MSc Chemistry
Analytical Sciences
Literature Thesis
Potential of Orbitrap mass spectrometry for
application in the newborn screening program
by
Bram Grob
July 2015
Supervisor:
dr. H. Lingeman
Clinical Chemistry - Metabolic Unit
VU University Medical Center
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Table of content
Abbreviations ... 3
Abstract ... 4
Introduction ... 6
1. Newborn screening – history and introduction TQMS ... 6
2. Criteria for inclusion of a disease into the NBS program ... 7
3. Sampling, Results and second tier testing ... 9
4. Cost-effectiveness ...10
5. Some disadvantages of NBS ...11
6. Research aim ...11
Metabolomics, Orbitrap MS and applications ...13
1. Potential new techniques for NBS ...13
2. Orbitrap MS – theory and description of operation ...14
3. Orbitrap MS - characteristics of performance ...18
4. Orbitrap MS - applications ...21
Discussion ...26
Conclusions ...30
Acknowledgement ...31
3
Abbreviations
APCI Atmospheric pressure chemical ionization CSF Cerebrospinal fluid
CID Collision induced dissociation C8 Octanoylcarnitine
C10 Decanoylcarnitine DBS Dried blood spots
DIMS Direct infusion mass spectrometry ESI Electrospray ionization
FT-ICR Fourier transform ion cyclotron resonance FWHM Full width at half maximum
HCD Higher energy collisional dissociation
1H-NMR 1H-nuclear magnetic resonance
HR-MS High-resolution mass spectrometry LC Liquid chromatography
MCADD Medium-chain acyl-Coenzyme A dehydrogenase deficiency MRM Multiple reaction monitoring
MS Mass spectrometry M/z Mass-to-charge NBS Newborn screening PKU Phenylketonuria RF Radio frequency
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Abstract
The newborn screening (NBS) is a population examination program, testing neonates in the first weeks after birth on a selection of, mostly treatable, inherited disorders which are asymptomatic at this early stage. Direct treatment following early diagnosis prevents the development of severe symptoms, such as irreversible mental or motor retardation. The current targeted approach of the tandem quadrupole mass spectrometry- (TQMS) based method limits the number of simultaneously measurable primary and secondary targets. Furthermore, the measurement of metabolite masses at unit resolution without a liquid chromatography (LC) separation might lead to false elevation of signals by equal mass compounds. The aim of this literature study was to examine whether application of Orbitrap mass spectrometry (MS) could lead to improved analytical performance of the current NBS by reducing the numbers of false positives and negatives, and the simultaneous measurement of confirmative secondary targets.
Orbitrap MS is a relatively new technology, and able to perform untargeted measurements at high resolution and (mass) accuracy, which might solve the drawbacks of TQMS. For two NBS included diseases, it was shown that the Orbitrap MS and the TQMS approach showed similar capacities in primary biomarker based discrimination between the positive and negative group. Possible secondary biomarkers were discovered by the untargeted method applied with Orbitrap MS improving the differentiation between positives and negatives, which appears to be a major benefit of this technology. Furthermore, both techniques showed comparable characteristics in terms of precision, accuracy, specificity and the lower limit of quantification for drug analysis. There are some considerations however, this highly sensitive technique can also be influenced by matrix induced ion suppression during direct infusion, moreover, measurements at high resolution might increase the scan cycle times.
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The Orbitrap MS appears to perform comparable to TQMS in terms of discrimination between true positives and negatives by primary target screening. However, the ability of the simultaneous measurement of secondary targets by Orbitrap MS might lead to improved analytical performances of the current TQMS-based NBS.
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Introduction
1. Newborn screening – history and introduction TQMS
Newborn screening (NBS) is a population test performed in the first weeks after the birth, examining the existence of inherited disorders which are asymptomatic under normal conditions in the neonatal period. Most of the examined disorders are treatable, if diagnosed quickly after the birth. Early diagnosis improves the patients quality of life, moreover, it could reduce costs compared to clinical symptom based diagnoses. In the beginning of the 1960s, NBS was initiated by testing neonates for phenylketonuria (PKU). PKU is an inborn error of metabolism, discovered in 1934, with an incidence of more than 1 in 13,000 births in the USA.[1] Due to an impaired intra cellular enzyme phenylalanine hydroxylase, phenylalanine is accumulated at the cellular level and subsequently elevated expressed in blood. In patients suffering from PKU, phenylalanine levels in blood of more than 20-fold higher than normal controls can be observed.[1] Exposure to high levels of phenylalanine causes neurotoxic effects, resulting in mental retardation of which the development is initiated after the birth. This process can be overcome if patients are treated with a phenylalanine-free diet from this early stage.[1] Therefore, a method using dried blood spots (DBS) was setup to determine blood phenylalanine levels, based on its bacterial growth inhibition.[2] Population-based NBS was successfully initiated in the USA with this assay, and screening on PKU is nowadays included in screening programs of all developed countries. In the following decades, tests for other treatable disorders like maple syrup urine disease, galactosemia, homocystinuria, congenital hypothyroidism, congenital toxoplasmosis, hemoglobinopathies, congenital adrenal hyperplasia, biotinidase deficiency, medium-chain acyl-Coenzyme A dehydrogenase deficiency (MCADD) and cystic fibrosis were added to the NBS program.[3]
Until the 1990s the NBS program was based on several tests for the diagnosis of the included diseases. Tandem quadrupole mass spectrometry (TQMS) was already in use in clinical laboratories at this time for mass based measurement of drugs and metabolites.
