QconCAT Standard for Calibration of Ion Mobility-Mass Spectrometry

Sep 17, 2012 - Manchester Institute of Biotechnology, Michael Barber Centre for Mass ... LGC, Teddington, Middlesex, TW11 0LY, United Kingdom. §...
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Technical Note pubs.acs.org/jpr

QconCAT Standard for Calibration of Ion Mobility-Mass Spectrometry Systems Ross Chawner,† Bryan McCullough,‡ Kevin Giles,§ Perdita E. Barran,∥ Simon J. Gaskell,⊥ and Claire E. Eyers*,† †

Manchester Institute of Biotechnology, Michael Barber Centre for Mass Spectrometry, School of Chemistry, University of Manchester, Manchester, M1 7DN, United Kingdom ‡ LGC, Teddington, Middlesex, TW11 0LY, United Kingdom § Waters U.K., Floats Road, Wythenshawe, Manchester, M23 9LZ, United Kingdom ∥ EastChem School of Chemistry, University of Edinburgh, Edinburgh, EH9 3JJ, United Kingdom ⊥ Queen Mary University of London, London, E1 4NS, United Kingdom S Supporting Information *

ABSTRACT: Ion mobility-mass spectrometry (IM-MS) is a useful technique for determining information about analyte ion conformation in addition to mass/charge ratio. The physical principles that govern the mobility of an ion through a gas in the presence of a uniform electric field are well understood, enabling rotationally averaged collision cross sections (Ω) to be directly calculated from measured drift times under well-defined experimental conditions. However, such “first principle” calculations are not straightforward for Traveling Wave (T-Wave) mobility separations due to the range of factors that influence ion motion through the mobility cell. If collision cross section information is required from T-Wave mobility separations, then calibration of the instruments using known standards is essential for each set of experimental conditions. To facilitate such calibration, we have designed and generated an artificial protein based on the QconCAT technology, QCAL-IM, which upon proteolysis can be used as a universal ion mobility calibration standard. This single unique standard enables empirical calculation of peptide ion collision cross sections from the drift time on a T-Wave mobility instrument. KEYWORDS: ion mobility, traveling wave, T-Wave, calibration, QconCAT, collision cross section, drift time

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protein complexes,8 peptide oligomers,9−11 covalently modified peptides,12 peptides with single point mutations,13 DNA secondary structures,2 chiral enantiomers14 and stereoisomers.15 The physical principles that govern the motion of ions traveling through drift tubes under the influence of uniform weak electric fields are well established and have been previously described.3,16 The relationship between the drift velocity v (cm s−1) of an ion species of mobility K (cm2 s−1 V−1), through a gas in the presence of an electric field, E (V cm−1), is given by the equation:

INTRODUCTION

Ion mobility (IM) measurements combined with mass spectrometry (MS) enable differentiation of an analyte by its size and shape in addition to its mass and charge.1−4 This extra dimension of sample characterization has potential for the study of gas phase protein and peptide structure (in addition to helping elucidate the conformation of different small molecule structural isomers), and these cross sectional area measurements can be compared with X-ray structures and NMR derived solution structures for proteins analyzed under native conditions,5,6 although some care must be taken with the interpretation of such measurements.7 Ion mobility-mass spectrometry (IM-MS) studies have been used in the structural characterization of a range of different species including large © XXXX American Chemical Society

(1)

v = KE Received: June 14, 2012

A

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As described using the Mason-Schamp equation (eq 2), K is dependent on both the properties of the ion (size, shape, mass, charge state) and the properties of the buffer gas through which it is traveling (composition, temperature, pressure): Ω=

3q 16N

2π 1 μkBT K

generates stoichiometric mixtures of the peptides of interest, suitable for use as reference quantification standards.33 This approach to “designer” protein production has also been used to generate QCAL, a standard for the optimization and calibration of mass spectrometric and reversed phase chromatographic instruments for proteome analysis.34 Here we describe a novel application of this designer protein technology with the development of QCAL-IM, an artificial protein that upon proteolytic cleavage generates a collection of peptides that can be used for calibration of ion mobility mass spectrometers and acts as a standard for assessing mobility separation. The peptides incorporated within QCAL-IM have been selected to provide a broad calibration range from a single protein digest, and are observed in charge states ranging from +1 to +4 when subjected to electrospray ionization (ESI). Collision cross section values for these peptides have been determined using linear field mobility analysis on two different platforms and these values then used to calibrate the Synapt T-Wave mobility instrument. Comparison of the Ω values of a selection of peptides calculated following instrument calibration with QCAL-IM demonstrate excellent agreement with the values published in the Clemmer database.30 Moreover, QCAL-IM can be readily used to calibrate the mobility of triply protonated peptide ions in addition to singly and doubly protonated ions, and in the assessment of mobility resolution.

