The Role of Ion Mobility Spectrometry–Mass Spectrometry in the

Jul 4, 2013 - and Alison E. Ashcroft. †. †. Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom. â...
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The Role of Ion Mobility Spectrometry−Mass Spectrometry in the Analysis of Protein Reference Standards Caroline Pritchard,*,†,‡ Gavin O’Connor,‡ and Alison E. Ashcroft† †

Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom LGC, Queens Road, Teddington TW11 0LY, United Kingdom

‡

ABSTRACT: To achieve comparability of measurement results of protein amount of substance content between clinical laboratories, suitable reference materials are required. The impact on measurement comparability of potential differences in the tertiary and quaternary structure of protein reference standards is as yet not well understood. With the use of human growth hormone as a model protein, the potential of ion mobility spectrometry−mass spectrometry as a tool to assess differences in the structure of protein reference materials and their interactions with antibodies has been investigated here.

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different to a real sample and raises the issue of the appropriateness of such a QC material. A greater knowledge of the tertiary and potential quaternary structure of the protein standards used as reference materials may further inform the debate surrounding the issue of commutability and attempt to explain why some materials may seem commutable on two different systems but not on a third. Structure is fundamental in determining the physiological role and activity a protein has. It has been shown previously that the presence of proteins such as bovine serum albumin (BSA) can have different effects on the behavior of two different recombinant standard materials of the same protein (natural and isotopically labeled materials),1 which is likely to be due to structural discrepancies between the two preparations. One way in which structural equivalence, or lack of it, can be assessed is by using ion mobility spectrometry−mass spectrometry (IMS-MS), a technique which has become widely used for protein structural analysis.12−15 IMS-MS can be used to separate co-populated protein conformers with the same m/ z ratio but that differ in physical size or shape to provide information on collision cross-sectional areas and isoform distribution. Additionally, it can be used to investigate noncovalently bound biomolecular species to identify which isoforms interact with, for example, antibodies or other proteins. Although it is a low resolution technique, and so cannot provide the atomic or molecular detail obtained using X-ray crystallography or NMR spectroscopy, IMS-MS provides a faster and simpler alternative. It requires only picomolar sample sizes, is amenable to varied buffer conditions, and has

he requirement for biological reference materials and certified reference materials is recognized within the clinical community and by the International Federation for Clinical Chemistry and Laboratory Medicine. Use of suitable reference materials supports results that are accurate, specific, and, most importantly, comparable between laboratories across both time and space. The first step to improve traceability and standardization of protein reference materials has been the development of methods for the traceable quantification of the total amount of substance content.1−3 Although this approach has been highly successful, amount of substance is only one component which must be considered in the production of protein reference standards. In a truly clinically relevant standard, a reference material should mimic exactly the naturally incurred protein being analyzed.4,5 Realistically, the current best-practice is that a certified reference material used as a quality control (QC) material should be commutable: that is, it should give the same measurement result as a real patient sample when analyzed using two different methods.6,7 The question of commutability has been addressed primarily by developing matrix-matched reference materials, e.g., in serum or plasma.8,9 The aim is to replicate the immediate environment of the naturally incurred protein analyte within the reference material. This would ensure that the immunochemical techniques, e.g., enzyme-linked immunosorbent assay (ELISA),10,11,9 used for most protein-based in vitro diagnostics recognize reference standards and patient samples in the same way. However, this does not provide a simple answer as either the matrix must be spiked with a recombinant protein which may behave differently to the patient sample or, even if a pooled patient material is used, any processing, e.g., freezedrying to ensure stability over time, immediately renders it © 2013 American Chemical Society

Received: March 28, 2013 Accepted: July 4, 2013 Published: July 4, 2013 7205

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Figure 1. ESI-IMS-MS Driftscope plots of the WHO (above) and labeled (below) rhGH materials at 10 μM in 25 mM ammonium acetate, pH 7, after dialysis. Two distinct monomer conformations, an extended (2800 Å2) and compact (2100 Å2) form, can be seen, as well as a dimer (3300 Å2). A small amount of trimer (4600 Å2) was observed for the WHO but not the labeled material. Both the EP and USP materials showed the same conformers as for the WHO. The corresponding mass spectra are shown on the left-hand side.