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Therefore, TQMS was proposed for application in the NBS program since for many of the screened diseases metabolite quantities were measured and this technique allows for the measurement of different analytes simultaneously.[4] Prior to a TQMS analysis, it is required to ionize the analytes, which made TQMS still a rather laborious technique as cleaning of the first ion source types was needed after every sample. The invention of electrospray ionization (ESI) allowed for the injection of a liquid phase, containing the sample metabolites, directly in the TQMS without the need of cleaning the source. ESI-TQMS was suitable to perform rapidly (2-3 minutes for 1 sample) highly sensitive and selective analyses, with low reagent costs.[5] The introduction of this technique in NBS was a major step towards in the change from several tests, to determine many diseases, to ‘one test’.[6],[7] Even though separately, many of the screened disorders have a low incidence, as a group the total amount of affected newborns is considerable, for instance around 1 on 800 births in the West Midlands, UK.[8] The ability of determining several diseases in one run by TQMS was a reason for inclusion of additional disorders in screening programs, the so called expanded NBS. In 2010, nearly all developed countries perform expanded NBS, including up to ~40 TQMS detectable inherited metabolic diseases, depending on the particular screening program.[9]
2. Criteria for inclusion of a disease into the NBS program
In 1968, 5 years after the start of NBS in the USA, the World Health Organization published a report written by Wilson and Jungner with principal criteria for screening.[10] These criteria, known as the principal Wilson and Jungner criteria, are widely accepted as general guidelines in deciding whether a disease should be included in a population screening program. The first criteria describes that the progression of a disease should be known without diagnosis prior to symptom exposure, which is derived from the cost effectiveness of a screening program. For instance diseases which expose symptoms prior to irreversible damaging of organs are often not considered to be cost-effective to be included in a screening program. Furthermore, the second criterion described for the
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admittance to a screening program is the requirement of proven therapy for a disease, preventing irreversible damage. Since a screening program such as NBS delivers a large amount of samples, of which the major part has a negative outcome, the third criteria is the availability of a simple and inexpensive test. This test should be highly sensitive and specific. The high sensitivity is required to obtain a minimal amount of false negatives, the high specificity on the other hand, is requested to reduce the amount of false positives. The fourth criterion is the possibility to confirm or disprove measured positives after screening with a second tier test. Furthermore, medical care should be available for managing treatment and possibilities to perform tests for families when a genetic disease is observed. The last criterion described by Wilson and Jungner is of a different category, mentioning the need of acceptance of the screening program by the subjected population. This criterion is vital for including diseases in the NBS program, as negative acceptance could lead to reduced participation of the society, subsequently making NBS less effective.
Even though the Wilson and Jungner criteria are still used as the classical criteria, some countries started with inclusion of untreatable disorders after the start of expanded NBS. The Health Council of the Netherlands published a report in 2015 with revised recommendations for the minister of health, welfare and sports.[11] In this report different categories are described to verify whether a disease should be included in the NBS program. In these categories a difference is made between disorders with a substantial improved health perspective for the patient if detected by NBS, and disorders with less impact on health or less effective proven interventions. Diseases of this latter category could still be added to the program after an assessment of pros, cons and the efficiency of NBS determination of these diseases compared to usual diagnosis. In the Netherlands, untreatable disorders are excluded from the NBS program. For these disorders NBS could deliver positive effects such as shortening of the diagnostic route, decreasing the possibility of wrong diagnosis or treatment and adapting of the patient and family lives to the particular disease. However, the Dutch Health Council has the
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opinion that a minor part of the pros of screening untreatable diseases concerns the affected patient. Table 1 shows the current TQMS screened biomarkers for the diseases included in the NBS program of the Netherlands.[12]
Table 1: TQMS screened diseases and biomarkers of the Dutch NBS program, including abbreviations corresponding to the disease. [12] Def.: deficiency
Disease Abbreviation Biomarker
Carnitine transporter (OCTN2) def. OCTN2 Free carnitine (C0)
Glutaric aciduria type I GA-I Glutarylcarnitine (C5DC)
Isovaleric academia IVA Isovalerylcarnitine (C5)
C2/C5 ratio (C2: acetylcarnitine) Long-chain hydroxyacylCoA
dehydrogenase deficiency
LCHADD C16-hydroxyacylcarnitine
(C16OH)
Maple syrup urine disease MSUD Leucine, Valine
Medium-chain acylCoA dehydrogenase deficiency MCADD Octanoylcarnitine (C8) C8/C10 ratio (C10: decanoylcarnitine) 3-methylcrotonyl-CoA- carboxylase1 def. 3-hydroxy-3-methylglutaryl2-
CoA lyase def.