(2)

where Ω is the collision cross section, q is the charge state of the ion, N is the number density of the drift gas, μ is the reduced mass of the ion and drift gas, kB is the Boltzmann constant and T is the temperature of the drift gas. The MasonSchamp equation, however, only applies when ions are in thermal equilibration with the background gas, and under conditions where ion heating can occur (such as during TWave mobility experiments), measures should be taken to minimize such heating in order for this equation to be relevant.17 Under low electric field conditions (where the energy imparted by the electric field is much less than the thermal energy of the system) mobility is independent of applied electric field and K is inversely proportional to Ω.16 Consequently, measurement of the drift time combined with knowledge of the electric field strength permits calculation of Ω for a given buffer gas type, temperature and pressure, as is routinely carried out in drift tube ion mobility analyses. However, such direct calculations are not straightforward for T-Wave mobility experiments,18 where the time-varying nonuniform electric field and mode of separation results in a nonlinear relationship between drift time and ion mobility, and hence Ω.19,20 Calibration of T-Wave instruments under identical conditions to those used for analysis is thus essential if drift time is to be used to empirically determine analyte ion Ω values. While many T-Wave studies have been undertaken to ascertain structural information of large proteins and protein complexes,8,11,21 an increasing number of researchers, including our group22−24 and others25,26 are interested in using T-Wave ion mobility to study the gas-phase conformation of peptides and their fragment ions, with a view to better understanding gas-phase peptide ion chemistry. As such, the availability of appropriate mobility calibration standards is crucial. Approaches toward T-Wave mobility calibration have been published,5,6,12,21,27,28 many of which rely on previously measured Ω values of multiple peptides/proteins included in the database published by Clemmer and co-workers.29,30 However, there is little published evidence validating these cross sections and use of the Clemmer database requires digestion of a number of proteins to provide a set of peptides with a range of Ω values suitable for calibration; calibration of doubly protonated peptide ions of Ω values between ∼173 and 428 Å2 can only be achieved using a minimum of 2 proteins (horse cytochrome c and sheep hemoglobin) and calibration of triply protonated ions is not addressed in detail. Development of single, well-characterized ion mobility standards appropriate to the analytes being investigated would be of great utility in reducing overall analysis time and potentially improving accuracy of derived Ω values. Indeed, while such standards would be of obvious value to a T-Wave based mobility system, they could also be used in calibrating other standard drift tube systems and/or providing a verification of instrument performance. The QconCAT strategy31,32 has previously been used to design and produce artificial proteins, proteolysis of which

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EXPERIMENTAL SECTION

Peptide Selection

Tryptic digestions of a selection of standard proteins (enolase from S. cerevisiae, horse myoglobin, human serum albumin, chicken lysozyme and carbonic anhydrase, QCAL34) were prepared and the resultant peptide mixtures analyzed by nanoESI (nESI) using glass emitters and a Synapt HDMS instrument (Waters, Manchester, U.K.). The drift time for each peptide ion was then extracted using DriftScope software (Waters, Manchester, U.K.), together with information regarding charge state and signal intensity to generate a library of candidate peptides for inclusion in the standard. Peptides were then ranked according to observed signal intensity and those seen with the maximum signal, while satisfying the broadest possible calibration range for each charge state (see Results and Discussion) and showing agreement with the global charge-dependent correlation between m/z and drift time were considered for inclusion within QCAL-IM. If two species at similar m/z and drift time were available then only the peptide with the higher relative signal intensity was included in order to avoid repetition of calibration points. QCAL-IM calibrant peptides were thus ultimately selected for inclusion in QCALIM based on their relative signal intensity and the chargedependent correlation between m/z and drift time (Figure 1S, Supporting Information). QCAL-IM Construction

The selected peptide sequences were concatenated in silico along with additional sequences to include the necessary initiator methionine (MGALR) and purification His6 tag (ALVALVHHHHHH) residues. DNA which encodes for the signature peptides was synthesized, concatenated and inserted into a bacterial expression vector (pET21a) by PolyQuant GmbH (Regensburg, Germany, http://www.polyquant.com/). B

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C

Carbonic anhydrase Enolase Enolase Enolase Enolase Enolase HSA Myoglobin Myoglobin Enolase Myoglobin HSA HSA QCAL Lysozyme Myoglobin HSA HSA Enolase Enolase Myoglobin Enolase QCAL QCAL

AATLLPK IGSEVYHNLK GNPTVEVELTTEK AVDDFLISLDGTANK GVLHAVK HLADLSK LVNEVTEFAK HGTVVLTALGGILK HPGDFGADAQGAMTK WLTGPQLADLYHSLMK YLEFISDAIIHVLHSK GVFR ALVLIAFAQYLQQCPFEDHVK GVNDNEEGFFSAR NLCNIPCSALLSSDITASVNCAK ALELFR YLYEIAR SHCIAEVENDEMPADLPSLAADFVESK SGETEDTFIADLVVGLR YGASAGNVGDEGGVAPNIQTAEEALDLIVDAIK VEADIAGHGQEVLIR TSPYVLPVPFLNVLNGGSHAGGALALQEFMIAPTGAK GGGVNDNEEGFFSAR GVNDNEEGFFSARNLCNIPCSALLSSDITASVNCAK

T1 T2/L5 T3 T4 T5/L8 T6 T7/L10 T8/L11 T9 T10 T11 T12 T13 T14 T15 T16 T17 T18 T19 T20 T21 T22 T23 L17

713.90 1160.32 1417.56 1579.75 723.89 783.90 1150.32 1379.69 1503.64 1874.23 1886.21 478.57 2491.77 1442.49 2509.40 748.91 928.08 2976.12 1823.02 3259.57 1607.82 3740.39 1556.60 3932.87

