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ImportanceUnderstanding Adverse Events (AEs) associated with SARS-CoV-2 vaccination has public health implications, especially with regards to vaccine hesitancy.ObjectiveTo establish whether individuals with prior history of COVID-19 were more likely to experience AEs after BNT162b2/Pfizer vaccination, than those without previous COVID-19, and whether COVID-19-vaccination interval influenced AE severity.DesignAn observational study explored AEs after vaccination. Participants were invited to complete an electronic survey, capturing self-reported COVID-19 symptoms, PCR/antibody results, and AEs following first dose of BNT162b2/Pfizer vaccine. In a subset where PCR/antibody results could be verified, a sensitivity analysis was conducted.SettingThree North-East England hospital Trusts in the United Kingdom.ParticipantsHealthcare workers formed an opportunistic sample – 265 of 974 reported prior positive SARS-CoV-2 PCR and/or antibody.ExposureAll participants had received their first dose of BNT162b2/Pfizer vaccine.Main Outcomes and MeasuresNature, severity, duration, and onset of self-reported AEs (reported via a modified version of the FDA Toxicity Grading Scale for vaccine-associated AEs), was compared between those with and without a prior history of COVID-19, using 2-way ANCOVA and logistic regression. Effects of age, gender, illness-vaccine interval, and ongoing symptoms (‘Long-COVID’) on AEs, were also explored.ResultsOf 974 respondents (81% female, mean age 48), 265 (27%) reported previous COVID-19 infection. Within this group (symptoms median 8.9 months pre-vaccination), 30 (11%) complained of Long-COVID. The proportion reporting one moderate/severe symptom was higher in the previous COVID-19 group (56% v 47%, OR=1.5 [95%CI, 1.1–2.0], p=.009), with fever, fatigue, myalgia-arthralgia and lymphadenopathy significantly more common. There was no significant relationship between illness-vaccine interval and symptom composite score (rs=0.09, p=.44). Long-COVID was not associated with worse AEs in comparison to the group without previous COVID-19. In the smaller sensitivity analysis cohort (412 people) similar findings were obtained although only myalgia and arthralgia remained significant.Conclusions and RelevancePrior COVID-19 infection but not ongoing Long-COVID symptoms were associated with an increase in the risk of self-reported adverse events following BNT162b2/Pfizer vaccination. COVID-19 illness-vaccination interval did not significantly influence AEs. This data can support education around vaccine-associated AEs and, through improved understanding, help to combat vaccine hesitancy.Key PointsQuestionDoes previous COVID-19 infection or ‘Long-COVID’ increase the frequency of Adverse Events (AEs) following first dose of BNT162b2/Pfizer vaccination?FindingsIn a survey-based observational study, healthcare workers in the United Kingdom reported AEs experienced after their first dose of BNT162b2/Pfizer vaccine. Prior COVID-19 infection, but not Long-COVID, were associated with increased risk of self-reported AEs including lymphadenopathy post-vaccination. Duration since COVID-19 infection did not affect severity of AEs.MeaningOur study can inform education and understanding of AEs associated with COVID-19 vaccination and help to combat vaccine hesitancy.

Apolipoproteins measured in plasma or serum are potential biomarkers for assessing metabolic irregularities that are associated with the development of cardiovascular disease (CVD). LC-MS/MS allows quantitative measurement of multiple apolipoproteins in the same sample run. However, the accuracy and precision of the LC-MS/MS measurement depends on the reproducibility of the enzymatic protein digestion step. With the application of an immobilized enzyme reactor (IMER), the reproducibility of the trypsin digestion can be controlled with high precision via flow rate, column volume and temperature. In this report, we demonstrate the application of an integrated IMER-LC-MS/MS platform for the simultaneous quantitative analysis of eight apolipoproteins. Using a dilution series of a characterized serum pool as calibrator, the method was validated by repeated analysis of pooled sera and individual serum samples with a wide range of lipid profiles, all showing intra-assay CV < 4.4% and inter-assay CV < 8%. In addition, the method was compared with traditional homogeneous digestion coupled LC-MS/MS for the quantification of apoA-I and apoB-100. Applied in large scale human population studies, this method can serve the translation of a wider panel of apolipoprotein biomarkers from research to clinical application. SIGNIFICANCE: Currently, the translation of apolipoprotein biomarkers to clinical application is impaired because of the high cost of large cohort studies using traditional single-analyte immunoassays. The application of on-line tryptic digestion coupled with LC-MS/MS analysis is an effective way to address this problem. In this work we demonstrate a high throughput, multiplexed, automated proteomics workflow for the simultaneous analysis of multiple proteins.