Multiple CoA carboxylase def.3
1 3-MCC
2 HMG
3 MCD
3-hydroxyisovalerylcarnitine (C5OH)
Phenylketonuria PKU Phenylalanine (PHE)
PHE/tyrosine ratio
Tyrosinemia type I TYR-I Succinylacetone
Very long chain acylCoA dehydrogenase deficiency
VLCADD Tetradecenoylcarnitine (C14:1)
C14:1/C2 ratio
3. Sampling, Results and second tier testing
In the Netherlands, collection of blood samples on filter paper for the NBS program is performed between 72 and 168 hours after the birth. All the spots on the card should be completely filled with blood collected from a heel prick. During collection the nurse should wear gloves and collect the blood without touching the skin. This to prevent
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contamination of the blood samples with for instance a lotion, which could affect the screenings results. After collection, the bloodspots should naturally dry before sending the card to the screening laboratory.[12]
For every screened disease, cut-off values are determined to discriminate between positives and negatives. In general, cut-off values are set to obtain a minimal number of false negatives.[7] Positive screens are usually repeated, and if this result is confirmed, a second tier test is performed in the same bloodspot, if such a test is available for this disorder. If the additional test also shows a positive result, a more sensitive and specific conformational test is required in a new sample to finally approve or disprove the positive screened disorder. A conformational test is often more laborious, however, the false positive rate is minimal or zero. Disproval of a positive screen is a false positive of the screened disorder. Although the primary objective of cut-off values is to obtain a minimal number of false negatives, a minimal number of false positives is also preferred.[13]
4. Cost-effectiveness
One of the most important objectives of NBS is the rapid diagnosis of diseases with clinically severe perspectives, the subsequent increased quality of life of the affected patients and the reduced parental stress. However, for population based screening programs, such as the NBS, the cost-effectiveness is another important aspect. Several parts of the process are generating costs, like for instance the required personnel, analytical and medical equipment, reagents, second tier tests, and treatment and medical follow-up of true positive screened patients. On the other hand, diagnosing patients suffering from metabolic diseases by their clinical symptoms, without NBS, can also be expensive. For instance diagnostic routes can be long and the chance of irreversibly damaged organs leading to a lifelong healthcare-dependency is present. In a study of Venditti et al. a cost-effectiveness study was performed on NBS for MCADD indicating that costs would remain underneath a certain accepted range.[14]
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5. Some disadvantages of NBS
Even though (expanded) NBS programs in different countries yields considerable advantages such as the prevention of many newborns from the effects of a selection inherited disorders by diagnosis at early stage, negative aspects have also been observed. One of these disadvantages is the time schedule of screening and the turnaround time for patients with diseases which can be fatal in the first days of life. For instance: a risk of sudden death is present in the first 48 hours after the birth of newborns with carnitine-acylcarnitine translocase deficiency.[15] Another drawback was reported of NBS programs including diseases without severe, or not completely disentangled clinical perspectives. Bleicher et al. described that screening on such diseases can be less beneficial for the screened patient compared to the advantages of NBS.[16] Furthermore, it is possible to obtain false negatives if the acquired results are not clearly representative for the disease. This occurred for instance with neonates suffering from tyrosinemia type I.[17] Contradictory, as described in paragraph 3, false positives might be encountered with NBS in addition to true diagnoses. False positive screens cause additional work for a metabolic laboratory due to the subsequent confirmative tests, moreover, it unnecessary disrupts family lives and cause parental stress. Nevertheless, the intensity of parental stress is showing a decrease for diseases picked up by NBS compared with (later) clinical diagnosis.[13]
6. Research aim
Current TQMS-based NBS programs are designed to obtain a minimal number of false negatives.[7] A major drawback of this approach is the considerable number of false positives, causing unnecessary parental stress[13], additional workload and healthcare costs. At present, TQMS measurements are obtained in unit resolution, which might lead to elevated signals by interfering compounds of a slightly different mass. Furthermore, high sensitivity and selectivity is obtained via a targeted approach during TQMS.
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Recently, other analytical techniques have been applied during metabolic studies, such as
1H-nuclear magnetic resonance (1H-NMR) spectroscopy and high-resolution mass
spectrometry (HR-MS).[18], [19] The Orbitrap mass spectrometer, a relatively new technique to perform HR-MS, is based on a different mass spectrometric mechanism compared to TQMS, which results in altered analytical characteristics.[20] Orbitrap MS allows for the performance of untargeted high accurate mass scans, and therefore may be a technique of interest for NBS.
The aim of this literature study is to investigate whether Orbitrap MS could improve the analytical performance of TQMS-based NBS in terms of reducing false positive and negative numbers by enhanced resolution and simultaneous determination of secondary targets.