[M + H]+

202.3 (0.1) 229.7

197.7 200.3 261.7

Ω (Å2) 357.45 580.66 709.28 790.38 362.45 392.45 575.66 690.34 752.32 937.62 943.61 239.79 1246.39 721.75 1255.20 374.96 464.54 1488.56 912.01 1630.29 804.41 1870.70 778.80 1966.94

[M + 2H]2+

(0.6) (0.1) (1.0) (1.0)

284.2 435.9 200.2 230.6

349.1 (0.6)

(1.2) (2.2) (1.4) (1.8) (1.1) (0.4) (2.1) (2.1)

197.0 261.8 289.0 312.6 204.9 212.9 256.8 297.6

Ω (Å2) 238.63 387.44 473.19 527.25 241.96 261.96 384.11 460.56 501.88 625.41 629.40 160.19 831.26 481.50 837.13 250.30 310.03 992.71 608.34 1087.19 536.61 1247.46 519.53 1311.62

[M + 3H]3+

616.7 (2.3)

599.0 (0.6)

535.7 (1.1)

496.1 (0.4)

407.1

353.4 (5.8)

300.8 (3.3)

Ω (Å2)

Average Ω values obtained from Linear Field Mobility analysis using the modified Synapt instrument are also shown. The values in parentheses following Ω indicate the standard deviation (or range when only two measurements are available) attributed to each measurement (n = 2−7). When parentheses are absent, n = 1. Peptides with no assigned Ω were not observed during analysis.

a

originating protein

sequence

peptide

Table 1. Sequence and m/z Values of QCAL-IM Tryptic/Lys-C Peptidesa

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QCAL-IM Expression and Sample Preparation

The QCAL-IM protein (Figure 2S, Supporting Information) was expressed in E. coli BL21(DE3)pLysS cells and purified as described previously.32 Extracted protein was then diluted to 10 pmol/μL in 50 mM NH4HCO3, reduced with dithiothreitol (4 mM, 60 °C, 45 min) and alkylated (iodoacetamide, 14 mM, room temperature, dark, 1 h). The protein was digested overnight with trypsin or Lys-C proteases (2% [w/w], 37 °C) and the resultant peptide mixtures desalted using C18 ZipTips (Millipore, Watford, U.K.) prior to analysis.

Technical Note

RESULTS AND DISCUSSION

Design of QCAL-IM

The peptides to comprise the QCAL-IM standard (Figure 2S, Supporting Information) were selected to ensure that the widest possible range of drift times (and hence Ω) was covered for each charge state (+1 to +3) using peptides which gave good gas-phase ion yields.36 Those selected cover a mass range of 470−3740 Da and drift time range of 2750−9470 μs (using the conditions described for T-Wave analysis). During design of the artificial protein construct, the selected tryptic peptides were concatenated in such a way as to potentially extend the range over which the drift time calibration could be performed with the application of an alternate protease: Lys-C (Figure 2S). The coding sequence for this artificial protein was then inserted into a bacterial expression plasmid enabling expression and subsequent affinity purification of QCAL-IM.

Traveling-Wave Ion Mobility Analysis

Samples were diluted to 1 pmol/μL with 50% (v/v) acetonitrile in water, containing 0.3% (v/v) formic acid and infused into a Synapt HDMS instrument using gold coated nanospray emitter tips (Proxeon, Odense, Denmark). The capillary voltage, cone voltage and source temperature were typically set at 1.9 kV, 40 V and 80 °C respectively. The IM traveling wave speed was set to 300 m/s and wave height ramped from 4.2 to 16.0 V over the full drift time. Data were collected for species between 100 and 4000 m/z with the TOF pusher operating at a 64 μs interval. The nitrogen gas pressure in the T-Wave cell was approximately 4.4 × 10−1 Torr. For higher mobility resolution IM-MS data, QCAL-IM was infused at 1 μL/min into a Synapt G2 HDMS instrument (Waters) using PicoTip Emitters (Waters) with the capillary voltage set to 2.1 kV, cone voltage at 30 V and source temperature at 120 °C. The IM wave was operated at 300 m/s with a constant wave height of 37.5 V. The N2 pressure in the mobility cell was 1.9 Torr for these experiments.

Determination of Collision Cross Section Values for QCAL-IM Derived Peptides

To establish QCAL-IM as a calibration standard, the absolute Ω value of each peptide, as detailed in Table 1, was determined by replicate analyses (multiple injections on different days) of the proteolyzed purified QCAL-IM on the modified Synapt instrument described above. The geometry of this modified Synapt instrument mimics that of the T-Wave Synapt and was therefore optimal for assigning Ω values to the calibrant peptides in QCAL-IM, as potential differences in ion energy and experimental time scales that can contribute toward the calculated cross section were minimized. Such factors can make the use of calculated collision cross sections from drift time data acquired on disparate instrumentation problematic for T-Wave calibration.17 Peptide ions derived from QCAL-IM using trypsin and Lys-C proteases give a range of Ω values from 197.0 to 616.7 Å2, as shown in both Table 1 and Figure 1. The

