In an ideal FPOP experiment, each protein, and the solution immediately surrounding it, would be irradiated no more than once. This is because once a protein has been irradiated, it has the possibility, although not certainty, of being labelled by a nearby hydroxyl radical, and the introduction of such non-native oxidations may shift the protein structure into a non-native conformation. As a result, continued oxidative labelling of the protein increases the chance of probing non-native structures generated as artefacts of FPOP labelling, rather than the protein in its native state.
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However, the experimental setup typically used in FPOP, both in the literature and our laboratory (described in detail in Section 3.5), makes the determination of exactly how many irradiation events, or βshotsβ, a protein has undergone quite challenging. The FPOP analyte solution is passed, at a fixed flow rate, through a capillary that intersects orthogonally the beam of a UV excimer laser, necessary to liberate hydroxyl radicals from hydrogen peroxide. Due to the nature of the laser itself, this beam is not constant and is instead pulsed at a constant frequency, for a fixed duration of 20 ns (Figure 5.1). Although this extremely short pulse duration can be assumed as instantaneous, for most practical purposes, this setup is such that many other parameters are involved in determining how many times a protein molecule is irradiated including: laser firing frequency, beam width, diffusion of the protein in solution, and time spent in the irradiation window, the latter of which is directly proportional to the flowrate of the solvent in the capillary.
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Figure 5.1 Schematic diagram of a typical FPOP experimental setup. Solution containing the protein sample, hydrogen peroxide and a scavenger amino acid is passed through a capillary intersecting an irradiation window where 248 nm UV light causes photolysis of the hydrogen peroxide, generating hydroxyl radicals. These hydroxyl radicals then covalently label the protein sample, before the sample is diverted into a quench solution to prevent further radical oxidation.
The beam width (~3 mm), firing frequency (15 Hz) and the flowrate (20 Β΅l min-1) of
the analyte solution have been optimised previously in our laboratory to approximate each bolus of liquid in the capillary experiencing a single shot, which can be demonstrated, knowing the inner radius of the capillary (50 Β΅m), by showing that the irradiated volume per second is approximately equal to the flow rate used [231].
147 πΌπππππππ‘ππ π£πππ’ππ πππ π πππππ = ππ2 Γ π€ Γ π = π(50 Γ 10β6)2Γ (3 Γ 10β3) Γ 15 = 3.54 Γ 10β10 π3π β1 = 21 Β΅π πππβ1 (5.1)
Equation 5.1 Calculating irradiation volumes in FPOP capillary experimental setups assuming a plug flow model. Area of the capillary (units = m2) multiplied by beam width (denoted w, units = m)
multiplied by laser firing frequency (denoted f, units = s-1). Units of m3 s-1 converted to Β΅l min-1 by
multiplying by 109 (units = Β΅l s-1) and multiplying again by 60 (Β΅l min-1).
Although others have used more conservative experimental parameters, opting for a brief exclusion volume with no irradiation between each shot [227], which has been
achieved by increasing the flowrate or reducing the firing frequency, both of these calculations are flawed in that they assume a βplug flowβ model where all solvent across the radius of the capillary is flowing at the same rate. In reality, the flow regime in the capillary is more accurately described by a laminar flow model, where solvent travels faster at the centre of the capillary, and slower at the capillary walls, a scenario typically visualised as a series of concentric rings of varying flow rates around the capillary centre (Figure 5.2). The velocity of solvent at any given radius from the centre of the capillary can be calculated using Equation 5.2 [268].
π£(π) = 2 ( π
ππ2) (1 β
π2 π 2)
(5.2)
Equation 5.2 Calculating the velocity of solvent at different radii from the capillary centre in laminar flow. Where v(r) is the velocity at a given radius (r) from the capillary centre (units = m s-1), V is flow
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Figure 5.2 Visualising laminar flow in capillaries. Side view (left) and end view (right) of laminar flow shows solvent flows faster at the capillary centre and slower at the capillary walls. R and r are annotated as in Equation 5.2.
