OVERCOMING PEAK CAPACITY LIMITATIONS IMPOSED BY HYDROGEN EXCHANGE QUENCH CONDITIONS Bradley B. Stocks1, Thomas E. Wales1, Martha Stapels2, Scott J. Berger2, Keith Fadgen2, Michael Eggertson2, Geoff Gerhardt2, John R. Engen1 1 Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA 2Waters Corporation, Milford MA RESULTS • • Objective: Incorporate ion mobility into the hydrogen exchange mass spectrometry workflow and investigate effects on sequence coverage and deuterium retention within complex samples Methods: Hydrogen exchange (HX); ion mobility mass spectrometry (IMS-MS); nanoACQUITYTM with HDX technology Results: Ion mobility increased peptide coverage without sacrificing deuterium label or cycle time for complex samples. INTRODUCTION Continuous labeling hydrogen exchange experiments 1 were carried out at room temperature. Peptide exchange samples were analyzed using a Waters nanoACQUITY with HDX technology3 coupled to a Waters SYNAPT G2 equipped with an ESI source. Ion mobility separation was performed in the IMS cell pressurized with argon. All samples were digested online prior to RP-HPLC separation. Peptic peptides were identified using Waters ProteinLynx Global Server with deuterium incorporation data generated using Waters DynamX 2.0 software. Sample: Peptic digest of phosphorylase B (~90 kDa) The SYNAPT HDMS applied for HX studies supports three dimensions of peptide separation : MS only TIC 14 min gradient XIC m/z 800.14 MS: mass-tocharge (m/z) Funding for this work at Northeastern University was provided by the NIH (R01 GM101135) and the Waters Corporation MS only + IMS 97% 98% 80% 95% 271 peptides 315 peptides 43 peptides 65 peptides Enabling the Ion Mobility Separation (IMS) within the SYANAPT HDMS did not affect deuterium recovery in HXMS experiments. MS only + IMS 0s 10 s 1 min 10 min 8 723-731 DQRGYNAQE 541 542 543 544 545 m/z m/z Experimental Methodology This improvement occurs due to the ability of the ion mobility separation dimension to resolve peptide ions not resolved by m/z or chromatographic dimensions. 6 LC Retention Mass Spectra IMS MS Equilibrate 25°C, formulated pH same pH and temperature Backbone amides become deuterated Quench exchange reaction at various times, 0°C, pH 2.5 Pepsin digestion, 0°C, pH 2.5 MS for each peptide UPLC of pepsin fragments, 0°C Compare sequence coverage and deuterium levels 602 605 608 m/z 3.0 5.0 7.0 Time (min) MS + IMS The HXMS workflow4 602 605 608 m/z TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS 1 801 TIC 6 min gradient XIC m/z 800.14 802 m/z 803 804 800 801 802 m/z 803 804 + 2.88 Da + ??? Da 30 min 0s 800 2 0 403.0 0.1 1 10 403.5 404.0 404.5 4.55 4.65 4.75 30 40 50 60 100 m/z 691.5 692.0 692.5 tR (min) 693.0 5.45 5.55 5.65 40 55 70 85 m/z 691.34 10 s 1 min 10 min 100 min IMS -3 Peptide Number 9 12 802 m/z 803 804 800 801 802 m/z 803 804 IMS-MS with the shorter gradient reduced back-exchange by 0.65 Da , and improved the quality of the deuterium uptake measurement CONCLUSIONS 1+ • No significant difference in deuterium uptake level was measured for peptides analyzed by MS or IMS-MS. m/z 691.372+ REFERENCES 3.Wales, T.E.; et al., (2008) Anal. Chem., 80, 17, 6815-6820 -1 6 801 100 2.Iacob, R.E.; et al., (2008) Rapid Commun. Mass Spectrom., 22, 2898-2904 MS 3 Time (min) Time (min) Drift time (ms) 1.Wales, T.E.; Engen, J.R. (2006) Mass Spectrom. Rev., 25, 1, 158-170 0 -2 800 m/z 403.251+ 3 2 0s 4 Consistent deuterium retention for all phosphorylase b peptic peptides was demonstrated at various labeling times, as measured with and without IMS. Dashed lines represent +/- 0.5 Da. Peak capacity is greatly improved by IMS. 6 times in D2O shown Electrospray MS + 2.23 Da Ion Mobility m/z 691.693+ D2O buffer + 2.23 Da m/z 402.913+ LabelingTime time (min) Labeling (min) 541 542 543 544 545 Difference (Da) ACKNOWEDGEMENTS + IMS 100 min deuterium In this work, we examined the effects of IMS on peak capacity in a complex protein mixture. HX was done on two large proteins, and peptide coverage and deuterium retention are compared in experiments with and without IMS. MS only Relative ESI-MS Intensity Ion Mobility Separations (IMS) can separate coeluting peptides in the gas-phase, prior to MS detection, based on peptide charge and crosssectional area. In previous published work, it was demonstrated that IMS increased the number of useful peptides in HXMS experiments on a 8.2 kDa protein, by resolving co-eluting species.2 + IMS 30 min LC: Peptide hydrophobicity IMS: Peptide ion charge and shape (collisional cross section) The extra dimension of IMS permits the use of shorter gradients that facilitate greater deuterium recovery, and longer gradients that facilitate peptide-level HX analysis of larger proteins, more complex protein mixtures, or lower protein purity. Sample: 150 kDa mAb Acquisition Methodology HXMS is a powerful method for determining protein structure and dynamics.1 The need to analyze samples under quench conditions (low pH and Temp) negatively affects LC peak capacity. Extending the LC gradient can improve peak capacity, however the increased separation time leads to lower deuterium recovery. Enabling IMS improved data quality for a 292 AA protein in 1:28 molar ratio mixture with a 645 AA protein contaminant. Relative ESI-M ESI-MSSIntensity Relative Intesity • Enabling the Ion Mobility Separation (IMS) within the SYANAPT HDMS maintained high sequence coverage with improved coverage redundancy. Relative ESI-MS Intensity Relative Deuterium Level METHODS OVERVIEW 4.Engen, J.R.; et al., (2011) Ency. Anal. Chem., ISBN 978047002731 5.Kavan, D.; Man, P. (2011) Int. J. Mass Spectrom., 302, 53-58 • Peak capacity, protein coverage, and HX measurement quality increased for complex protein mixtures or larger proteins with IMS-MS. • Addition of IMS to the HXMS workflow allowed for the use of shorter LC gradients, which leads to increased deuterium retention on peptides. ©2014 Waters Corporation
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