Linear Field Mobility Analysis

Analysis was undertaken on a modified Synapt HDMS instrument (Waters) in which the T-Wave mobility cell has been replaced by a linear drift field mobility cell with radio frequency (RF) voltage ion confinement, a detailed description of which is presented elsewhere.21 Samples were prepared at a final concentration of 2 pmol/μL, in 50% (v/v) acetonitrile in water containing 0.3% (v/v) formic acid, and sprayed using nESI tips prepared in-house. Analyses were conducted in helium drift gas at ∼2.1 Torr and ∼300 K with voltages ranging between 50 and 200 V across the 18 cm long drift cell. The TOF pusher was operated at a 70 μs interval and data acquired over 100 to 2700 m/z range. The mobility of each of the ions (and subsequently the Ω) were obtained from the gradient of the straight lines obtained by plotting ion drift times for each species as a function of reciprocal drift voltage. Further linear mobility analysis was undertaken on a modified commercial Q-TOF instrument, which has been described in detail previously.35 In brief, the instrument incorporates a drift cell and ancillary optics postsource that enables measurement of linear field ion mobility in addition to m/z ratio data. Samples in this instance were at a concentration of ∼35 pmol/μL in acetonitrile/water (1/1, v/v) containing 0.3% (v/v) formic acid and sprayed using glass capillary nESI tips prepared in-house. Analysis was conducted in helium drift gas at ∼2.7 Torr and ∼300 K with voltages ranging between 15 and 60 V across the 5.1 cm long drift cell. This secondary determination of Ω was conducted to provide confirmatory values for a range of peptides included within QCAL-IM.

Figure 1. Plot of QCAL-IM peptide ion Ω values versus m/z: Ω values were obtained on the modified Synapt with a linear field mobility cell, using helium drift gas.

consequent Ω calibration range for each observed charge state is thus 197.7−261.7 Å2 (singly charged), 197.0−435.9 Å2 (doubly charged) and 300.8−616.7 Å2 (triply charged). It is interesting to note that peptides observed in both +1 and +2 charge states exhibit minimal difference in Ω values. However, there is a notable increase in Ω between the +2 and +3 species of the same sequence: Ω values for IGSEVYHNLK are 261.8 ± 2.2 Å2 and 300.8 ± 3.3 Å2 for +2 and +3 charge states respectively, with HGTVVLTALGGILK exhibiting Ω values of D

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297.6 ± 2.1 and 353.4 ± 5.8 Å2 for the +2 and +3 charge states. We attribute these differences to the more significant charge repulsion in the triply charged peptides, causing expansion/ opening of the gas-phase structure and hence increased Ω values. Measurement of the peptide ion Ω values using more than one instrumental platform improves confidence in the derived values for the standards and critically provides confidence in the process of interlaboratory cross section determination. Table 2 shows a comparison of Ω values Table 2. Comparison of Calculated Ω for [M + 2H]2+ Peptide Ions from Studies Undertaken on the Modified Synapt and Q-TOF Linear Field Mobility Instrumentsa peptide T2/L5 T3 T4 T6 T7/ L10 T8/ L11 T14 T15 T19

modified synapt Ω (Å2)

ΔΩ (Å2)

%Ω difference

m/z

modified QTOF Ω (Å2)

(2.2) (1.4) (1.8) (0.4) (2.1)

580.66 709.28 790.38 392.45 575.66

248.9 279.2 305.3 198.2 242.5

(14.0) (10.6) (15.3) (4.8) (10.2)

12.9 9.8 7.3 14.6 14.3

4.9 3.4 2.3 6.9 5.6

297.6 (2.0)

690.34

292.7 (10.1)

5.0

1.7

284.2 (0.6) 435.9 (0.1) 349.1 (0.6)

721.75 1255.20 912.01

26.1 −14.8 14.4

9.2 −3.4 4.1

261.8 289.0 312.6 212.9 256.8

258.1 450.7 334.6

a

The values in parentheses indicate the standard deviation (or range when only two measurements are available) associated with each measurement (n = 2−4). When parentheses are absent, n = 1.

calculated following measurements on the modified Synapt and a Q-TOF linear field mobility instrument. There is excellent correlation (R2 = 0.989, Figure 3S, Supporting Information) between the values determined on these two platforms and importantly good agreement, with a mean of 4.6% variation in the measured Ω values (maximally 9.2% for [M + 2H]2+ of GVNDNEEGFFSAR at m/z 721.74). This is within expected experimental variation as determined by the standard deviations detailed in Table 2, suggesting that potential effects arising from differing instrument geometries (and hence ion energies and/or experimental time scale) are not significantly influencing the collision cross section.

Figure 2. Plot of drift times versus m/z for QCAL-IM tryptic and LysC peptides following mobility separation on the T-Wave Synapt using nitrogen drift gas. (A) Driftscope plot; (B) extracted ion information.

