Optimization of Peptide Separations by Differential Ion Mobility Spectrometry

Differential ion mobility spectrometry (DIMS) has the ability to separate gas phase ions based on their difference in ion mobility in low and high electric fields. DIMS can be used to separate mixtures of isobaric and isomeric species indistinguishable by mass spectrometry (MS). DIMS can also be used as a filter to improve the signal-to-background of analytes in complex samples. The resolving power of DIMS separations can be improved several ways, including increasing the dispersion field and increasing the amount of helium in the nitrogen carrier gas. It has been previously demonstrated that the addition of helium to the DIMS carrier gas provides improves separations when the dispersion field is the kept constant as helium content is varied. However, helium has a lower breakdown voltage than nitrogen. Therefore, as the percent helium content in the nitrogen carrier gas is increased, the highest dispersion field accessible decreases. This work presents the trade-offs between increasing dispersion fields and using helium in the carrier gas by comparing the separation of a mixture of isobaric peptides. The maximum resolution for a separation of a mixture of three peptides with the same nominal molar mass was achieved by using a high dispersion field (~72 kV/cm) with pure nitrogen as the carrier gas within the DIMS assembly. The conditions used to achieve the maximum resolution also exhibit the lowest ion transmission through the assembly, suggesting that it is necessary to consider the trade-off between sensitivity and resolution when optimizing DIMS conditions for a given application

Quantitation of ortho-cresyl phosphate adducts to butyrylcholinesterase in human serum by immunomagnetic-UHPLC-MS/MS

Tri-ortho-cresyl phosphate (ToCP) is an anti-wear, flame retardant additive used in industrial lubricants, hydraulic fluids and gasoline. The neurotoxic effects of ToCP arise fromthe liver-activatedmetabolite 2-(o-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (cresyl saligenin phosphate or CBDP),which inhibits esterase enzymes including butyrylcholinesterase (BChE). Following BChE adduction, CBDP undergoes hydrolysis to formthe aged adduct ortho-cresyl phosphoserine (oCP-BChE), thus providing a biomarker of CBDP exposure. Previous studies have identified ToCP in aircraft cabin and cockpit air, but assessing human exposure has been hampered by the lack of a laboratory assay to confirm exposure. This work presents the development of an immunomagnetic- UHPLC-MS/MSmethod for the quantitation of unadducted BChE and the long-term CBDP biomarker, oCP-BChE, in human serum. Themethod has a reportable range from 2.0ng/ml to 150 ng/ml,which is consistent with the sensitivity ofmethods used to detect organophosphorus nerve agent protein adducts. The assay demonstrated high intraday and interday accuracy (≥85%) and precision (RSD≤ 15%) across the calibration range. The method was developed for future analyses of potential human exposure to CBDP. Analysis of human serum inhibited in vitro with CBDP demonstrated that the oCP-BChE adduct was stable for at least 72h at 4, 22 and 37 °C. Compared to a previously reported assay, this method requires 75% less sample volume, reduces analysis time by a factor of 20 and demonstrates a threefold improvement in sensitivity

Association of acute toxic encephalopathy with litchi consumption in an outbreak in Muzaffarpur, India, 2014: a case-control study

Background Outbreaks of unexplained illness frequently remain under-investigated. In India, outbreaks of an acute neurological illness with high mortality among children occur annually in Muzaffarpur, the country’s largest litchi cultivation region. In 2014, we aimed to investigate the cause and risk factors for this illness. Methods In this hospital-based surveillance and nested age-matched case-control study, we did laboratory investigations to assess potential infectious and non-infectious causes of this acute neurological illness. Cases were children aged 15 years or younger who were admitted to two hospitals in Muzaffarpur with new-onset seizures or altered sensorium. Age-matched controls were residents of Muzaffarpur who were admitted to the same two hospitals for a non-neurologic illness within seven days of the date of admission of the case. Clinical specimens (blood, cerebrospinal fluid, and urine) and environmental specimens (litchis) were tested for evidence of infectious pathogens, pesticides, toxic metals, and other non-infectious causes, including presence of hypoglycin A or methylenecyclopropylglycine (MCPG), naturally-occurring fruit-based toxins that cause hypoglycaemia and metabolic derangement. Matched and unmatched (controlling for age) bivariate analyses were done and risk factors for illness were expressed as matched odds ratios and odds ratios (unmatched analyses). Findings Between May 26, and July 17, 2014, 390 patients meeting the case definition were admitted to the two referral hospitals in Muzaffarpur, of whom 122 (31%) died. On admission, 204 (62%) of 327 had blood glucose concentration of 70 mg/dL or less. 104 cases were compared with 104 age-matched hospital controls. Litchi consumption (matched odds ratio [mOR] 9·6 [95% CI 3·6 – 24]) and absence of an evening meal (2·2 [1·2–4·3]) in the 24 h preceding illness onset were associated with illness. The absence of an evening meal significantly modified the effect of eating litchis on illness (odds ratio [OR] 7·8 [95% CI 3·3–18·8], without evening meal; OR 3·6 [1·1–11·1] with an evening meal). Tests for infectious agents and pesticides were negative. Metabolites of hypoglycin A, MCPG, or both were detected in 48 [66%] of 73 urine specimens from case-patients and none from 15 controls; 72 (90%) of 80 case-patient specimens had abnormal plasma acylcarnitine profiles, consistent with severe disruption of fatty acid metabolism. In 36 litchi arils tested from Muzaffarpur, hypoglycin A concentrations ranged from 12·4 μg/g to 152·0 μg/g and MCPG ranged from 44·9 μg/g to 220·0 μg/g. Interpretation Our investigation suggests an outbreak of acute encephalopathy in Muzaffarpur associated with both hypoglycin A and MCPG toxicity. To prevent illness and reduce mortality in the region, we recommended minimising litchi consumption, ensuring receipt of an evening meal and implementing rapid glucose correction for suspected illness. A comprehensive investigative approach in Muzaffarpur led to timely public health recommendations, underscoring the importance of using systematic methods in other unexplained illness outbreaks