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Metabolomics, Orbitrap MS and applications
1. Potential new techniques for NBS
In the last decennia several new omics-termed scientific fields appeared, one of these research topics is metabolomics. In contrast to metabolic investigation in which targeted metabolites are measured, metabolomics typically studies the entire representation of metabolites in an organism by identification and quantification. The obtained results are useful to examine fluxes of normal and disordered metabolism.[21] Due to next generation sequencing techniques, the amount of known metabolic disorders and less severe phenotype presentations is quickly increasing. By a metabolomics approach, it may be possible to verify the corresponding differences in metabolism. In the last years,
1H-NMR spectroscopy, and the relatively new HR-MS technique ion trap mass
spectrometry, has been applied to metabolomics studies, showing the analytical potency of these techniques for this field.[19], [22], [23]
1H-NMR is an analytical technique which can measure a major part of proton-containing
molecules, covering almost all metabolites. Measurements for metabolic investigation can be performed in all body fluids of interest, e.g. plasma, urine and cerebrospinal fluid (CSF).[22] This technique requires minimal sample preparation, it is non-destructive, and is applicable to metabolite identification. Furthermore, it is possible to acquire unbiased quantitative measurements, reaching typical limits of detection at the lower micro molar level.[24] This relatively high detection limit is a drawback of 1H-NMR since metabolites
at lower concentrations are excluded from the analysis, although these could be of interest. On the contrary, HR-MS applications are more sensitive, reaching limits of detection at the femtomole level.[25] HR-MS is compatible with all body fluids described for 1H-NMR, in general after some sample preparation steps. In general, mass
spectrometry (MS) is destructive for the sample, however, usually a minimal sample volume is required. TQMS can be applied for quantification of the metabolites and determination of their chemical structures.[26] Nevertheless, TQMS scans are obtained in
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unit resolution, therefore HR-MS such as ion trap technology is required if a more accurate approach of the exact mass is needed.[27] The mass range of metabolites is mainly <1000 Dalton, masses measured at unit resolution in this relatively low mass range are often corresponding with several compounds. Therefore, the application of unit resolution masses might lead to poor identification of a metabolite. Subsequently, it can influence the quantification of a certain metabolite if simultaneously several compounds with the same unit-mass are detected. However, by the application of a liquid chromatography (LC) separation and unique analyte specific mass transitions this influence is reduced, allowing for the performance of selective and sensitive TQMS measurements. Since highly accurate masses are obtained by the application of HR-MS such as ion trap MS, the corresponding number of chemical structures is considerable reduced.[28] Due to this mass accuracy, it is possible to perform highly selective and sensitive measurements, without the required preliminary separation or fragmentation necessary with TQMS. Since ion trap MS is currently an important analytical tool for the ‘omics’ research fields, it might also show potential for other research or diagnostically applications, like for instance the NBS.
Ion trap based MS techniques would have a major potential for application in NBS since the required sample volume is lower, detection levels are decreased and improved high-throughput compatibility compared to 1H-NMR. Orbitrap MS is a relative affordable ion
trap technique, therefore, this type of mass spectrometer could be a technique of interest for NBS.
2. Orbitrap MS – theory and description of operation
In the Orbitrap mass spectrometer, several equivalent principles of longer existing techniques are combined, such as the Kingdon trap, the Paul trap and the Fourier transform ion cyclotron resonance mass analyzer.[20] The Orbitrap mass spectrometer is commercially available since 2005. Nowadays, several Orbitrap variants have been
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developed. In this brief introduction for Orbitrap MS, the main elements of an Orbitrap mass spectrometer are described according to the schematic in figure 1.
Fig. 1: Schematic of an initial experimental Orbitrap mass spectrometer. On the left side, ionization of the analytes is performed. The ions are transported by several quadrupoles to the storage quadrupole, from this point the ions can be introduced via transfer lenses into the Orbitrap mass analyzer. (obtained from Hu et al. [20])
Two frequently applied, liquid chromatography compatible, ionization sources for Orbitrap analyses are atmospheric pressure chemical ionization (APCI) and ESI.[29], [30] The latter source is often used for metabolic investigations.[31], [32] After ionization, transport of ions with different mass-to-charge (m/z) ratios to the transport quadrupole is performed via a radio frequency- (RF) lens. The RF-lens and transport quadrupole transfers ions with different m/z ratios from atmospheric pressure into high vacuum.[20], [30] The transport quadrupole can operate as mass filter, by selecting a certain m/z ratio, or inversely a broad m/z range could be transported.[33] In the storage quadrupole, the ions velocity is reduced by collision with an inert gas and by the application of electrical fields. At this stage, ions are accumulated and focused towards the exit position, prior to the Orbitrap analysis. Subsequently, after pulsed opening of lens 1, a strong electrical field towards the Orbitrap mass analyzer is created. Spatial focused ion bundles are ejected in short periods (100-200 ns), after opening the lens to the Orbitrap mass analyzer. Acceleration of the ions is performed by a deflection lens
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system, followed by injection of the ions into the Orbitrap analyzer. During this movement ions with a higher m/z ratio reach the Orbitrap later compared with lower m/z ratios.[20], [30] The Orbitrap analyzer consists of an inner and outer electrode with oval shapes, as displayed in figure 2, creating an electrical field. The velocity of the entered ion bundles, in combination with the applied electrical field, causes the initiation of a trajectory around the inner electrode.[20] All injected ions show an equal amplitude during this movement. However, due to the injection at a point offset from the equator of both electrodes (z=0), an oscillation movement of the ions around this point over the z-axis can be initiated during administering a specific electrical field. The unique frequency of this axial oscillation is directly related with the m/z ratio of the ions. Circulating groups of a certain m/z ratio will form a thin ring shape, perpendicular to the z-axis, in which the ions are homogeneous spread over the collective orbit. The m/z dependent movement of these thin ion bands around z=0, is closely associated with a swung pendulum. Compared with radial frequency based ion trap techniques such as the Kingdon trap, the axial frequency in Orbitrap MS is not affected by chemical characteristics of the ions. Therefore, this frequency is applied for calculation of the m/z ratios, leading to highly accurate mass measurements and the high-resolution feature of this technique.[34] Recording and multiplying of these signals and frequencies is performed at the equatorial position, at which point the outer electrode is circular opened. The ion signals, measured in the outer electrode as a function of time, are Fourier-transformed into frequencies and subsequently converted to m/z ratios.[20]