Assessment of QCAL-IM Peptides for T-Wave Mobility Calibration

Having defined Ω for the multiple peptide ion species, the same proteolyzed samples were analyzed on a standard Synapt HDMS instrument with a T-Wave mobility cell. The drift times for each of the observed ions were plotted against m/z, showing separate trends for each ion charge state (Figure 2). Using the values from Table 1, Ω versus drift time calibration lines were produced for charge states +1 to +3, each yielding linear curve fits with R2 values of >0.99, as displayed in Figure 3. The observed linearity of these plots differs from the approximately square root dependence expected;19,20 this likely due to the use of a T-Wave voltage ramp (rather than a fixed voltage) and the range of drift times being analyzed, with the species in question comprising a near-linear subsection of this square root dependence between drift time and Ω. Figure 4 is a plot of Ω′ (determined from Ω adjusted for the charge state and reduced mass of the ions27) versus T-Wave drift time which shows good linear correlation (R2 = 0.982, Figure 4), and provides a more generic calibration curve. Plots such as those in

Figure 3. Drift time versus Ω charge dependent calibration lines for QCAL-IM peptides. Both vertical and horizontal error bars are smaller than any given data point.

Figures 3 and 4 therefore enable the Ω value of any analyte that falls within the covered calibration range to be calculated directly from the experimentally measured T-Wave drift time. Comparison with Literature Values

A potential concern when using peptide Ω values calculated in helium buffer gas (ΩHe) to calibrate the T-Wave drift times in nitrogen buffer gas are the drift gas specific effects on ion mobility. To test the validity of the calibration of the T-Wave mobility separation with QCAL-IM, a selection of peptides E

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and 4 for single and doubly charged ions respectively and demonstrate linear correlation (R2 = 0.997), with a mean difference of 3.9% across 18 peptides analyzed in triplicate. This excellent correlation between the calibrated T-Wave cross sections as calculated following experimentally determined drift times in nitrogen and those previously published from linear mobility experiments in helium provides a high degree of confidence in the method of cross section calibration. Importantly, it should be noted that this method of calibration permits calculation of the ΩHe values from arrival time distributions in a nitrogen buffer gas and potential drift gas effects are accounted for in the calibration process. Assessment of Mobility Separation Resolution

A number of the QCAL-IM peptide ions (particularly the quadruply protonated species generated following Lys-C digestion) are observed as heterogeneous populations with respect to drift time, indicating the presence of more than one conformation. While these ions cannot easily be used to generate a drift time calibration line (since the conformers may not always be readily distinguished, depending on mobility instrument resolution), they can be used as a means of assessing the relative resolution of the mobility separation either under a given set of experimental conditions or between different instruments (Figures 6 and 7). The degree of separation (or not) of these mixed conformers can thus be used to determine the ability of a given setup or mobility device to separate ions of similar m/z and drift time. For example, the distribution of drift times for the quadruply protonated T19/20 LysC peptide at m/z 1266.64, illustrates the resolution difference between the first and second generation Synapt instruments (Figure 6a and b, respectively). The broad distribution of drift times at the earlier arrival time (∼5120 μs, Figure 6a) suggests multiple closely related conformers that could not be resolved on the first generation Synapt instrument. However, two distinct species (Conformers 1 and 3) were resolved following analysis on the Synapt G2. Again, this is observed during analysis of the quadruply protonated T16/T17/T18 LysC peptide at m/z 1154.56 (Figure 7). A broad arrival time distribution was observed when analyzed on the first generation instrument (Figure 7a) from which multiple conformers were resolved during analysis on the second generation instrument (Figure 7b). Such analysis can permit calculation of the peak to peak resolution (Rpp) if required using the following equation:

Figure 4. Plot of charge and reduce mass corrected Ω (Ω′) versus drift time for QCAL-IM peptides.

included in the Clemmer database30 were analyzed on the TWave instrument and their Ω values (ΩHe) determined following extrapolation of the derived calibration lines (Figure 3). The comparative data are shown in Figure 5, and Tables 3

Figure 5. Linear regression of published Ω30 against average calculated Ω (n = 3) following T-Wave mobility calibration with QCAL-IM, for a selection of [M + H]+ and [M + 2H]2+ peptides.

Table 3. Comparison of Published Ω30 with the Average QCAL-IM Calculated Ω (n = 3) for a Selection of [M + H]+ Peptidesa peptide

MW (Da)

EK LR QR FPK DTHK VASLR AWSVAR ATEEQLK HLK ALELFR ELGFQG NDIAAK

275.16 287.20 302.18 390.23 499.25 544.34 688.37 817.43 396.26 747.44 649.31 630.34

literature Ω (Å2) 97.89 110.03 104.07 123.14 142.74 163.7 185.37 206.40 133.05 202.99 172.29 173.78

(1.14) (1.44) (1.92) (0.85) (1.62) (0.92) (0.81) (1.81) (0.71) (0.33) (0.14) (0.84)

QCAL-IM Ω (Å2)

ΔΩ (Å2)

%Ω difference

91.3 102.9 97.7 115.6 136.2 154.3 181.3 204.8 129.8 199.1 168.7 170.9

6.6 7.1 6.4 7.6 6.6 9.4 4.0 1.6 3.3 3.9 3.6 2.9

6.7 6.4 6.1 6.2 4.6 5.7 2.2 0.8 2.5 1.9 2.1 1.7

R pp =

2Δtd (wb1 + wb2)

(3)

where Δtd is the difference in drift time between the peaks of interest, and wb1 and wb2 are the measured peak widths at half height. Analysis of these highly charged peptide ions with multiple conformers derived from QCAL-IM thus enables the resolution performance of an ion mobility instrument to be monitored, with individual laboratories able to define a set of species that should be resolved with a given mobility cell under a defined set of conditions.