Laminar flow regimes have been described previously in the context of FPOP experiments by Konermann et al., where the authors concluded that diffusion of the protein both axially along the capillary, and laterally between flow regimes were negligible factors in the resulting number of irradiation events experienced by protein molecules in the sample. However, the authors also concluded that due to the slower moving proteins in solvent nearer the capillary walls, multiple shots for a certain proportion of the sample, and only minimal radiation exposure for proteins in faster flow regimes in the capillary centre, are inevitable consequences of laminar flow in these experiments [268]. Nevertheless, based on these findings, the FPOP method used
previously in our laboratory was re-evaluated to determine the degree to which multiple exposure events were occurring under the present experimental conditions. Using Equation 5.2 and experimental parameters listed in Section 3.5, the velocity of solvent, and time spent inside the irradiation window, at various different radii from the capillary centre, were calculated (Figure 5.3). By assuming this velocity is consistent across short widths of the capillary, we can simplify the problem by splitting the flow in the capillary into multiple, smaller βplug flowβ models, where each
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concentric ring of flow, of arbitrary width, is approximated to have the same average velocity. This approximation is most accurate closest to the capillary centre, where the dependence of velocity on radial position is the lowest (Figure 5.3). As such, the calculation was split into multiple 5 Β΅m wide rings from the capillary centre (0-5 Β΅m, 5-10 Β΅m etcβ¦) where, smaller, 1 Β΅m intervals were used after 45 Β΅m from the capillary centre, where radial position affects velocity most significantly.
Figure 5.3 Velocities and time spent in the irradiation zone calculated for different flow regimes in the FPOP capillary. Calculated using Equation 5.2 and experimental parameters listed in Section 3.5. A radius of 0 on the x axis is the capillary centre. The work of Prof. Nik Kapur (Applied Fluid Mechanics, School of Mechanical Engineering, University of Leeds) is gratefully acknowledged for his help in these calculations.
Knowing the firing frequency of the laser (15 Hz) and the time spent in the irradiation window for each concentric ring of flow, or flow regime, we can determine the average number of shots and, consequently, the maximum and minimum number of shots, a protein could experience under each flow regime, maximum being a scenario where a protein is irradiated the instant it enters the beam width, and the minimum being a protein that follows one that was instantly irradiated (equivalent to one fewer than the maximum number of hits). As the proportion of protein receiving the maximum and minimum number of shots under each flow regime must sum to 1, we can determine the fraction of protein, under each flow regime, that receives the maximum and minimum number of shots (Equation 5.3, Table 5.1).
150 π₯ + π¦ = 1
πΌπ₯ + π½π¦ = ππ£πππππ π βππ‘π
(5.3)
Equation 5.3 Simultaneous equations calculating the maximum and minimum number of irradiation events for different flow regimes in FPOP. Where x and y are the proportions of protein receiving the maximum and minimum number of shots, and Ξ± and Ξ² are the maximum and minimum number of shots for a given flow regime.
Calculating the cross sectional area of each flow regime ring, we can determine the mean flow rate of solvent in each regime, by multiplying by the mean velocity of the solvent at this radius (Table 5.1). Dividing this by the mean flow across the whole capillary, we can estimate the fraction of the sample subjected to each flow regime. Knowing how much of the sample is in each flow regime, and how much of each flow regime experiences different numbers of irradiation events, we can determine, overall, the proportion of the sample that is under the desired single exposure conditions (Figure 5.4).
Table 5.1 Determining the number of irradiation events under different flow regimes of a laminar flow model in FPOP experiments.
Radius (Β΅m) Velocity (m s-1) Time in zone (s) Max hits Average hits Min hits fraction Max hits fraction
Mean flow at this radius (m3 s-1) Fraction flow at this radius (%) 0 0.084883 0.035343 1 0.530144 0.469856 0.530144 0-5 0.084034 0.0357 1 0.535499 0.464501 0.535499 6.6E-12 2.210133 5-10 0.081487 0.036816 1 0.552233 0.447767 0.552233 1.92E-11 6.429478 10-15 0.077243 0.038838 1 0.582576 0.417424 0.582576 3.03E-11 10.15768 15-20 0.071301 0.042075 1 0.631124 0.368876 0.631124 3.92E-11 13.12685 20-25 0.063662 0.047124 1 0.706858 0.293142 0.706858 4.5E-11 15.06909 25-30 0.054325 0.055223 1 0.82835 0.17165 0.82835 4.69E-11 15.7165 30-35 0.04329 0.0693 2 1.039498 0.960502 0.039498 4.42E-11 14.8012 35-40 0.030558 0.098175 2 1.472622 0.527378 0.472622 3.6E-11 12.05527 40-45 0.016128 0.186015 3 2.79023 0.20977 0.79023 2.15E-11 7.210839 45-46 0.013038 0.230097 4 3.451457 0.548543 0.451457 3.73E-12 1.248176 46-47 0.00988 0.303633 5 4.5545 0.4455 0.5545 2.89E-12 0.966672 47-48 0.006655 0.450803 7 6.762038 0.237962 0.762038 1.99E-12 0.665094 48-49 0.003361 0.892498 14 13.38747 0.612531 0.387469 1.02E-12 0.343013
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Figure 5.4 Exposure events per protein in the current FPOP experimental arrangement.