The number of caesarean sections at maternal request without medical indication is increasing. We aimed to explore the views of pregnant women, midwives and doctors using six hypothetical clinical scenarios and compare group views on: (a) perceived appropriateness of requests for caesarean section and (b) the reasons underlying these requests.A questionnaire was distributed to 166 pregnant women, 31 midwives and 52 doctors within maternity units at two hospitals in the North East region of England. Six hypothetical clinical scenarios for maternal requests were used: (1) uncomplicated first pregnancy, (2) one previous normal delivery, (3) one previous instrumental delivery, (4) one previous caesarean section, (5) one previous caesarean section with vaginal delivery since and (6) uncomplicated twin pregnancy. To highlight the differences in group responses, two main questions were asked for each scenario: 1. Should women be able to request a caesarean section? 2. What do you feel are the reasons for requesting a caesarean section? Data was analysed using Chi-squared or likelihood ratio as appropriate.In scenarios 1-3, professional groups were 'less likely' than pregnant women to always support a request (2.4% vs. 19.4%), (2.6% vs. 15.6%), (4.6% vs. 22%), (p0.001). No significant differences were shown between doctors and midwives except for scenario 6 (twins), where midwives more often felt maternal requests should be declined (26.1% vs. 1.9%) (p=0.001). Multiparous women (n=95) were more likely to agree 'sometimes' to maternal requests in scenarios 1, compared to nulliparous women (n=71) (21.1% vs. 4.2%) (p=0.04). 'Safety of the baby' was ranked highly with pregnant women in scenarios 1-3 (mean 24.4%, range [15.8-38%]) compared with healthcare professionals (7.6% [3.4-12.8%]). However in scenario 3, healthcare professionals attributed 'fear of injury to self' (29.6%) as the most likely reason compared to 14.6% of pregnant women.Healthcare professionals and pregnant women's views differ significantly. Multiparous patients' views differ from those who have not had children before. We should provide clearer information on risks and benefits which encompass areas that concern women most.

Mass spectrometry (MS) coupled with 1-D and 2-D electrophoresis can be utilized to detect and identify immunogenic proteins, but these methods are laborious and time-consuming. We describe an alternative, simple, rapid gel-free strategy to identify multiple immunogenic proteins from Bordetella pertussis (Bp). It couples immunoprecipitation to nano liquid chromatography- tandem mass spectrometry (IP-nLC-MS/MS) and is significantly both time- and labor-saving. We developed a gel-free magnetic bead-based immunoprecipitation (IP) method using different NP-40/PBS concentrations in which solubilized proteins of Bp Tohama I membrane fractions were precipitated with polyclonal rabbit anti-Bp whole cell immune sera. Immune complexes were analyzed by MS and Scaffold analysis (>95% protein identification probability). Total immunoproteins identified were 50, 63 and 49 for 0.90%, 0.45% and 0.22% NP-40/PBS buffer concentrations respectively. Known Bp proteins identified included pertactin, serotype 2 fimbrial subunit and filamentous hemagglutinin. As proof of concept that this gel-free protein immunoprecipitation method enabled the capture of multiple immunogenic proteins, IP samples were also analyzed by SDS-PAGE and immunoblotting. Bypassing gels and subjecting immunoprecipitated proteins directly to MS is a simple and rapid antigen identification method with relatively high throughput. IP-nLC-MS/MS provides a novel alternative approach for current methods used for the identification of immunogenic proteins.

In late February 2008, law enforcement officials in Las Vegas, Nevada, discovered in a hotel room, a copy of The Anarchist Cookbook, suspected castor beans and a "white powder" thought to be a preparation of ricin. Ricin is a deadly toxin from the seed of the castor bean plant (Ricinus communis). The United States regulates the possession, use, and transfer of ricin and it is the only substance considered a warfare agent in both the Chemical and the Biological Weapons Conventions. Six samples obtained from the hotel room were analyzed by laboratories at the Centers for Disease Control and Prevention using a panel of biological and mass spectrometric assays. The biological assays (real time-PCR, time resolved fluorescence and cytotoxicity) provided presumptive evidence of active ricin in each of the samples. This initial screen was followed by an in-depth analysis using a novel, state-of-the-art mass spectrometry-based ricin functional assay and high sensitivity tandem mass spectrometry for protein identification. Mass spectrometric analysis positively identified ricin and confirmed that in each of the samples it was enzymatically active. The tandem mass spectrometry analysis used here is the most selective method available to detect ricin toxin. In each sample, ricin was unequivocally identified along with other R. communis plant proteins, including the highly homologous protein RCA120. Although database searches using tandem mass spectra acquired from the samples indicated that additional controlled substances were not present in these samples, the mass spectrometric results did provide extensive detail about the sample contents. To the best of our knowledge following a review of the available literature, this report describes the most detailed analysis of a white powder for a public health or forensic investigation involving ricin.