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Fig. 2: Overview of the Orbitrap mass analyzer. Introduction of the ions occurs orthogonal to the z-axis at a point deviated from the center (z=0), producing an oscillating movement. Moreover, the ions velocity under influence of electrical fields produced by the inner- and outer electrode result in a unique orbit around the inner electrode. Finally, homogeneous spread equal m/z ratio ions circulate in a collective orbit perpendicular to the z-axis, oscillating around z=0 at an unique m/z dependent frequency. (obtained from Hu et al. [20])
In more recent Orbitrap mass spectrometers, additional or improved devices such as an octopole collision cell and a C-trap are often encased, as shown in figure 3. The C-trap, for instance, is an advanced iontrap, comparable with the described storage quadrupole. The performance of the Orbitrap after injection with a storage quadrupole can be restricted, since the capacity to trap high amounts of ions is limited for this device. Due to a different construction of the C-trap, the space-charge capacity has been improved. This leads to improved injection conditions, and subsequently, advanced Orbitrap performances for an increased number of ions.[35] Furthermore, an octopole collision cell, in figure 3 the higher energy collisional dissociation (HCD) cell, can be applied to obtain improved TQMS spectra. Collision induced dissociation (CID) is basically also possible in the C-trap device. However, several configurations can be applied differently in the octopole compared to the C-trap. For instance the pressure and type of collision gas is adjustable, without influencing the Orbitrap parameters. Furthermore, the octopole
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allows for the administration of a higher collision energy compared to for instance the C-trap.[36]
Fig. 3: Schematic of a Thermo Scientific Q Exactive Plus Orbitrap mass spectrometer, compared to preliminary Orbitrap mass spectrometers, this instrument included improved devices such as the ‘C-trap’ ion trap. Furthermore, a HCD cell is enclosed into this model. (obtained from
http://planetorbitrap.com, 2015 [37])
The Orbitrap is, compared to techniques such as TQMS, an affordable high sensitive mass spectrometer obtaining higher mass accuracy and resolution.[35] Therefore, this is an valuable technique for analyses of complex samples, for instance for the proteomics and metabolomics research fields.
3. Orbitrap MS - characteristics of performance
Sensitive mass based measurements of analytes in complex samples, such as bio fluids, demand highly sensitive and selective MS strategies.[28] During TQMS methods, this can be achieved by measuring unique mass transitions of precursor ions to an analyte-specific fragment after CID.[7] Although, technically TQMS allows for the performance of untargeted mass screening without fragmentation, the highly sensitive and selective characteristics of this technique are mainly based on the fragmentation strategy.
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In high-resolution techniques such as Fourier transform ion cyclotron resonance- (FT-ICR) and Orbitrap MS, high sensitivity and selectivity is also reached during untargeted analysis.[38] While FT-ICR technology is an expensive technique, Orbitrap MS has become more affordable. The Orbitrap mass spectrometer possesses improved characteristics such as mass accuracy and resolving power, compared to for instance TQMS. Mass accuracy for Orbitrap MS is described in parts per million (ppm), and is calculated by dividing the mass error (exact analyte mass minus the measured mass) by the exact mass, multiplied by 106.[39] A mass accuracy below 5 ppm can be reached by
Orbitrap mass analyzers, and even below 1 ppm under certain calibrated conditions.[40] Application of HR-MS, with a mass accuracy below 5 ppm, facilitates the opportunity of simultaneously quantification and qualification during a single measurement.[31] Furthermore, high accurate measurements reduce the amount of possible chemical structures which can influence the quantification. Another important parameter in MS is the (mass) resolution, which is the capacity of the instrument of acquiring separated signals of ions with a minimal mass difference.[33] The resolution is closely related with the data acquisition time, increasing with a longer acquisition time, and also depends on the height of the m/z ratio. In figure 4, an example is given of an insecticide, Pirimicarb, measurement at different resolution levels.[41] Orbitrap MS can reach a mass resolution up to 150,000.[20] Together, the high mass accuracy and resolution characteristics can lead to a reduction of false positive and negative altered signals during measurements.[42]
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Fig. 4: Example of a mass scan of Pirimicarb, an insecticide, obtained at a resolution of 15,000 and 80,000. In contrast to the low resolution measurement, a matrix component is separated from the analyte peak at high resolution. (obtained from Thermo Scientific [41])
Other parameters that show the performance of a mass spectrometer are the mass range and dynamic range. The dynamic range of a mass spectrometer is the ratio of the minimal and maximal detectable signal. Both the number of ions detectable by the detector and the digitization ability of certain signals influence the dynamic range value. Subsequently, the mass range covers the measurable m/z ratios with the mass analyzer, for Orbitrap MS with an upper limit approximately at 4000.[33] Hu et al. described a dynamic range of 102 – 104 during Orbitrap measurements of reserpine, a drug, obtained
with certain parameters as electrospray ionization at 150ºC, 3000V and 5 µL/min, and an ion collection period of 70 ms in a storage quadrupole.[20] In a publication of Marakov a detection level in de attomole range was described.[35] In figure 5, an overview is displayed of several mass spectrometric technique investment costs versus the achieved average resolutions. In this overview, Orbitrap MS is presented at the higher resolution level together with Magnetic Sector MS and FT-ICR at the high-end.