monoclonal anti-growth hormone antibody (MAb) was purchased from SigmaAldrich (Gillingham, U.K., catalogue number G8523). Each rhGH material was prepared at 1 mg/mL in 25 mM ammonium acetate and dialyzed into the same buffer using 3.5 kDa cellulose membrane cassettes (3 mL, Thermo Scientific, Loughborough, U.K.). Standard samples were diluted to 10 μM in 25 mM ammonium acetate. For samples containing BSA (SigmaAldrich, catalogue number B4287), stock standards were diluted to 10 μM in 0.2% BSA, 25 mM ammonium acetate. Negative controls were prepared at 0.2% alcohol dehydrogenase (ADH), 25 mM ammonium acetate. The MAb was prepared at 20 μM in 100 mM ammonium acetate and buffer exchanged via 7 kDa spin columns (0.5 mL, Thermo Scientific). Samples were mixed at a 1:1 molar ratio with rhGH standards at a final concentration of 10 μM. Negative controls were prepared with β-lactoglobulin at the same ratio and concentration.

the capacity to be used for highly complex samples, making IMS-MS a potentially highly valuable approach for the comparison of reference standards. To assess this potential, a comparison of commercially available human growth hormone (hGH) reference standards and a custom synthesized, isotopically labeled, hGH internal standard was performed using IMS-MS, and the interactions of these hGH standards with a hGH-specific antibody was investigated.

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EXPERIMENTAL SECTION Sample Preparation. The rhGH reference standards used were the following; World Health Organization (WHO) WHO 98/574 (NIBSC, Potters Bar, U.K.), European Pharmacopeia (EP) CRS S0947000 (LGC Standards, Teddington, U.K.) and United States Pharmacopeia (USP) USP1615708 (LGC Standards), all 22 kDa isoforms, MW 22124.8 Da. The K/Risotopically labeled protein was a requested synthesis by PROMISE Advanced Proteomics, MW 22305.5 Da (Grenoble, France). For further details, see Pritchard et al.1 The 7206

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For destabilizing experiments, the reducing agent dithiothreitol (DTT) was added at 50 nM to 10 μM rhGH standards and incubated for 30 min. Mass Spectrometry. Experimental measurements were performed on a Synapt HDMS mass spectrometer (Micromass UK Ltd./Waters, Manchester, U.K.) equipped with a NanoMate (Advion Biosystems, Inc., Ithaca, NY) nanoESI autosampling device. Positive ion nanoESI with a capillary voltage of 1.69 kV, a nitrogen nebulizing gas pressure of 0.7 psi, and a source temperature of 60 °C were used throughout. For mass spectral analysis of the rhGH alone, a cone voltage of 20 V, a trap voltage of 6 V, and a transfer voltage of 4 V were applied. Ion mobility separation was performed using a static wave height of 7 V at a speed of 300 ms−1. Data were acquired over the m/z range 500−5000. For analysis of MAb with the rhGH, a cone voltage of 100 V was applied, with the trap and transfer T-wave devices set at 150 and 40 V, respectively. Data were acquired over the m/z range of 500−13000. Drift times were corrected for mass-dependent and mass-independent times,16 and the drift time cross-sectional function was calibrated as reported previously.17 An aqueous solution of CsI was used for m/z calibration, and the raw data were processed by use of MassLynx v.4.1 and Driftscope v.3.0 software (Micromass UK Ltd./Waters, Manchester, U.K.). Equations. For determining the theoretical maximum charge of a protein while maintaining its native structure, the equation z = 0.077 × m1/2 has been used, where z is the theoretical maximum charge and m is the mass of the protein.18 For isotropic multimer formation calculations of the collision cross-sectional area (CCS), the formula CCS = CCSmon·n2/3 has been used, where CCSmon is the monomer cross section and n is the oligomeric state.19

Figure 2. (a) Collision cross-sectional area (Å2) of each of the ESI-MS charge state ions originating from the labeled rhGH showing a compact monomer (lowest charge states, 7+ to 11+), extended monomer (11+ to 14+) and a dimer (10+ to 14+) with no trimer detected in this case. (b) Showing how semiquantitative analysis was performed. At each charge state, the maximum intensity of the extracted drift peak was plotted and a Gaussian curve fitted. To give a relative intensity of each conformer, the area underneath each curve was calculated as a percentage of the total observed signal, here {compact monomer + extended monomer + dimer}.

