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Resolution ladder for high resolution mass spectrometry Matthias Schittmayer, and Ruth Birner-Gruenberger Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02042 • Publication Date (Web): 28 Aug 2017 Downloaded from http://pubs.acs.org on August 30, 2017

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Analytical Chemistry

RESOLUTION LADDER FOR HIGH RESOLUTION MASS SPECTROMETRY

Matthias Schittmayer1,2,3, Ruth Birner-Gruenberger1,3,* 1 Research Unit Functional Proteomics and Metabolic Pathways, Institute of Pathology, Medical University of Graz, Stiftingtalstrasse 24, 8010 Graz, Austria 2 Institute of Molecular Systems Biology, ETH Zürich, 8093 Zürich, Switzerland 3 Omics Center Graz, BioTechMed-Graz, 8010 Graz, Austria

*Corresponding author: Ruth Birner-Gruenberger, Phone: +43 (0)316 385-72962, Email: [email protected]

Abstract High resolution mass spectrometry has become a key technology in life sciences. Since it is often unfeasible to find pairs of analytes with an appropriate mass difference to actually quantify the resolution experimentally, resolution is usually calculated from the shape of a single mass peak. In this study we show that the commonly employed strategy yields a poor measure of true resolution since it does not account for interactions that take place between ions of very similar mass and might be further distorted by signal processing effects. We present a straightforward and easily adaptable method to create a ladder of mass pairs to experimentally quantify actual mass resolution over a wide m/z range, compare the experimental resolution to the single peak based calculated resolution and demonstrate the applicability of mass resolution ladders to study interactions of similar ions in various types of widely used mass spectrometers.

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High resolution mass spectrometry has become a dominant method in life sciences. The ability to reliably discriminate isobaric analytes and determine their accurate mass enables non-targeted analysis of thousands of molecular features in a single analysis. While mass accuracy and resolution are independent in theory, this only holds true when it can be ensured that only a single elemental composition contributes to the mass spectral peak in question1. Non-resolved peaks can therefore lead to misassigned peaks by impacting mass accuracy. Another potential detrimental effect of failing to resolve two peaks is impaired quantification since peak areas are accumulative and non-resolved peaks will appear as one larger peak. To be able to estimate these effects, mass resolution has to be considered when interpreting high resolution mass spectrometry data. Mass resolution is defined by the IUPAC as mass (m) divided by mass difference (Δm) of two peaks which can be resolved (Eq. 1). Equation 1

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ܴ݁‫∆ = ݊݋݅ݐݑ݈݋ݏ‬௠

resolution definition by the IUPAC

The de facto standard to measure mass resolution is to calculate it from the shape of a single peak, since feasible pairs requiring exactly the resolution determined by the mass spectrometer to be resolved are usually not readily available. The single peak approach, however, does not take into account interactions between ions of very similar mass and signal processing effects. This potentially leads to an overestimation of experimental resolution. In this study we present the concept of a mass resolution ladder and show how it can be applied to experimentally measure and study effects impacting mass resolution.

Experimental Section A mixture of glycine oligomers with n = 5 – 8 (equal mass fraction) was purchased from piChem (Raaba-Grambach, Austria). All other chemicals and solvents used were purchased from Sigma Aldrich (Vienna, Austria). Polyglycine based mass resolution ladder Individual glycine oligomers (n = 1 - 4) were dissolved as stock solutions at 0.1 mg / mL and the glycine oligomer mix (n = 5 - 8) at 0.4 mg / mL in 50 mM sodium hydroxide, respectively. 10 µL of each stock solution were mixed and the mixture was diluted with 50 % acetonitrile to a final volume of 1 mL. The sample was divided and subjected to reductive methylation over night at 37 °C by adding either formaldehyde-d2 (final concentration 0.2 %) and sodium cyanoborodeuteride (final concentration 30 mM, heavy) or formaldehyde-13C, d2 and sodium cyanoborohydride (same concentrations, light, also see Fig. S1). The resulting light and heavy oligomer mix was either diluted 1:10 and measured separately or mixed one plus one and the resulting mixture was diluted 1:5.