s Toxins 2011 / Toxicon 68 (2013) 60–123 102 Use of multiple enzymes in sequential in-gel digestions improves sequence coverage of botulinum neurotoxins for confident subtyping by mass spectrometry D. Wang, J. Baudys, J. Rees, S.R. Kalb, J.R. Barr Division of Laboratory Science, National Center for Environmental Science, Centers for Disease Control and Prevention, Atlanta, GA, USA E-mail address: dov2@cdc.gov (D. Wang). Purpose of study: To develop a method for subtyping of botulinum neurotoxin (BoNT) by mass spectrometry with high sequence coverage. Methods used: Samples were separated by gel electrophoresis. The bands of interest were sliced, reduced, and alkylated followed by in-gel digestion with individual enzymes of various specificities. Sequential digestions were performed by re-digesting each of the gel bands with a select number of enzymes. The peptide mixtures were analyzed by LC-MS/MS on a FT-LTQ instrument. Summary of results: A SDS-PAGE purified protein, BoNT/A1, was initially examined with six commonly used enzymes. Four of these enzymes, including trypsin, chymotrypsin, Asp-N, and Lys-C resulted in substantial sequence coverage individually and yielded unique peptides that contributed to extended sequence coverage after a combination of the results. The data demonstrate that sequential digestion of up to four times on the same gel piece with different enzymes led to increased sequence coverage. A systematic study assessed the effect of different combinations of enzymes, sequential order and reaction conditions. As a result, 1285 of 1295 total amino acid residues of the BoNT/A1 protein were covered by a pool of detected peptides under one specific combination using a few micrograms of the protein. This nearcomplete sequence coverage should enable highly confident determination of known or unknown subtypes of BoNT. The strategy was applied to all serotypes of botulinum neurotoxins and significant improvements were observed in comparison with conventional single digestion methods. In addition, the primary sequence of a botulinum neurotoxin present in a carrot juice sample was almost completely determined (99% sequence coverage) and thus allowed the identification of its subtype and specific strain. Conclusions: Our study demonstrates in-gel digestion with the combination of different enzymes and various sequential orders improves the subtyping of botulinum neurotoxins by mass spectrometry. http://dx.doi.org/10.1016/j.toxicon.2012.07.118 Effects of botulinum-neurotoxin-complexing proteins on lymphatic cells L. Wang , Y. Sun , B.R. Singh a,b a BBTech Inc. Dartmouth, MA, USA Botulinum Research Center, University of Massachusetts Dartmouth

A multiple reaction monitoring liquid chromatography method with tandem mass spectrometric detection for quantitation of Staphylococcus aureus via phage amplification detection is described. This phage amplification detection method enables rapid and accurate quantitation of viable S. aureus by detecting an amplified capsid protein from a specific phage. A known amount of metabolically labeled 15N reference bacteriophage, utilized as the input phage and as the internal standard for quantitation, was spiked into S. aureus samples. Following a 2-h incubation, the sample was subjected to a 3-min rapid trypsin digest and analyzed by high-throughput liquid chromatography tandem mass spectrometric detection targeting peptides unique to both the 15N (input phage) and 14N (progeny phage) capsid proteins. Quantitation was achieved by comparing peak areas of target peptides from the metabolically labeled 15N bacteriophage peptide internal standard with that of the wild-type 14N peptides that were produced by phage amplification and subsequent digestion when the host bacteria was present. This approach is based on the fact that a labeled species differs from the unlabeled one in terms of its mass but exhibits almost identical chemical properties such as ion yields and retention times. A 6-point calibration curve for S. aureus concentration was constructed with standards ranging from 5.0 × 104 colony forming units (CFU) ml−1 to 2.0 × 106 CFU ml−1, with the 15N reference phage spiked at a concentration of 1.0 × 109 plaque forming units (PFU) ml−1. Amplification with 15N bacteriophage coupled with LC-MS/MS detection offers speed (3 h total analysis time), sensitivity (LOD: < 5.0 × 104 CFU ml−1), accuracy, and precision for quantitation of S. aureus.