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RESULTS AND DISCUSSION Standard Analysis. All four standard materials, the three natural reference standards (World Health Organization, WHO; European Pharmacopeia, EP; United States Pharmacopeia, USP) and the labeled rhGH, were analyzed using IMSMS. The WHO and labeled rhGH results are shown in Figure 1. For each material, two different monomer forms of the recombinant protein were observed: a compact monomer populating lower charge states (7+ to 11+, collision crosssectional area (CCS) approximately 2100 Å2) and a more extended monomer populating higher charge states (11+ to 14+, CCS approximately 2800 Å2). The measured CCS for each conformer was highly reproducible between the different recombinant preparations, within 3% relative standard deviation (RSD, Figure 2a). The observation of larger CCS for the higher charge states (12+ to 14+) is consistent with the hypothesis that increased Coulombic repulsion occurs with greater numbers of charges,20 increasing the CCS. The theoretical maximum charge a protein of this mass can carry while still retaining its native structure18 is 12+, with only charges lower than 12+ observed for the compact monomer (Figure 2a). A dimer (CCS approximately 3300 Å2) was observed for all four materials with a small amount of trimer (CCS approximately 4600 Å2) present for the natural reference standards but not observed with the labeled material. The narrow charge state distributions for the dimer (11+ to 15+) and the trimer (15+ to 16+) and the observed maxima (dimer 16+, trimer 20+) suggest that the dimer and trimer are also native-like,18 and measured CCS values agree well with

isotropic multimer formation calculations.19 Accordingly, multimer formation appears to be occurring without collapse to the molten globule state but by maintaining native-like structure, as we would expect under these conditions. The CCS of each conformer observed agreed well across all four rhGH standards (Table 1). The structures observed here may be interpreted with reference to previous work21 where denaturing experiments have shown the presence of a folding intermediate of hGH in solution. This is likely to correspond to a molten globule state as found for bovine GH.21 In the molten globule state, most of the secondary structure is retained, with some unfolding of the tertiary structure.22 The approximate radius shift between the native and molten globule intermediate was found to be ∼15%,21 which translates to a shift in CCS of ∼30%. This agrees well with the difference in CCS values between the compact and extended hGH monomer conformers measured here using IMS-MS and suggests that the extended monomer may correspond to the molten globule folding intermediate. As the primary difference between the standards appeared to be in the distribution of the conformers, as described below, the relative distributions of each conformer within the respective preparations was determined. The intensities of the extracted drift peak for each charge state (Figure 2a) were plotted across the series for each conformer and a Gaussian curve was fitted (Figure 2b). The areas underneath each of these curves were compared to give relative abundances of each species, shown in Table 2. The WHO and EP materials showed very similar distributions, as to be expected of samples from the same 7207

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Table 1. CCS (Å2) of Dialyzed Individual Standard Preparationsa WHO compact monomer extended monomer dimer trimer

2077 2774 3369 4605

± ± ± ±

230 258 163 230

USP (11%) (9%) (5%) (5%)

2115 2850 3395 4691

± ± ± ±

218 240 166 239

EP (10%) (8%) (5%) (5%)

2119 2693 3358 4685

± ± ± ±

224 214 159 250

lab.rhGH (11%) (8%) (5%) (5%)

average (%rsd)

2069 ± 119 (6%) 2774 ± 175 (6%) 3323 ± 192 (6%) 

2095 2773 3361 4660

(1.2%) (2.3%) (0.9%) (1.0%)

a

Uncertainty values shown here encompass the variability associated with the stability of each conformer across the observed charge states and the variability as a result of repeat (n = 3) measurements; each uncertainty value is the combined standard error of the mean across the observed charge states and the average uncertainty of the CCS of each observed charge state for a particular conformer. Percentage uncertainties are shown in brackets. Samples were analyzed from solution at 10 μM in 25 mM ammonium acetate, pH 7.