Polyethylene glycol based mass resolution ladder (Δm = 12.2 mmu) To 1 mL of a solution of either glycine-2-13C,15N (3 equivalents, 13 µmol, light 1 mg / mL) or glycine2,2-d2 (3 eq., 13 µmol, heavy, 1 mg / mL) in 50 % acetonitrile, 50 µL of a solution of O-[(Nsuccinimidyl)succinyl-aminoethyl]-O′-methylpolyethylene glycol (750) (1 eq., 4.3 µmol, 64 mg / mL) in 50 % acetonitrile was added. After addition of 1.1 µL (1.5 eq., 6.5 µmol) of diisopropylethylamine the mixture was incubated under shaking for 1 h at 37 °C. The resulting light and heavy labeled polyethylene glycols were either diluted 1:10 for separate measurement or mixed one plus one and the mixture diluted 1:5.


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Polyethylene glycol based mass resolution ladder (Δm = 18.0 mmu) To 1 mL of a solution of either glycine-2-13C2 (light, 3 equivalents, 13 µmol, 1 mg / mL) or glycine2,2-d2 (heavy, 3 eq., 13 µmol, 1 mg / mL) in H2O, 50 µL of a solution of O-[(N-succinimidyl)succinylaminoethyl]-O′-methylpolyethylene glycol (750) (1 eq., 4.3 µmol, 64 mg / mL) in H2O was added. After addition of 1.1 µL (1.5 eq., 6.5 µmol) of diisopropylethylamine the mixture was incubated under shaking for 1 h at 37 °C. The reaction mixture was loaded onto an Agilent C18 Bond Elut (50 mg / 1 mL) SPE cartridge and washed with 1 mL H2O to remove excess glycine. The single glycine labeled PEG was then eluted with 1 mL acetonitrile, the solvent was removed in a vacuum concentrator and the PEG was resuspended in 200 µL H2O. The carboxy terminus of the bound glycine was activated with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (1 eq., 4.3 µmol, 0.8 mg) / Nhydroxysuccinimide (3 eq., 13 µmol, 1.5 mg) and either glycine-2-13C, 15N (light, 1 eq., 4.8 µmol, 1 mg / mL) or glycine -2,2-d2 (heavy, 1 eq., 4.8 µmol, 1 mg / mL) was added. The sample was incubated again for 1h at 37 °C yielding PEG labeled with 2 glycines.

Detailed instrument parameters are available in the supplementary information and from the original data files which can be downloaded from http://omicscentergraz.at/resources.html

Results and Discussion The major practical hindrance of experimental measurement of mass resolution is the lack of feasible peak pairs which require a resolution predetermined by the mass spectrometer to be resolved. We have devised a simple yet effective way to cover a wide range of resolutions in a single experiment. Crafting molecular labels with a given mass difference on a low molecular weight polymer with high polydispersity will result in a ladder of mass pairs with fixed ∆m. As evident from Eq. 1, resolution requirements increase stepwise for each increase in polymer number, resulting in a mass resolution ladder. Small mass differences can be introduced through the different mass defects of stable isotopes2 (Table S1). Combining several of these differences allows to conveniently cover a wide ∆m range for the resulting pairs. Another important aspect in creating a mass spectrometric resolution ladder is the backbone carrying the mass labels. Low molecular weight polymers with high polydispersity are ideal carriers for the mass labels, however solubility in solvents commonly used in mass spectrometry has to be ensured. The monomer mass defines the spacing between the individual peaks and therefore also the resolution steps. Groups that can be easily ionized in positive and negative mode add sensitivity and flexibility. Finally, the backbone should have functional groups which can be specifically targeted for mass labelling, ideally by simple aqueous chemistry. We examined whether we can reproduce resolutions calculated from single peak FWHM experimentally on three types of high resolution mass spectrometers available in our laboratory. The m/z and resolution range of each mass resolution ladder was adapted to match the resolution characteristics of the used mass spectrometer. Time of Flight Time of flight (TOF) mass spectrometers achieve their highest resolution at higher m/z. The maXis II (Bruker Daltonics) is a very high resolution TOF platform with a single peak FWHM specification of 80,000 at 1200 m/z. Plotting the theoretical TOF resolution characteristic (Eq. S1) versus the theoretical resolution requirements of mass resolution ladders with various ∆m allows estimation of ACS Paragon Plus Environment