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CONCLUSIONS QCAL-IM (Figure 2S, Supporting Information) is a designer protein which yields a stoichiometric mixture of specific peptides for ion mobility calibration upon proteolysis. Together these peptides enable calibration of T-Wave ion mobility

Values in parentheses following the literature Ω indicate the associated standard deviation. a

F

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Table 4. Comparison of Published Ω30 with the Average QCAL-IM Calculated Ω (n = 3) for a Selection of [M + 2H]2+ Peptidesa

a

peptide

MW (Da)

HLVDEPQNLIK RHPEYAVSVLLR LFTGHPETLEK HGTVVLTALGGILK VEADIAGHGQEVLIR GLSDGEWQQVLNVWGK

1304.72 1438.81 1270.66 1377.84 1605.85 1814.90

literature Ω (Å2) 290.36 313.44 286.20 312.22 340.95 352.48

(0.83) (2.35) (4.02) (0.49) (1.0) (0.52)

QCAL-IM Ω (Å2)

Δ Ω (Å2)

% Ω difference

272.7 291.6 282.5 296.9 333.1 343.3

17.6 21.8 3.7 15.3 7.9 9.2

6.1 7.0 1.3 4.9 2.3 2.6

Values in parentheses following the literature Ω indicate the associated standard deviation.

published by Clemmer.30 Data have been obtained for each of the constituent calibrant peptides included within the standard from both linear field and T-Wave mobility instruments. Application of QCAL-IM as an ion mobility standard thus enables direct calculation of peptide ion Ω for charge states +1 to +3 with any employed T-Wave experimental conditions. In addition, the standard can be used to monitor the performance of the IMS cell toward separation of species with similar mobility by assessing separation of different conformers of multiply protonated species. While differences in size, number of bonds and associated degrees of freedom between peptides (as analyzed here) and other classes of small molecules (e.g., lipids) may result in differing effects of any ion heating on the measured cross section, we anticipate that such differences may not be large;37 therefore it is likely that the QCAL-IM could be used for calibration of other species which have CCS values within the appropriate range, although additional studies would be needed to confirm this. Finally, due to the simple manner in which the recombinant protein standard is produced, QCALIM is available in limitless supply and can be kept as a stock solution, obtained from commercial sources, to be analyzed alongside the analyte of interest.

Figure 6. Mobility separation on the (A) first generation Synapt and the (B) higher resolution Synapt G2 of the [M + 4H]4+ peptide T19/ 20 at m/z 1266.64 produced from Lys-C proteolysis of QCAL-IM.

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ASSOCIATED CONTENT

* Supporting Information S

Supplementary figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Manchester Institute of Biotechnology, Michael Barber Centre for Mass Spectrometry, School of Chemistry, University of Manchester, 131 Princess Street, Manchester, M1 7DN. Email: [email protected]. Tel.: +44 161 306 5168. Fax: +44 161 306 5201.

Figure 7. Mobility separation on the (A) first generation Synapt and the (B) higher resolution Synapt G2 of the [M + 4H]4+ peptide T16/ T17/T18 at m/z 1154.56 produced from Lys-C proteolysis of QCALIM.

Notes

separations, thus providing a simple method of empirically determining peptide ion Ω values using a single standard. Unlike previous attempts at calibrating drift times through TWave mobility cells that involve the analysis of multiple protein digests,2,3,9,15,16 the strategy described only requires analysis of a single digested standard and additionally can readily be used for calibration of triply protonated species. QCAL-IM is commercially available (Entelechon GmbH) to laboratories wishing to perform T-Wave mobility calibration for the determination of ΩHe values of peptides and peptide fragment ions, making it a significantly more convenient alternative to the widely used calibration standard peptides, derived from multiple sources and detailed in the database previously

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS C.E. acknowledges the Royal Society for a Dorothy Hodgkin Research Fellowship. This work was supported in part by a Biotechnology and Biological Sciences Research Council Grant (BB/C007735/1) to S.J.G.. R.C. is supported by an EPSRC Case Studentship in collaboration with Waters UK Ltd. We thank PolyQuant GmbH for gene design and construction. The authors thank Professor Carol Robinson for access to the modified Synapt instrument along with Zoe Hall, Dr. Martin De Cecco and Ewa Jurneczko for their assistance. G