Table 2. Relative Proportions of Each rhGH Conformer Observed (10 μM, 25 mM Ammonium Acetate, pH 7)a compact monomer extended monomer dimer trimer a

WHO

USP

EP

lab.rhGH

48% 41% 10% 1%

55% 38% 6% 1%

43% 41% 15% 1%

63% 26% 11% 

Table 3. Relative Proportions of Each rhGH Conformer Observed in the Presence of 0.2% BSA (rhGH at 10 μM, 25 mM Ammonium Acetate, pH 7)a compact monomer extended monomer dimer trimer

Uncertainties on all these measurements are ±5%.

a

original recombinant preparation. The USP material, from a different original source, was biased slightly toward the compact monomer and the labeled material, from a third source, sat predominantly in the compact monomer (63%). IMS-MS has been used here to highlight differences in recombinant preparations that, on any other level (cysteine bridges, primary sequence, amounts of substance content, molecular mass. etc.), have been shown to be identical.1 The presence of multiple structural conformers of identical primary sequence in purified standards raises the questions as to whether a number of structural conformers are likely to be present in vivo and what impact that has on the protein’s functional behavior and definition of the measurand. As the protein structure is of primary importance in the interaction with antibodies raised for immunoassay kits and the interactions within serum, the behavior of these recombinant preparations in the presence of serum albumin and a hGHspecific monoclonal antibody has been investigated.23 Analysis in the Presence of BSA. The effects on the tertiary structure of all four rhGH preparations was analyzed when in the presence of BSA. Previous experiments1 have shown that the presence of BSA has an impact on the efficacy of digestion of rhGH, potentially due to a-specific binding effects between the serum albumin and rhGH. A ratio of 1:30 (rhGH:BSA) was investigated here; this ratio is significantly lower than that of hGH to serum albumin in serum samples (∼1:1 000 000) but still enables the rhGH to be observed in the mass spectrometer and provides an indication of the likely behavior in serum. No direct binding was found to occur between the BSA and the rhGH standards, but the presence of BSA stabilized the compact monomer dramatically for the WHO, EP and USP materials (see Table 3, Figure 3). The relative distribution of the three natural standards showed an average shift toward the compact monomer of 25%, from 49% to 74%, with a loss of both the dimer and extended monomer. No trimer was observed, but this might also be due to loss of signal intensity from ion suppression caused by the presence of BSA. Intriguingly, the distribution of the labeled material was almost identical in the presence and absence of BSA, with a high (13%) proportion of dimer still observed. This suggests that,

WHO

USP

EP

lab.rhGH

73% 24% 3% 

74% 23% 3% 

74% 26%  

63% 24% 13% 

Uncertainties on all these measurements are ±5%.

although the CCS values are similar (within 5%) to the natural materials, the equilibrium in the label between the two monomer conformations and the dimer is much more stable. This same stabilizing effect was also observed when ADH, which exists as a dimer of similar size to BSA, was added to the rhGH standards. This suggests that in serum, where numerous other proteins are present, a spiked hGH reference standard is likely to populate preferentially in the more compact conformer. This clearly demonstrates the value of performing a more detailed assessment of the structure of reference standards. Without the understanding of whether the amounts of different monomers are equivalent in individual preparations, the amount of substance being detected by routine techniques may not be consistent between samples and reference standards, or even between different standards. Denaturing Effects. Having discovered the stabilizing effect of BSA, the effect of adding a reducing agent was investigated. Disulfide bond reduction can lead to loss of threedimensional structure and it has previously been shown that in the case of rhGH some destabilization of the native structure does occur.24,25 The aim here was to determine how the loss of three-dimensional structure would impact the compact conformer and whether the apparently more stable labeled form would be affected differently. As the WHO, USP and EP materials had all behaved so similarly in previous experiments, subsequent experiments were performed using only the WHO and labeled materials. The reducing agent DTT was added at 50 nM to the dialyzed WHO and labeled hGH materials to reduce the two disulfide bonds. The compact and extended monomers were still detected for both materials and no multimers were seen, as to be expected on reduction (Figure 4). The continuing presence of the compact form is consistent with previous literature which has shown hGH to be a highly stable protein, with the helical regions of the protein remaining stable across extreme denaturing conditions.26 In addition to the extended monomer detected when analyzing the proteins alone (2827 ± 179 Å2), a further extended monomer was observed for both the WHO (3292 ± 78 Å2) and labeled (3032 ± 121 Å2) material, along with a second further extended monomer 7208

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Figure 3. ESI-MS mass spectrum of a mixture of 10 μM EP rhGH material (open circles) and 0.2% BSA (open stars) in 25 mM ammonium acetate, pH 7, showing the stabilizing effect of BSA in a shift to lower charge states for rhGH. No direct binding between the proteins was observed.