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suitable ∆m for this instrument with an intersection of the two functions in the preferred m/z range of the mass spectrometer (Fig. 1A). The slope of the solid line is defined by ∆m. Heavy isotope labelled amino acids are a great source to introduce various ∆m as they are commercially available in a wide variety at comparably low cost. Moreover, they can be combined arbitrarily by standard peptide synthesis, adding great flexibility whenever needed. Finally, coupling chemistry for amino, carboxy and thiol groups is very well established. We initially opted to use either glycine-2,2-d2 or glycine-213 C, 15N as mass label for the TOF resolution ladder, resulting in a ∆m of 12.2 mmu. As backbone we chose a commercially available polyethylene glycol 750 (PEG) carrying a succinic acid linker preactivated with N-hydroxy succinimide (Fig. 1C). This combination of mass label and backbone results in an effective mass range of 700 to 1300 m/z and resolution requirements of 5.3 x 104 to 1.1 x 105. We first examined single peak calculated FWHM resolution for individual measurements of glycine2,2-d2 and glycine-2-13C, 15N labelled samples. As expected, the values reported by the vendor software exceeded the theoretically required values (from 6.7 to 7.8 x 104) for all peaks ≤ 895.5 m/z. We then proceeded to test whether all peak pairs up to this m/z limit can be resolved in a mixed sample. The labelled samples were mixed at equal concentration and diluted to match individual concentrations in the separate samples. Signal intensities were in the range of 2 x 106, well below the theoretical saturation limit of the detector (7.8 x 106). However, none of the peak pairs of the mixed sample was resolved and no signs of separation were observed upon manual inspection. The resolution values reported for the mixed samples were 20 to 40 % lower and well below the required resolution for separation of all the mass pairs. To pinpoint the experimental resolution we modified the mass resolution ladder by adding either a second deuterated glycine or a 13C labelled glycine, increasing the ∆m to 18.0 mmu. The mass pair at m/z 1130 was the last resolved one of this ladder, corresponding to an experimental resolution of 6.3 x 104 (Fig. 1A) while the lowest reported single peak calculated resolution of individual measurements was 7.5 x 104. We therefore conclude that the resolution calculated from single peak FWHM is considerably overestimated. Fourier transformation based mass spectrometers. Fourier transform ion cyclotron resonance (FT-ICR) and Orbitrap based mass spectrometers achieve their highest resolutions at low m/z. For the low m/z range preferred by FT based mass spectrometers we used a homo-oligomer of glycine (n = 1-8) as a backbone. Modifying the N-terminal amine by different reductive methylation3, 4 schemes (Fig. S1) results in a twin-series of m/z pairs with a Δm of approximately 5.8 mmu (Fig. 1B,D) and a mass range of 110 to 509 m/z. We first compared single peak based FWHM with experimental resolution of an FT-ICR type instrument (LTQ FT Ultra, 7 Tesla, Thermo Fisher Scientific, set resolutions 25 k, 50 k and 100 k at 400 m/z). The resolution of the individual peaks was determined by measuring the light and heavy labelled oligo-glycine separately as well as in an equimolar mixture. The resolution as reported by the vendor software was then compared to the ability to resolve peaks in the mixed samples. Interestingly, the resolution reported in the mixed sample was slightly higher almost up to the theoretical resolution limit for each set instrument resolution. Exceeding the theoretical resolution resulted, as expected, in a not resolved, wide peak. For the FT-ICR, single peak based full width at half maximum (FWHM) resolution was in good agreement with the actual ability to separate mass pairs. The same oligo-glycine based mass resolution ladder was also analysed on an Orbitrap Velos Pro (Thermo Fisher Scientific, set resolutions 30 k, 60 k and 100 k at 400 m/z). Despite very high resolutions reported by the vendor software, none of the mass pairs in the mixed sample was resolved in our initial experiment. For example, using a 1 Da scan window the mass pair at m/z 167 was ACS Paragon Plus Environment