Oral abstracts 3: RA Treatment and outcomesO13. Validation of jadas in all subtypes of juvenile idiopathic arthritis in a clinical setting

Background: Juvenile Arthritis Disease Activity Score (JADAS) is a 4 variable composite disease activity (DA) score for JIA (including active 10, 27 or 71 joint count (AJC), physician global (PGA), parent/child global (PGE) and ESR). The validity of JADAS for all ILAR subtypes in the routine clinical setting is unknown. We investigated the construct validity of JADAS in the clinical setting in all subtypes of JIA through application to a prospective inception cohort of UK children presenting with new onset inflammatory arthritis. Methods: JADAS 10, 27 and 71 were determined for all children in the Childhood Arthritis Prospective Study (CAPS) with complete data available at baseline. Correlation of JADAS 10, 27 and 71 with single DA markers was determined for all subtypes. All correlations were calculated using Spearman's rank statistic. Results: 262/1238 visits had sufficient data for calculation of JADAS (1028 (83%) AJC, 744 (60%) PGA, 843 (68%) PGE and 459 (37%) ESR). Median age at disease onset was 6.0 years (IQR 2.6-10.4) and 64% were female. Correlation between JADAS 10, 27 and 71 approached 1 for all subtypes. Median JADAS 71 was 5.3 (IQR 2.2-10.1) with a significant difference between median JADAS scores between subtypes (p < 0.01). Correlation of JADAS 71 with each single marker of DA was moderate to high in the total cohort (see Table 1). Overall, correlation with AJC, PGA and PGE was moderate to high and correlation with ESR, limited JC, parental pain and CHAQ was low to moderate in the individual subtypes. Correlation coefficients in the extended oligoarticular, rheumatoid factor negative and enthesitis related subtypes were interpreted with caution in view of low numbers. Conclusions: This study adds to the body of evidence supporting the construct validity of JADAS. JADAS correlates with other measures of DA in all ILAR subtypes in the routine clinical setting. Given the high frequency of missing ESR data, it would be useful to assess the validity of JADAS without inclusion of the ESR. Disclosure statement: All authors have declared no conflicts of interest. Table 1Spearman's correlation between JADAS 71 and single markers DA by ILAR subtype ILAR Subtype Systemic onset JIA Persistent oligo JIA Extended oligo JIA Rheumatoid factor neg JIA Rheumatoid factor pos JIA Enthesitis related JIA Psoriatic JIA Undifferentiated JIA Unknown subtype Total cohort Number of children 23 111 12 57 7 9 19 7 17 262 AJC 0.54 0.67 0.53 0.75 0.53 0.34 0.59 0.81 0.37 0.59 PGA 0.63 0.69 0.25 0.73 0.14 0.05 0.50 0.83 0.56 0.64 PGE 0.51 0.68 0.83 0.61 0.41 0.69 0.71 0.9 0.48 0.61 ESR 0.28 0.31 0.35 0.4 0.6 0.85 0.43 0.7 0.5 0.53 Limited 71 JC 0.29 0.51 0.23 0.37 0.14 -0.12 0.4 0.81 0.45 0.41 Parental pain 0.23 0.62 0.03 0.57 0.41 0.69 0.7 0.79 0.42 0.53 Childhood health assessment questionnaire 0.25 0.57 -0.07 0.36 -0.47 0.84 0.37 0.8 0.66 0.4

Case Reports1. A Late Presentation of Loeys-Dietz Syndrome: Beware of TGFβ Receptor Mutations in Benign Joint Hypermobility