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stereoisomers of ephedra alkaloids in natural health products using flow injection-electrospray ionization-high field asymmetric waveform ion mobility spectrometry-mass spectrometry. Anal. Chem. 2003, 75 (11), 2538−42. (16) Mason, E. A.; McDaniel, E. W. Transport properties of ions in gases; Wiley: New York, 1988. (17) Merenbloom, S. I.; Flick, T. G.; Williams, E. R. How Hot are Your Ions in TWAVE Ion Mobility Spectrometry? J. Am. Soc. Mass Spectrom. 2012, 23 (3), 553−62. (18) Giles, K.; Pringle, S. D.; Worthington, K. R.; Little, D.; Wildgoose, J. L.; Bateman, R. H. Applications of a travelling wavebased radio-frequency only stacked ring ion guide. Rapid Commun. Mass Spectrom. 2004, 18 (20), 2401−14. (19) Shvartsburg, A. A.; Smith, R. D. Fundamentals of traveling wave ion mobility spectrometry. Anal. Chem. 2008, 80 (24), 9689−99. (20) Wildgoose, J. L.; Giles, K.; Pringle, S. D.; Koeniger, S.; Valentine, S.; Bateman, R. H.; Clemmer, D. E. In ASMS Proceedings, Seattle, 2006. (21) Bush, M. F.; Hall, Z.; Giles, K.; Hoyes, J.; Robinson, C. V.; Ruotolo, B. T. Collision Cross Sections of Proteins and Their Complexes: A Calibration Framework and Database for Gas-Phase Structural Biology. Anal. Chem. 2010, 82 (22), 9557−65. (22) Riba Garcia, I.; Giles, K.; Bateman, R. H.; Gaskell, S. J. Studies of peptide a- and b-type fragment ions using stable isotope labeling and integrated ion mobility/tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 2008, 19 (12), 1781−7. (23) Riba-Garcia, I.; Giles, K.; Bateman, R. H.; Gaskell, S. J. Evidence for structural variants of a- and b-type peptide fragment ions using combined ion mobility/mass spectrometry. J. Am. Soc. Mass Spectrom. 2008, 19 (4), 609−13. (24) Chawner, R.; Gaskell, S. J.; Eyers, C. E. Proposal for a common nomenclature for peptide fragment ions generated following sequence scrambling during collision-induced dissociation. Rapid Commun. Mass Spectrom. 2012, 26 (2), 205−6. (25) Shvartsburg, A. A.; Creese, A. J.; Smith, R. D.; Cooper, H. J. Separation of peptide isomers with variant modified sites by highresolution differential ion mobility spectrometry. Anal. Chem. 2010, 82 (19), 8327−34. (26) Xuan, Y.; Creese, A. J.; Horner, J. A.; Cooper, H. J. High-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled with high-resolution electron transfer dissociation mass spectrometry for the analysis of isobaric phosphopeptides. Rapid Commun. Mass Spectrom. 2009, 23 (13), 1963−9. (27) Ruotolo, B. T.; Benesch, J. L. P.; Sandercock, A. M.; Hyung, S. J.; Robinson, C. V. Ion mobility-mass spectrometry analysis of large protein complexes. Nat. Protoc. 2008, 3 (7), 1139−52. (28) Williams, J. P.; Scrivens, J. H. Coupling desorption electrospray ionisation and neutral desorption/extractive electrospray ionisation with a travelling-wave based ion mobility mass spectrometer for the analysis of drugs. Rapid Commun. Mass Spectrom. 2008, 22 (2), 187− 96. (29) Valentine, S. J.; Counterman, A. E.; Clemmer, D. E. A database of 660 peptide ion cross sections: Use of intrinsic size parameters for bona fide predictions of cross sections. J. Am. Soc. Mass Spectrom. 1999, 10 (11), 1188−211. (30) Clemmer, D. E. Cross Section Database, http://www.indiana. edu/∼clemmer/Research/cross%20section%20database/ cs%20database.htm. (31) Beynon, R. J.; Doherty, M. K.; Pratt, J. M.; Gaskell, S. J. Multiplexed absolute quantification in proteomics using artificial QCAT proteins of concatenated signature peptides. Nat. Methods 2005, 2 (8), 587−9. (32) Pratt, J. M.; Simpson, D. M.; Doherty, M. K.; Rivers, J.; Gaskell, S. J.; Beynon, R. J. Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes. Nat. Protoc. 2006, 1 (2), 1029−43. (33) Carroll, K. M.; Simpson, D. M.; Eyers, C. E.; Knight, C. G.; Brownridge, P.; Dunn, W. B.; Winder, C. L.; Lanthaler, K.; Pir, P.; Malys, N.; Kell, D. B.; Oliver, S. G.; Gaskell, S. J.; Beynon, R. J.

ABBREVIATIONS (n)ESI, (nano) electrospray ionization; IM, ion mobility; MS, mass spectrometry; RF, radio frequency; T-Wave, Traveling wave.