Figure 4. ESI-IMS-MS data of WHO hGH material reduced with DTT (50 nM) showing presence of two further extended monomers. No dimer or trimer observed. Some evidence of further unfolding in ESI spectrum but no real distinction between extended and further extended conformers. The corresponding mass spectrum is shown on the left-hand side.

rhGH-specific antibody have been investigated, first to determine whether there were any observable differences in binding between the natural and labeled materials, and second to investigate the impact on the populations of the two monomer conformations on the binding. The hGH binding epitope spans most of the length of the helical bundle and so any differences in tertiary structure which affect this bundle, which may be the case for the extended monomer, may prevent binding. The MAb (expected MW 147 kDa) was first analyzed alone using ESI-MS, giving a clean signal of predominantly monomer at the expected MW, together with a small amount of dimer. On IMS-MS analysis (Figure 5), three monomer conformations with CCSs ranging from 6100 to 7600 Å2 and two dimer conformations (10 600−11 500 Å2) were observed. The MAb sits primarily (∼80%) in the monomer 2 conformation (6807 ± 280 Å2). The MAb was mixed with each of the WHO and labeled standard materials separately at a molar ratio of 1:1. Binding occurred at 1:1 for all of the standards with the bound rhGH− MAb, free rhGH and free MAb present in each case (Figure 6). Varying ratios of MAb to rhGH were investigated but binding was only ever observed at an equimolar ratio. As a negative

detected only for the WHO sample (3828 ± 237 Å2). If the original extended monomer is the molten globule state, retaining secondary structure but with unfolded tertiary structure, these further extended monomers may be due to the unfolding of the secondary structure. Again this shows the increased stability of the labeled recombinant preparation over the WHO standard and the different behavior of two apparently identical materials. This resistance to denaturation may also explain the differences in digestion behavior between the natural and labeled materials observed in previous work.1 It also underlines the importance of fully understanding structure when using a labeled protein internal standard. The stabilizing effect of BSA was further confirmed by the addition of 0.2% BSA to the two reduced, denatured samples. The most extended monomer of the WHO was no longer observed and the relative conformeric distributions of both the WHO and labeled materials were shifted back in favor of the compact monomer (data not shown). Antibody Experiments. Routine protein measurements are performed typically using ELISA, which relies on the use of specific antibodies to identify the protein of interest. Here, the interactions between the standards and a commercially available 7209

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Figure 5. ESI-IMS-MS Driftscope plots of 10 μM MAb in 25 mM ammonium acetate, pH 7. Three distinct monomer conformations can be seen, ranging from 6100 to 7600 Å2, as well as two distinct dimers (10 649 Å2, 11 443 Å2). The MAb sits preferentially (80%) in the monomer 2 (6807 Å2) conformation. The corresponding mass spectrum is shown on the left-hand side.

Figure 6. ESI-MS mass spectrum of a mixture of WHO rhGH and MAb showing free MAb (closed stars) and MAb bound to rhGH at 1:1 (closed circles), highlighting the region m/z > 5500 to show the presence of the {MAb + rhGH} complex. The free rhGH charge states are observed at m/z < 5500 and so not seen here.

control, another protein, β-lactoglobulin, was added to the WHO and labeled rhGH materials, and no binding was observed, demonstrating that the MAb binding was indeed specific and not a gas-phase adduction phenomenon. None of the compact rhGH monomer was observed in the free rhGH detected in the presence of MAb. Two monomers with the same CCSs as the extended (∼2800 Å2) and further extended (∼3300 Å2) monomers seen earlier for the rhGH standards in the presence of DTT were present. The extended monomer form has been shown to be likely to involve unfolding of the α-helical core.21 As the MAb epitope sits across the helix bundle, binding to this form was not anticipated. It may however act as an intermediate to the further extended conformer which appears to be involved in MAb−rhGH binding. Most of the observed free rhGH (63%) was present in the further extended form. The presence of BSA or other proteins has been shown to stabilize the compact form,

so this suggests that the further extended form may be an intermediate necessary for MAb−rhGH binding rather than the MAb binding exclusively to the compact form. The presence of the two highly compact MAb conformers present at low relative intensities (total ∼10%) provides further evidence for an intermediate transition state as these conformers were not present in the free MAb and both are seen to interact with the rhGH. These preliminary data show the impact IMS-MS can have on the understanding of the behavior of reference standards in the presence of an antibody and underline the potential of IMS-MS for reference material characterization in the future.