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reported as a single peak with an alleged resolution of 2 x 105 while a true resolution of 2 x 104 FWHM suffices for separation. The same effect was also observed for the most intense peaks in full scan mode with scan window sizes up to 350 m/z. The cause was quickly revealed to be a rather extreme example of ion coalescence resulting from phase-locking of ion clouds of similar m/z in FT type analysers. Gorshkov et al.5 reported ion coalescence effects in an Orbitrap Elite for a single mass pair consisting of bradykinin and a custom synthesized bradykinin isobar with a nominal mass of 1059 and Δm of 22.5 mmu (22 ppm). The onset of ion coalescence for their specific mass pair was determined at a fill target of 7.5 x 104. Accordingly, changing fill targets from 1 x 106 (recommended for full scan MS) to 5 x 104 (recommended for SIM) resulted in effective separation of the peaks. We want to point out that the initially used fill target (1 x 106) is widely employed in shotgun proteomics6 and metabolomics7 experiments. Especially disconcerting was the high resolution reported for fully coalesced peaks, which was far in excess of the theoretically required resolution of the peak pair (Fig. 2D). This can easily lead to misinterpretation of data. While lowering fill targets markedly reduced coalescence effects (Fig. 2A-C) it also potentially results in a loss of sensitivity and decreased dynamic range. It has to be noted, however, that in complex samples the same total number of ions is distributed to a multitude of individual ion packets resulting in lower absolute ion numbers for most ion packets, short of the most intense ones. Our mass resolution ladder approach allows for the first time to systematically investigate the ion coalescence effect over a wide m/z range and in dependence of relative Δm. Mass pairs were measured at stepwise increased fill targets. Exceeding certain fill targets leads to interaction of ion packets in the Orbitrap analyser and peaks move closer to each other on the mass axis. We defined the onset of peak coalescence as the point where the peaks’ movement towards each other exceeded the mass accuracy limits specified for internal calibration (1 ppm, Δmmeasured < Δmtheoretical – 2 x mass accuracy). As can be seen in Fig. 2E, higher m/z pairs show onset of coalescence at lower signal intensities because their relative mass difference is smaller. The peak pair at 224 m/z showed a deviation from the general trend which might either be explained by increased transmission in this m/z range or by the limited number of steps for the fill targets tested. Interestingly, while ion coalescence effects were initially described on FT-ICR instruments8, we did not observe any negative effects on mass accuracy or mass resolution at the vendor recommended fill target (5 x 105) in our FT-ICR experiments. One potential explanation for this is the far more efficient ion transmission in the Orbitrap system. The highest observed summed signal to noise ratio for a peak pair observed was around 1.5 x 103 for the FT-ICR while the Orbitrap signal to noise ratios at the vendor recommended fill target (1x 106) reached up to 1.4 x 105. After tracing out the limits of ion coalescence with respect to relative mass difference we examined whether these effects also play a role at standard fill targets in more routine applications such as bottom up proteomics. Werner et al.9 reported ion coalescence effects at MS2 level for 10-plex TMT reporter ions and Tarasova et al.10 used a set of synthetic peptides to investigate coalescence under typical bottom-up proteomics settings. They concluded that in a general label-free proteomics experiment coalescence should not be an issue. In contrast when we reanalysed a complex proteomics data set (200 ng HeLa digest), acquired at the manufacturer’s recommended fill target (1 x 106), we did find signs of coalescence. Roughly 5 % of high confidence peptides co-eluted within 0.2 min of an isobar in a 50 ppm distance. Co-eluting isobars had a significantly higher mass error variance (p < 1038 ) and absolute mass error compared (p < 10-4) to the other peptides. The fraction of peptides outside the specified mass accuracy with external calibration (3 ppm) for co-eluting and other peptides was 18 % and 7 %, respectively. Finally, the average mass error of the lower ion of each pair was 0.28 ppm while the error of the upper ion was -1.60 ppm on average, both significantly different from the average mass error of all peptides in the dataset of -0.33 ppm (p < 0.001). Peptides overlapping with more than one co-eluting isobar were excluded from the last analysis. The shift of the ion pairs



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towards each other clearly indicates that ion coalescence is at least partially responsible for the decreased mass accuracy of co-eluting isobars.

Conclusion Despite the fact that ion clouds of similar characteristics interact in the limited space of a mass spectrometer, it has become de facto standard to calculate instrument resolution from peaks originating from a single, isolated ion cloud. In this study we demonstrate that single peak calculated FWHM based resolution insufficiently reflects true resolution of mass spectrometers. We provide a blueprint on how to create a mass resolution ladder for experimental resolution determination with emphasis on simple synthesis and high adaptability. This new tool can be used to validate FWHM resolution values, to reveal unexpected links of resolution to other instrument parameters and to study space charge effects. We therefore think that our concept of a mass resolution ladder will be highly valuable for scientists and instrument developers alike.