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Fig. 5: Comparison of several mass spectrometric technologies, based on the investment costs (American dollars) versus the resolution (FWHM). (obtained from http://www.chromacedemy.com, 2015 [43])
4. Orbitrap MS - applications
In the last years, Orbitrap MS has been used in several fields of interest such as environmental studies, drug metabolism, metabolite specific applications and especially for proteomics and metabolomics studies.[25], [31], [32], [44]–[47] In a study of Dénes et al., a direct (chip based) infusion Orbitrap MS method was developed for amino acid and acylcarnitine analysis in dried blood spots for the diagnosis and screening of inborn metabolic disorders.[25] This type of DBS analysis is worldwide applied during NBS programs by application of ESI-TQMS. The aim of this study was to investigate whether the application of HR-MS could improve the sensitivity and selectivity to reduce the number of false positives.
Direct infusion of 5 µL of filtered methanolic DBS extracts, containing stable isotope labeled standards at a certain concentration, was performed with a TriVersa NanoMate ion source. The ionization was switched from the positive to negative mode during data acquisition. Signals were determined at a resolution of 50,000 – 100,000 full width at half maximum (FWHM) and with a mass accuracy range of 1.0 ppm.
Measurements of DBS samples resulted in the detection of amino acids and acylcarnitines that are relevant for NBS. Other metabolite groups like for instance organic- and fatty acids and carbohydrates were also observed. Comparison of this new method with the
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conventional (ESI-TQMS) method was established by analyses of two well known NBS diseases, PKU and MCADD. In general, Orbitrap MS measured concentrations phenylalanine (marker PKU), octanoylcarnitine and decanoylcarnitine (resp. C8 and C10:1; markers MCADD) were lower compared to ESI-TQMS, as illustrated in figure 6. The lack of a derivatization step, which may hydrolyze proteins into amino acids, could explain the higher phenylalanine levels obtained with ESI-TQMS (butyl esterification required). Furthermore, metabolites with a similar molecular weight and fragmentation properties are possibly discriminated by the Orbitrap method. Although, obtained concentrations with the high-resolution method are possibly more accurate, the separation between the patient and normal concentration range is comparable.[25]
Fig. 6: Comparison of box charts for markers of PKU (a, b, e, f) and MCADD (c, d, g, h) markers in healthy and affected patients, obtained with TQMS (left) and Orbitrap MS (right). Markers: phenylalanine (a, e), phenylalanine/tyrosine ratio (b, f), octanoylcarnitine (C8; c, g), and decenoylcarnitine (C10:1; d, h) (obtained from Dénes et al. [25])
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An improvement of the Orbitrap- over the ESI-TQMS-method is the simultaneous measurement of additional metabolites showing abnormal levels of true patients. For instance, patients suffering from PKU also show elevated levels of phenylpyruvic acid and phenyllactic acid, even the separation between the healthy and disordered group seems to be larger with these metabolites in comparison to phenylalanine. Orbitrap MS could also be beneficial for the diagnosis of MCADD since the corresponding confirmatory test is the measurement of urinary organic acids.[25] In contrast with ESI-TQMS, these metabolites are also detectable in DBS samples with negative mode Orbitrap MS, shown in figure 7 for octenedioic acid and hexanoylglycine measured in the MCADD and healthy group.