Figure 5.4 indicates that ~two thirds (66%) of the sample is under single exposure conditions using these experimental parameters, just over 25% of the sample experiences no irradiation events at all, leaving less than 8% of the sample to experience multiple exposures. This is slightly more oxidation than was suggested as optimal by Konermann et al., [268] and as such, the effects of multiple exposure events
on wild-type Ξ²2m were evaluated to determine the significance of these events with
regards to their effect on protein conformation and downstream analysis.
Native MS experiments showed no significant change in the charge state distribution following FPOP compared with the control protein, with the most notable change being a significant decrease in signal intensity for the FPOP sample, presumably due to signal splitting between the various different oxidised species (Figure 5.5).
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Figure 5.5 Native MS of FPOP oxidised wild-type Ξ²2m. (a) Wild-type Ξ²2m oxidised by FPOP (as per
experimental procedures described in section 3.5.1) compared with (b) the control without FPOP or exposure to hydrogen peroxide. Inset shows a zoom of the 7+ charge state ions highlighting the
observable +16 Da oxidations in the oxidised sample and no oxidations, although some salt adducts, on the non-oxidised sample. The ESI-MS solution was 150 mM ammonium acetate, pH 7.4.
ESI-IMS-MS of the native protein showed identical ATDs for the unmodified and oxidised versions of the protein for up to three oxidations, which is the maximum number resolvable using native MS (Figure 5.6a), thus indicating no significant conformational changes result from FPOP oxidation for wild-type Ξ²2m under the
conditions employed.
To determine the effect, if any, of multiple oxidations on proteolytic digestion of the wild-type protein, samples of wild-type Ξ²2m contained in Eppendorf tubes, rather
than the typical capillary flow setup, were irradiated between one and ten times, reduced, alkylated and digested with trypsin, before the tryptic peptides were analysed by LC-MS/MS. Figure 5.6b shows that as the number of irradiation events
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increases, the percentage of the protein sequence covered from the peptides identified decreases. A likely explanation for this is that multiple irradiation events cause significant oxidation which splits the peptide signal among many different oxidised species eluting at different retention times, which fall below the limit of detection.
Figure 5.6 The effect of FPOP oxidation on native IMS ATDs and sequence coverage in proteolytic digests of wild-type Ξ²2m. a) ATD extracted from the 6+ charge state ions of wild-type Ξ²2m for the
unmodified, and up to three oxidative FPOP modifications. b) Sequence coverage from tryptic digests of Ξ²2m following FPOP oxidation.
Although some evidence suggests that FPOP oxidations can partially unfold proteins
[269], others have found limited oxidation to be relatively benign [226, 270], with some
evidence suggesting that certain enzymes can even retain activity following oxidation by FPOP [271]. This is consistent with the fact that FPOP labels primarily solvent
exposed side chains, often hydrophilic structures with a high degree of conformational freedom, and thus are unlikely to be significantly structurally perturbed by the effect of a small, hydrophilic modification such as hydroxylation. Overall, these data suggest that the current FPOP experimental setup is unlikely to generate significant detectable structural artefacts, given only ~8% of the sample experiences multiple exposure events (Figure 5.4), and such exposure has no significant effect on the native protein conformation, to the resolution of an IMS experiment. Additionally, those molecules that do experience multiple exposure events contribute less to the analysis of oxidations in LC-MS/MS data, as the higher
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levels of oxidation split the peptide signal further across multiple oxidised species, increasing the likelihood that such species fall below the limit of detection.