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Associated Content Supporting Information: Instrumental parameters, equations for mass analyser type characteristics; table of isotopic masses; synthetic scheme dimethylation, chemical list Sigma Aldrich

Acknowledgements This work was supported by the Austrian Science Fund (FWF) project P 26074 and the FWF Erwin Schrödinger Fellowship J-3983. The authors thank Nicola Zamboni for valuable comments and discussion and Stefan Spoerk for excellent technical assistance.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Marshall, A. G.; Blakney, G. T.; Chen, T.; Kaiser, N. K.; McKenna, A. M.; Rodgers, R. P.; Ruddy, B. M.; Xian, F., Mass Spectrom. 2013, 2 , S0009. Blaum, K., Phys. Rep. 2006, 425 , 1–78. Hsu, J. L.; Huang, S. Y.; Chow, N. H.; Chen, S. H., Anal. Chem. 2003, 75 , 6843-6852. Schittmayer, M.; Fritz, K.; Liesinger, L.; Griss, J.; Birner-Gruenberger, R., J. Proteome Res. 2016, 15, 1222-1229. Gorshkov, M. V.; Fornelli, L.; Tsybin, Y. O., Rapid Commun. Mass Spectrom. 2012, 26, 1711-1717. Kalli, A.; Hess, S., Proteomics 2012, 12 , 21-31. Lu, W.; Clasquin, M. F.; Melamud, E.; Amador-Noguez, D.; Caudy, A. A.; Rabinowitz, J. D., Anal. Chem. 2010, 82 , 3212-3221. Naito, Y.; Inoue, M., J. Mass Spectrom. Soc. Jpn. 1994, 42 , 1-9. Werner, T.; Sweetman, G.; Savitski, M. F.; Mathieson, T.; Bantscheff, M.; Savitski, M. M., Anal. Chem. 2014, 86 , 3594-3601. Tarasova, I. A.; Surin, A. K.; Fornelli, L.; Pridatchenko, M. L.; Suvorina, M. Y.; Gorshkov, M. V., Eur. J. Mass Spectrom. 2015, 21, 459-470.



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Fig. 1: A: Mass spectrometer resolution characteristics of TOF based mass spectrometers (Eq. S3). Dashed line: Resolution calculated from single peak at m/z = 1130. Dotted line: Experimental resolution determined from peak pair at m/z = 1130. Solid line: mass resolution ladder with ∆m = 18.0 mmu. Vertical lines: mass pairs of mass resolution ladder shown in C. B: Mass resolution characteristics of FT based mass spectrometers (Eq. S1 and S2). Dashed line: FT-ICR at resolution 5 x 104 at m/z 400. Dashed and dotted line: FT-ICR at resolution of 1 x 105 at m/z = 400. Dotted line: Orbitrap at a resolution of 1 x 105 at m/z = 400. Solid line: mass resolution ladder with ∆m = 5.8 mmu. Vertical lines: mass pairs of mass resolution ladder shown in D. C: Chemical structure of polyethylene glycol based mass resolution ladder, Δm = 12.8 mmu or 18.0 mmu. * indicates isotope labelled positions. D: Chemical structure of polyglycine based mass resolution ladder, ∆m = 5.8 mmu.


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Fig. 2: A-D: Ion coalescence effect in an Orbitrap mass spectrometer at a set resolution of 1 x 105 in dependence of fill target at a mass window of 1 Da. Mass pair 281 of polyglycine based mass resolution ladder, Δm = 5.8 mmu or 21 ppm for specific mass pair. A: fill target 5 x 104; B: fill target 1 x 105, peaks start to move towards each other; C: fill target 2 x 105, onset of coalescence, peaks outside mass accuracy limits; D: fill target 4 x 105, full coalescence. E: Signal intensity threshold for onset of ion coalescence effects as a function of m/z and relative mass difference (set resolution 1 x 105).



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for TOC only

Words 3445 Fig. 1: 123 words Fig. 2: 306 words Total: 3874


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