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REFERENCES

(1) Pringle, S. D.; Giles, K.; Wildgoose, J. L.; Williams, J. P.; Slade, S. E.; Thalassinos, K.; Bateman, R. H.; Bowers, M. T.; Scrivens, J. H. An investigation of the mobility separation of some peptide and protein ions using a new hybrid quadrupole/travelling wave IMS/oa-ToF instrument. Int. J. Mass Spectrom. 2007, 261 (1), 1−12. (2) Baker, E. S.; Dupuis, N. F.; Bowers, M. T. DNA hairpin, pseudoknot, and cruciform stability in a solvent-free environment. J. Phys. Chem. B 2009, 113 (6), 1722−7. (3) Bohrer, B. C.; Mererbloom, S. I.; Koeniger, S. L.; Hilderbrand, A. E.; Clemmer, D. E. Biomolecule Analysis by Ion Mobility Spectrometry. Annu. Rev. Anal. Chem. 2008, 1, 293−327. (4) Wyttenbach, T.; Grabenauer, M.; Thalassinos, K.; Scrivens, J. H.; Bowers, M. T. The effect of calcium ions and peptide ligands on the relative stabilities of the calmodulin dumbbell and compact structures. J. Phys. Chem. B 2010, 114 (1), 437−47. (5) Scarff, C. A.; Thalassinos, K.; Hilton, G. R.; Scrivens, J. H. Travelling wave ion mobility mass spectrometry studies of protein structure: biological significance and comparison with X-ray crystallography and nuclear magnetic resonance spectroscopy measurements. Rapid Commun. Mass Spectrom. 2008, 22 (20), 3297−304. (6) Smith, D. P.; Knapman, T. W.; Campuzano, I.; Malham, R. W.; Berryman, J. T.; Radford, S. E.; Ashcroft, A. E. Deciphering drift time measurements from travelling wave ion mobility spectrometry-mass spectrometry studies. Eur. J. Mass Spectrom. 2009, 15 (2), 113−30. (7) Barran, P. E.; Jurneczko, E. How useful is ion mobility mass spectrometry for structural biology? The relationship between protein crystal structures and their collision cross sections in the gas phase. Analyst 2011, 136 (1), 20−8. (8) Erba, E. B.; Ruotolo, B. T.; Barsky, D.; Robinson, C. V. Ion Mobility-Mass Spectrometry Reveals the Influence of Subunit Packing and Charge on the Dissociation of Multiprotein Complexes. Anal. Chem. 2010, 82 (23), 9702−10. (9) Cole, H. L.; Kalapothakis, J. M.; Bennett, G.; Barran, P. E.; Macphee, C. E. Characterizing early aggregates formed by an amyloidogenic peptide by mass spectrometry. Angew. Chem., Int. Ed. 2010, 49 (49), 9448−51. (10) Bernstein, S. L.; Dupuis, N. F.; Lazo, N. D.; Wyttenbach, T.; Condron, M. M.; Bitan, G.; Teplow, D. B.; Shea, J. E.; Ruotolo, B. T.; Robinson, C. V.; Bowers, M. T. Amyloid-beta protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer’s disease. Nat. Chem. 2009, 1 (4), 326−31. (11) Smith, D. P.; Radford, S. E.; Ashcroft, A. E. Elongated oligomers in beta(2)-microglobulin amyloid assembly revealed by ion mobility spectrometry-mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 2010, 107 (15), 6794−8. (12) Thalassinos, K.; Grabenauer, M.; Slade, S. E.; Hilton, G. R.; Bowers, M. T.; Scrivens, J. H. Characterization of Phosphorylated Peptides Using Traveling Wave-Based and Drift Cell Ion Mobility Mass Spectrometry. Anal. Chem. 2009, 81 (1), 248−54. (13) De Cecco, M.; Seo, E. S.; Clarke, D. J.; McCullough, B. J.; Taylor, K.; MacMillan, D.; Dorin, J. R.; Campopiano, D. J.; Barran, P. E. Conformational preferences of linear beta-defensins are revealed by ion mobility-mass spectrometry. J. Phys. Chem. B 2010, 114 (6), 2312−8. (14) Mie, A.; Jornten-Karlsson, M.; Axelsson, B. O.; Ray, A.; Reimann, C. T. Enantiomer separation of amino acids by complexation with chiral reference compounds and high-field asymmetric waveform ion mobility spectrometry: Preliminary results and possible limitations. Anal. Chem. 2007, 79 (7), 2850−8. (15) McCooeye, M.; Ding, L.; Gardner, G. J.; Fraser, C. A.; Lam, J.; Sturgeon, R. E.; Mester, Z. Separation and quantitation of the H

dx.doi.org/10.1021/pr3005327 | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

Technical Note

Absolute Quantification of the Glycolytic Pathway in Yeast: Deployment of a Complete QconCAT Approach. Mol. Cell. Proteomics 2011, 10 (12), M111 007633. (34) Eyers, C. E.; Simpson, D. M.; Wong, S. C.; Beynon, R. J.; Gaskell, S. J. QCAL–a novel standard for assessing instrument conditions for proteome analysis. J. Am. Soc. Mass Spectrom. 2008, 19 (9), 1275−80. (35) McCullough, B. J.; Kalapothakis, J.; Eastwood, H.; Kemper, P.; MacMillan, D.; Taylor, K.; Dorin, J.; Barran, P. E. Development of an ion mobility quadrupole time of flight mass spectrometer. Anal. Chem. 2008, 80 (16), 6336−44. (36) Eyers, C. E.; Lawless, C.; Wedge, D. C.; Lau, K. W.; Gaskell, S. J.; Hubbard, S. J. CONSeQuence: prediction of reference peptides for absolute quantitative proteomics using consensus machine learning approaches. Mol. Cell. Proteomics 2011, 10 (11), M110 003384. (37) Williams, J. P.; Scrivens, J. H. Coupling desorption electrospray ionisation and neutral desorption/extractive electrospray ionisation with a travelling-wave based ion mobility mass spectrometer for the analysis of drugs. Rapid Commun. Mass Spectrom. 2008, 22, 187−96.

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