Fig. 7: Box charts for proposed (secondary) markers for PKU (a, b) and MCADD (c, d), obtained with Orbitrap MS in healthy and affected patients. Markers: phenylpyruvic acid (a), phenyllactic acid (b), octenedioic acid (c), and hexanoylglycine (d) (obtained from Dénes et al. [25])
In a study of Henry et al., LC-coupled to Orbitrap MS was compared to TQMS for the quantitative analysis of drugs in plasma samples.[38] The examined drugs were divided in 3 groups, requiring different sample preparation and separation procedures. Detection with TQMS was performed in the SRM mode. Orbitrap MS was performed in a
‘high-24
resolution full scan’ and ‘all-ion fragmentation’ mode with a m/z range of 100 to 2000 and a mass window of 5 ppm and a resolution of 50,000 FWHM (m/z 200). For both techniques, ESI was applied in the positive ionization mode. Obtained analytical characteristics such as precision, accuracy, specificity, equations of lower limit of quantification calibration curves were relatively similar for both MS approaches. An advantage of Orbitrap MS was the possibility to perform simultaneous quantitative, semi-quantitative and qualitative measurements. Furthermore, the author described Orbitrap MS as an attractive instrument for quantitative measurements in clinical laboratories, since it is exchangeable with TQMS without required alteration of sample preparation or chromatographic method.[38]
Besides LC coupled to Orbitrap MS, also direct infusion mass spectrometry (DIMS) methods have been applied with this MS technique. Although DIMS is fast and relatively easy due to the lack of a separation method, also some disadvantages have been published.[31] Since no separation is applied, one of the drawbacks of such approaches is ion suppression induced by matrix components, reducing the sensitivity and precision of a method. The effect of matrix on the analyte signal was for instance examined during a study of Madalinski et al. Measurements of a selection 14N-labeled metabolites spiked in
yeast cell extracts were performed at different cell concentrations.[31] In table 2 the percentages are shown relative to metabolite signals measured in solvent (water/methanol, 50/50, containing 0.1% formic acid). These results show that sample matrix can substantial affect the intensity of the analyte signals, however the exact origin of suppression was not investigated in this study. Furthermore, non separated samples may cause formation of product, isotopic and adduct ions during the ionization-evaporation process. For instance in the study of Madalinski et al. it was shown that differentiating between γ-glutamyl-cysteine and cysteine in extracts of yeast cells is not possible with DIMS, since fragmentation of the former into cysteine occurs during the
25
ionization process. Although, fragmentation in the source can also take place in LC-MS methods, separation of these metabolites allows for the selective detection of cysteine.
Table 2: Results of a matrix effect determination for several metabolites at increasing concentrations of yeast extracts. (obtained from Madalinski et al. [31])
In order to reach high resolution (100,000) during Orbitrap MS, a large scan period of 1 s is required.[28] This scan time is relatively high for coupling a separation method, therefore applied resolutions during LC-Orbitrap MS are frequently around 30,000 in these methods.[48]
Even though TQMS allows for sensitive and selective measurements of many compounds simultaneous[49], this method is only focused on targets of its multiple reaction monitoring (MRM) program. Analytes which are not included, are not visible in this mode, and addition of other interesting compounds for quantitative objectives can be time-consuming due to method development or required validation. Moreover, additional mass transitions cause a reduction in sensitivity of the method since cumulative dwell times for individual compounds will decrease. An increased number of analytes in the MRM method can also lead to high scan cycle times, and subsequent, a reduced number of measuring points during acquiring data. Application of Orbitrap MS might therefore be an improvement for quantitative methods addressing to the measurement of additional analytes.[28]
26
Discussion
During NBS neonates are tested for a selection of, mostly treatable, disorders, which display no symptoms in this period of live. Determination and treatment of these diseases at early stage prevents the development of severe symptoms, and thereby increases the patients quality of life.[1] Current TQMS-based NBS causes a relatively high number of false positives, causing i.a. unnecessary parental stress. Additional second tier tests are required to confirm or disprove a positive screened result.[7] For some diseases a primary biomarker-based false positive result can be disproved by the measurement of secondary biomarker concentrations. The analytical performance of TQMS to measure many different types of primary and secondary biomarkers simultaneous is limited, since high sensitivity and selectivity is achieved via a targeted approach. HR-MS, such as the relatively new Orbitrap technology, can perform untargeted high accurate (mass) measurements[20], allowing for the determination of many primary and secondary biomarkers simultaneous. Furthermore, the high mass accuracy obtained with this technique might lead to increased selectivity towards biomarker measurements, compared to the TQMS-based method.
Orbitrap MS can detect accurate masses up to 5 digits behind the comma instead of masses at unit resolution obtained with TQMS. In biological material, such as blood, urine and CSF, hundreds of different metabolites are present, in which some metabolites have a minimal mass difference. Consequently, if these metabolites are measured in unit resolution following direct infusion, the examined metabolite signal might be falsely elevated due to the simultaneous measurement of metabolites with nearly equal masses.[25] During TQMS-based NBS, an incorrect elevated signal can lead to the determination of false positives or negatives. Therefore, the research question of this study was to theoretically investigate whether Orbitrap technology could improve the current TQMS-based NBS. Based on the currently applied MRM-mode TQMS method during NBS, false elevation of a signal should be only possible with equal mass compounds showing a similar mass transition after CID.
27
As shown in the previous chapter, Dénes et al. obtained a reduction of concentrations with Orbitrap MS compared to TQMS results, for PKU and MCADD biomarkers in DBS of healthy and affected patients.[25] Separation between these groups was approximately similar to the TQMS results. However, the measurement of secondary biomarkers showed an improved separation between the healthy and patient group. In order to maintain the sensitivity and low cycle times, currently targeted TQMS in the MRM-mode is restricted to a certain number of mass transitions. The untargeted approach of the Orbitrap technique is therefore a major improvement and allows the simultaneous measurement of primary and secondary biomarkers. Furthermore, in contrast to TQMS, metabolites like acylglycines, fatty- and dicarboxylic acids can be measured in DBS with Orbitrap MS.[25] At present, the measurement of these metabolites is performed in urine by a gas chromatography coupled to MS analysis. The difference in results obtained with TQMS and Orbitrap MS during this study might not be exclusively caused by switching to another MS technique. Additional parameters were altered, omitting of derivatization for the Orbitrap analysis and the measurement of intact ionized molecules instead of mass transitions after CID. Therefore additional studies, determining the influence of CID and derivatization on Orbitrap measurements could clarify the currently reduced concentrations compared to TQMS and the similar separation between the healthy and patient group. Even though, results obtained by Orbitrap analysis of PKU and MCADD biomarkers show similar discrimination between the positive and negative group compared to TQMS, inclusion of other NBS disorders in a future study might show improved differentiation of these groups.