Background: Thoracic aortic aneurysms (TAA) and dissections are not uncommon causes of sudden death in young adults. Loeys-Dietz syndrome (LDS) is a rare, recently described, autosomal dominant, connective tissue disease characterized by aggressive arterial aneurysms, resulting from mutations in the transforming growth factor beta (TGFβ) receptor genes TGFBR1 and TGFBR2. Mean age at death is 26.1 years, most often due to aortic dissection. We report an unusually late presentation of LDS, diagnosed following elective surgery in a female with a long history of joint hypermobility. Methods: A 51-year-old Caucasian lady complained of chest pain and headache following a dural leak from spinal anaesthesia for an elective ankle arthroscopy. CT scan and echocardiography demonstrated a dilated aortic root and significant aortic regurgitation. MRA demonstrated aortic tortuosity, an infrarenal aortic aneurysm and aneurysms in the left renal and right internal mammary arteries. She underwent aortic root repair and aortic valve replacement. She had a background of long-standing joint pains secondary to hypermobility, easy bruising, unusual fracture susceptibility and mild bronchiectasis. She had one healthy child age 32, after which she suffered a uterine prolapse. Examination revealed mild Marfanoid features. Uvula, skin and ophthalmological examination was normal. Results: Fibrillin-1 testing for Marfan syndrome (MFS) was negative. Detection of a c.1270G > C (p.Gly424Arg) TGFBR2 mutation confirmed the diagnosis of LDS. Losartan was started for vascular protection. Conclusions: LDS is a severe inherited vasculopathy that usually presents in childhood. It is characterized by aortic root dilatation and ascending aneurysms. There is a higher risk of aortic dissection compared with MFS. Clinical features overlap with MFS and Ehlers Danlos syndrome Type IV, but differentiating dysmorphogenic features include ocular hypertelorism, bifid uvula and cleft palate. Echocardiography and MRA or CT scanning from head to pelvis is recommended to establish the extent of vascular involvement. Management involves early surgical intervention, including early valve-sparing aortic root replacement, genetic counselling and close monitoring in pregnancy. Despite being caused by loss of function mutations in either TGFβ receptor, paradoxical activation of TGFβ signalling is seen, suggesting that TGFβ antagonism may confer disease modifying effects similar to those observed in MFS. TGFβ antagonism can be achieved with angiotensin antagonists, such as Losartan, which is able to delay aortic aneurysm development in preclinical models and in patients with MFS. Our case emphasizes the importance of timely recognition of vasculopathy syndromes in patients with hypermobility and the need for early surgical intervention. It also highlights their heterogeneity and the potential for late presentation. Disclosures: The authors have declared no conflicts of interes

Differential Ion Mobility Spectrometry Coupled to Tandem Mass Spectrometry Enables Targeted Leukemia Antigen Detection

Differential ion mobility spectrometry (DIMS) can be used as a filter to remove undesired background ions from reaching the mass spectrometer. The ability to use DIMS as a filter for known analytes makes DIMS coupled to tandem mass spectrometry (DIMS–MS/MS) a promising technique for the detection of cancer antigens that can be predicted by computational algorithms. In experiments using DIMS–MS/MS that were performed without the use of high-performance liquid chromatography (HPLC), a predicted model antigen, GLR (FLSSANEHL), was detected at a concentration of 10 pM (20 amol) in a mixture containing 94 competing model peptide antigens, each at a concentration of 1 μM. Without DIMS filtering, the GLR peptide was undetectable in the mixture even at 100 nM. Again, without using HPLC, DIMS–MS/MS was used to detect 2 of 3 previously characterized antigens produced by the leukemia cell line U937.A2. Because of its sensitivity, a targeted DIMS–MS/MS methodology can likely be used to probe for predicted cancer antigens from cancer cell lines as well as human tumor samples

Quantitation of ortho-cresyl phosphate adducts to butyrylcholinesterase in human serum by immunomagnetic-UHPLC-MS/MS

Tri-ortho-cresyl phosphate (ToCP) is an anti-wear, flame retardant additive used in industrial lubricants, hydraulic fluids and gasoline. The neurotoxic effects of ToCP arise fromthe liver-activatedmetabolite 2-(o-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (cresyl saligenin phosphate or CBDP),which inhibits esterase enzymes including butyrylcholinesterase (BChE). Following BChE adduction, CBDP undergoes hydrolysis to formthe aged adduct ortho-cresyl phosphoserine (oCP-BChE), thus providing a biomarker of CBDP exposure. Previous studies have identified ToCP in aircraft cabin and cockpit air, but assessing human exposure has been hampered by the lack of a laboratory assay to confirm exposure. This work presents the development of an immunomagnetic- UHPLC-MS/MSmethod for the quantitation of unadducted BChE and the long-term CBDP biomarker, oCP-BChE, in human serum. Themethod has a reportable range from 2.0ng/ml to 150 ng/ml,which is consistent with the sensitivity ofmethods used to detect organophosphorus nerve agent protein adducts. The assay demonstrated high intraday and interday accuracy (≥85%) and precision (RSD≤ 15%) across the calibration range. The method was developed for future analyses of potential human exposure to CBDP. Analysis of human serum inhibited in vitro with CBDP demonstrated that the oCP-BChE adduct was stable for at least 72h at 4, 22 and 37 °C. Compared to a previously reported assay, this method requires 75% less sample volume, reduces analysis time by a factor of 20 and demonstrates a threefold improvement in sensitivity