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CONCLUSIONS Understanding the tertiary structure of a protein reference standard is crucial to an understanding of the physiological role that it performs. With the use of traditional structural 7210

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If significant structural differences are observed between recombinant preparations from different sources, the differences between a recombinant preparation and the naturally incurred protein and are likely to be even greater. The need for standardizing protein structure has been recognized within the biopharmaceutical field as small changes in tertiary structure may have dramatic impacts on the biological activity of a protein.30 There is now significant regulation for the production of biosimilars (follow-on versions of biopharmaceuticals) which requires the detailed assessment and comparison of protein tertiary and quaternary structure. The requirements are constantly being updated and revised with the development of new technologies and improved understanding of the implications of structural differences. To improve protein reference standard production, a similar approach which uses multiple analytical techniques to provide structural information combined with, e.g., ELISA measurements to give the most comprehensive picture of a material may be necessary. However, purifying or refining protein measurements based on the tertiary structure as observed by mass spectrometry may not actually be biologically relevant as it may not reflect the structural environment found in vivo.

techniques, growth hormone has been shown to be a highly stable protein, with very few differences seen between the solution and crystal structures.27 A high proportion of the helical structure is maintained across a wide pH range (2.5− 11.0) and only removed under extreme denaturation conditions (>6 M Gnd·HCl).26 Even on removal of the cysteine bridges, little reduction in the biological activity has been observed previously, although this did result in increased population of stable intermediate states.25 It would be expected that the structure of multiple recombinant preparations would be identical. Mass spectrometric methods have shown differences in the tertiary structure between recombinant preparations from different sources.1 The differences observed here are subtle differences in the equilibrium between conformers which could not be seen by traditional techniques, rather than large shifts in the structure. However, these differences could have major consequences for the standardization of immunoassay measurements for the analysis of hGH in particular and proteins in general. Typically, immunoassay kits from different suppliers will use different monoclonal antibodies to determine biological activity. Depending on the site of the chosen epitope, the conformers present in a reference material may have a significant impact on the interaction of the protein with the antibody.28 As a consequence, the amount of substance being measured is not the total amount of hGH, as is currently defined,29 but rather is the amount of a particular conformer present in the sample and hence is not traceable to the total amount of the standard used. If the reference standards used to calibrate immunoassays are themselves not structurally identical, or adopt different conformations depending on the buffers or stabilizers present, every time a different material or set of conditions is used to perform calibration, the variability of measurements may be increased. This raises the question as to whether standardization of the tertiary structure of reference materials is the next step in improving comparability of measurements. If this were to be the case, the production of a pure protein of a single conformation would present a significant challenge to producing an appropriate reference standard for use with immunoassays. However, purifying or refining a protein based on tertiary structure as defined by mass spectrometry measurements may not be biologically relevant, as demonstrated by the highly similar behavior of the natural and labeled standard materials in the presence of the antibody. Although tertiary structure provides an indication of biological activity it is not a conclusive measure. Different conformers of the same protein may retain the same degree of activity if the differences occur in a region of the structure that is not involved in the relevant protein− protein interactions. Furthermore, the multitude of active conformers present in real patient samples may not be represented accurately, preventing true commutability between the reference standard and real patient samples. The work carried out here on structural analysis of purified protein standards is merely a first step. The reference standard is at its most simple and yet still presents a highly complex picture which is not fully understood and will require significant further investigation. However, this increased awareness of the structural disparity of different recombinant protein standard preparations can be used to further inform the debate as to what definition of the measurand is the most appropriate for a protein reference standard.

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AUTHOR INFORMATION

Corresponding Author

*Address: LGC, Queens Road, Teddington TW11 0LY, United Kingdom. Phone: 0208 943 7522. E-mail: caroline.pritchard@ lgcgroup.com. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work described was supported by the UK National Measurement System Chemical and Biological Metrology Programme. The Synapt HDMS mass spectrometer was purchased with a Research Equipment Initiative grant from the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/E012558/1).

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REFERENCES

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dx.doi.org/10.1021/ac400927s | Anal. Chem. 2013, 85, 7205−7212