceuticals Using High-Resolution Ion Mobility Mass Spectrometry.

The identification and localization of isomeric peptide modifications is a critical requirement of the biopharmaceutical industry. Despite the ability of liquid chromatography-mass spectrometry to identify many of the common post translational modifications, the identification of isobaric or racemized peptides is confounded by modern mass spectrometry-based techniques. Here, we present a novel approach combining liquid chromatography with a high-resolution ion mobility mass spectrometry system to differentiate peptide and peptide fragments based upon their mobility and mass.

Longitudinal study indicating antecedent psychosocial vulnerability as predictor of anxiety disorders post-treatment in people with head and neck cancer.

OBJECTIVE: This study aimed to: (1) determine the contribution of pre-cancer psychosocial vulnerability as an independent predictor of anxiety disorder (AD) onset immediately post-treatment in patients diagnosed with a first occurrence of head and neck cancer (HNC), controlling for sociodemographics and medical variables; and (2) estimate prevalence of AD and identify trajectories from the moment of diagnosis to the immediate post-treatment (i.e., over a period of 3 months) in this population. METHODS: Two-hundred twenty-four consecutive patients (participation rate = 72%) newly diagnosed with a primary HNC were assessed with a structured clinical interview for a mental disorder, validated psychometric measures, and medical chart reviews. RESULTS: Twenty-five percent of patients presented a lifetime AD, 19.4% within 2 weeks of HNC diagnosis, and 16.6% immediately post-treatment; representing 26.7% of patients with AD at any timepoint from the moment of diagnosis to immediately post-treatment. Patients were more likely to present an AD immediately post-treatment when they: were diagnosed with advanced-stage cancer (OR = 3.40, p = 0.006), presented a upon cancer diagnosis AD (OR = 2.45, p = 0.008) and/or experienced childhood abuse (OR = 1.96, p = 0.03). CONCLUSIONS: Several AD trajectories may arise when patients are diagnosed with primary HNC. Health professionals should address AD and screen for risk factors (i.e., advanced stage cancer, AD upon cancer diagnosis, history of childhood abuse) as early as possible to assure optimal mental health care in this vulnerable population.

Coupling Droplet Microfluidics with Mass Spectrometry for Ultrahigh-Throughput Analysis of Complex Mixtures up to and above 30 Hz.

High- and ultrahigh-throughput label-free sample analysis is required by many applications, extending from environmental monitoring to drug discovery and industrial biotechnology. HTS methods predominantly are based on a targeted workflow, which can limit their scope. Mass spectrometry readily provides chemical identity and abundance for complex mixtures, and here, we use microdroplet generation microfluidics to supply picoliter aliquots for analysis at rates up to and including 33 Hz. This is demonstrated for small molecules, peptides, and proteins up to 66 kDa on three commercially available mass spectrometers from salty solutions to mimic cellular environments. Designs for chip-based interfaces that permit this coupling are presented, and the merits and challenges of these interfaces are discussed. On an Orbitrap platform droplet infusion rates of 6 Hz are used for analysis of cytochrome c, on a DTIMS Q-TOF similar rates were obtained, and on a TWIMS Q-TOF utilizing IM-MS software rates up to 33 Hz are demonstrated. The potential of this approach is demonstrated with proof of concept experiments on crude mixtures including egg white, unpurified recombinant protein, and a biotransformation supernatant.

Recommendations for reporting ion mobility Mass Spectrometry measurements.

Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values (K0 ) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E/N; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method-dependent results) only if the gas nature, temperature or E/N cannot match those of the primary method. Our analysis highlights the urgency of a community effort toward establishing primary standards and reference materials for ion mobility, and provides recommendations to do so. © 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc.

Treatment de-escalation for HPV-associated oropharyngeal squamous cell carcinoma with radiotherapy vs. trans-oral surgery (ORATOR2): study protocol for a randomized phase II trial.

BACKGROUND: Patients with human papillomavirus-positive (HPV+) oropharyngeal squamous cell carcinoma (OPC) have substantially better treatment response and overall survival (OS) than patients with HPV-negative disease. Treatment options for HPV+ OPC can involve either a primary radiotherapy (RT) approach (± concomitant chemotherapy) or a primary surgical approach (± adjuvant radiation) with transoral surgery (TOS). These two treatment paradigms have different spectrums of toxicity. The goals of this study are to assess the OS of two de-escalation approaches (primary radiotherapy and primary TOS) compared to historical control, and to compare survival, toxicity and quality of life (QOL) profiles between the two approaches. METHODS: This is a multicenter phase II study randomizing one hundred and forty patients with T1-2 N0-2 HPV+ OPC in a 1:1 ratio between de-escalated primary radiotherapy (60 Gy) ± concomitant chemotherapy and TOS ± de-escalated adjuvant radiotherapy (50-60 Gy based on risk factors). Patients will be stratified based on smoking status (< 10 vs. ≥ 10 pack-years). The primary endpoint is OS of each arm compared to historical control; we hypothesize that a 2-year OS of 85% or greater will be achieved. Secondary endpoints include progression free survival, QOL and toxicity. DISCUSSION: This study will provide an assessment of two de-escalation approaches to the treatment of HPV+ OPC on oncologic outcomes, QOL and toxicity. Results will inform the design of future definitive phase III trials. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT03210103. Date of registration: July 6, 2017, Current version: 1.3 on March 15, 2019.

Immediate post-treatment supportive care needs of patients newly diagnosed with head and neck cancer.

OBJECTIVE: This study aimed at identifying supportive care needs of patients with head and neck cancer (HNC-P) immediately post-treatment, finding early predictors of unmet needs, and contrasting how immediate post-treatment needs differed from needs in longer-term survivorship. METHODS: Prospective longitudinal study of 223 consecutive adults (313 approached; 72% participation) newly diagnosed with a first occurrence of primary HNC. Patients completed the Supportive Care Needs Survey-Short Form (SCNS), the Structured Clinical Interview for DSM-IV, and other outcomes. Medical chart reviews were conducted. RESULTS: A total of 68% of patients (n = 145/223) completed the SCNS. The multiple linear regression indicated that when controlled for medical variables, patients presented higher levels of unmet needs when they presented with higher level of anxiety upon HNC diagnosis (p = 0.03), higher neuroticism (p = 0.03), and more stressful life events in the year pre-diagnosis (p = 0.01). Patients immediately post-treatment had a wider variety of unmet needs compared with those in extended survivorship, with psychological unmet needs most prevalent at both time points. Immediately post-treatment, patients needed more support regarding pain (p = 0.04) and worries about treatment results (p = 0.05), whereas patients in longer-term survivorship needed more support regarding anxiety (p = 0.02), changes in sexual relationships (p = 0.04), and fear of death and dying (p = 0.001). CONCLUSION: This study identifies areas needing further development to improve quality of care for HNC-P in the immediate post-treatment period, as well as early determinants of unmet needs. HNC clinics may want to routinely screen for anxiety, neuroticism, and burden from other life events, to pro-actively address needs upon treatment completion and alleviate disease burden.