Quantification of Metabolites for Assessing Human Exposure to Soapberry Toxins Hypoglycin A and Methylenecyclopropylglycine

Ingestion of soapberry fruit toxins hypoglycin A and methylenecyclopropylglycine has been linked to public health challenges worldwide. In 1976, over 100 years after Jamaican vomiting sickness (JVS) was first reported, the cause of JVS was linked to the ingestion of the toxin hypoglycin A produced by ackee fruit. A structural analogue of hypoglycin A, methylenecyclopropylglycine (MCPG), was implicated as the cause of an acute encephalitis syndrome (AES). Much of the evidence linking hypoglycin A and MCPG to these diseases has been largely circumstantial due to the lack of an analytical method for specific metabolites. This study presents an analytical approach to identify and quantify specific urine metabolites for exposure to hypoglycin A and MCPG. The metabolites are excreted in urine as glycine adducts methylenecyclopropylacetyl-glycine (MCPA-Gly) and methylenecyclopropylformyl-glycine (MCPF-Gly). These metabolites were processed by isotope dilution, separated by reverse-phase liquid chromatography, and monitored by electrospray ionization tandem mass spectrometry. The analytical response ratio was linearly proportional to the concentration of MCPF-Gly and MCPA-Gly in urine from 0.10 to 20 μg/mL with a correlation coefficient of <i>r</i> > 0.99. The assay demonstrated accuracy ≥80% and precision ≤20% RSD across the calibration range. This method has been applied to assess exposure to hypoglycin A and MCPG as part of a larger public health initiative and was used to provide the first reported identification of MCPF-Gly and MCPA-Gly in human urine

High-Confidence Qualitative Identification of Organophosphorus Nerve Agent Adducts to Human Butyrylcholinesterase

In this study, a data-dependent, high-resolution tandem mass spectrometry (ddHRMS/MS) method capable of detecting all organophosphorus nerve agent (OPNA) adducts to human butyrylcholinesterase (BChE) was developed. After an exposure event, immunoprecipitation from blood with a BChE-specific antibody and digestion with pepsin produces a nine amino acid peptide containing the OPNA adduct. Signature product ions of this peptic BChE nonapeptide (FGES*AGAAS) offer a route to broadly screen for OPNA exposure. Taking this approach on an HRMS instrument identifies biomarkers, including unknowns, with high mass accuracy. Using a set of pooled human sera exposed to OPNAs as quality control (QC) materials, the developed method successfully identified precursor ions with <1 ppm and tied them to signature product ions with <5 ppm deviation from their chemical formulas. This high mass accuracy data from precursor and product ions, collected over 23 independent immunoprecipitation preparations, established method operating limits. QC data and experiments with 14 synthetic reference peptides indicated that reliable qualitative identification of biomarkers was possible for analytes >15 ng/mL. The developed method was applied to a convenience set of 96 unexposed serum samples and a blinded set of 80 samples treated with OPNAs. OPNA biomarkers were not observed in convenience set samples and no false positive or negative identifications were observed in blinded samples. All biomarkers in the blinded serum set >15 ng/mL were correctly identified. For the first time, this study reports a ddHRMS/MS method capable of complementing existing quantitative methodologies and suitable for identifying exposure to unknown organophosphorus agents

Quantification of Toxins in Soapberry (Sapindaceae) Arils: Hypoglycin A and Methylenecyclopropylglycine

Methylenecyclopropylglycine (MCPG) and hypoglycin A (HGA) are naturally occurring amino acids found in some soapberry fruits. Fatalities have been reported worldwide as a result of HGA ingestion, and exposure to MCPG has been implicated recently in the Asian outbreaks of hypoglycemic encephalopathy. In response to an outbreak linked to soapberry ingestion, the authors developed the first method to simultaneously quantify MCPG and HGA in soapberry fruits from 1 to 10 000 ppm of both toxins in dried fruit aril. Further, this is the first report of HGA in litchi, longan, and mamoncillo arils. This method is presented to specifically address the laboratory needs of public-health investigators in the hypoglycemic encephalitis outbreaks linked to soapberry fruit ingestion