Another advantage of Orbitrap MS, compared to the TQMS based NBS method, is the possibility to perform high selective measurements without the need of fragmentation of the analyte, as shown in the study of Dénes et al.[25] During the current TQMS method, a derivatization step is included to obtain improved fragmentation properties for amino acids and acylcarnitines. A reduction in sample preparation steps, by the absence of derivatization, might lead to an improved representation of the real sample composition.
28
In the study of Henry et al., comparing Orbitrap MS with a TQMS application, similar results were shown in terms of accuracy, precision, sensitivity and linearity.[38] This study also describe the major advantage of Orbitrap MS of measuring additional analytes simultaneous to the targets during full scan measurements. For some diseases, the ratio between a primary and secondary target, e.g. octanoylcarnitine (C8) and decanoylcarnitine (C10) for MCADD, is an additional evidence for diagnosis. The measurement of biomarkers in DBS compared to e.g. plasma samples can be less reliable due to for instance stability issues or incorrect filling of the spots, which might influence a primary biomarker value. Obtained ratios are sometimes less affected compared to the primary biomarker value. Therefore for certain diseases, obtaining of ratios by secondary target measurement could possibly improve the diagnostic accuracy of the current method, especially if primary target concentrations are affected.
Obtaining additional analytes by TQMS applied in the MRM mode is achieved by extra mass transitions, affecting the methods analytical performances for instance by a decreased number of data points if the cycle time increases. In contrast, Orbitrap MS in full scan mode requires no alteration of the MS method. Although, Orbitrap MS can perform high-resolution measurements, this also leads to elevated cycle times. In the study of Madalinski et al., mass spectra of direct infused biological samples were obtained at a resolution of 100,000 at m/z 400.[31] Acquired mass spectra were the averages of 4 scans. In order to obtain an increased number of data points, measuring at a lower resolution (50,000 – 100,000) as performed for DBS analysis in the study of Dénes et al. might be advisable for a future application in NBS.[25]
Since the possibilities of injecting liquid phases into an Orbitrap mass spectrometer are comparable to TQMS, this technique could relatively replace the current NBS. Furthermore, in contrast to for instance FT-ICR HRMS, Orbitrap mass spectrometers are becoming increasingly affordable.[43] The opportunity to obtain secondary targets, and the subsequent reduction of required second tier tests, might lead to a reduction of costs additional to the basic screening method. This in combination with the advantage of
29
measuring additional compounds in the full scan mode without concessions addressing the analytical performance might show the added value of a future application of Orbitrap MS during NBS, replacing TQMS.
Prior to a potential future implementation of Orbitrap MS, replacing current TQMS, additional studies would be required. All screened disorders should be examined, addressing the determination of positive and negative cut-off values of primary and secondary targets, and the correlation with the presently applied methods. The diagnostically value of new secondary biomarkers, like phenylpyruvic acid and phenyllactic acid for PKU, need to be validated prior to implementation. Furthermore, several parameters should be determined according to a validation procedure for new analytical methods, such as the linearity, sensitivity, selectivity, accuracy, precision, repeatability and reproducibility.
Since Orbitrap MS also can suffer from matrix induced ionization suppression, which affect the analytical performance, it might be valuable to study the influence of this effect. During studying the ion suppression, the performance of other ionization interface types could be examined, such as the nanospray ionization applied during the study of Dénes et al.[25]
30
Conclusions
The application of an untargeted Orbitrap HR-MS approach for NBS in DBS could improve current TQMS based method by the simultaneously measurement of secondary targets.[25], [38] This could lead to a reduced amount of required second tier tests to confirm or disprove abnormal values. In terms of primary target-based separation between the true positive and negative groups, both techniques currently seem to perform comparable.[25]
Although, the simultaneous measurement of secondary targets by Orbitrap MS is a major advantage compared to TQMS, additional studies are required to determine cut-off values for primary and secondary biomarkers of NBS included diseases. Furthermore, studies would be required to examine ion suppression effects and the analytical performance of the new method.
31
Acknowledgement
This literature thesis is the final of my master study, and therefore the right moment to thank some people who have helped me throughout this period.
I would like to express my gratitude for all the help I received during my literature study from dr. Desirée Smith. I really appreciate all the effort you made in guiding me throughout this study. Your feedback always helped me a lot to get more progression in writing my thesis. Next, I like to thank dr. Henk Lingeman for his supervision and the nice meetings during question and answer sessions.
I have followed this master study next to my daily work, therefore I want to thank prof. dr. ir. Cornelis Jakobs, prof. dr. Henk Blom and prof. dr. Gajja Salomons for giving me this opportunity. Furthermore, following this master study would not have been possible without the help of all colleagues from the VUmc metabolic laboratory. Thank you all for your flexibility, support and social talks during this time!
32
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