TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2013 Waters Corporation INTRODUCTION The use of mass spectrometry in the characterisation of high molecular weight species has been an area of considerable growth over the past ten to fifteen years. Key areas of investigation have been on non-covalently bound protein complexes of many hundreds of kilodaltons to virus capsids of many megadaltons. An area of more recent and significant growth has been in biopharmaceutical characterisation. The high mass capability of time-of flight analysers has made them the instrument of choice for the study of such systems. Here we investigate the high mass performance characteristics of quadrupole/ion mobility/time-of-flight instrument (Q-IM-TOF) incorporating a novel conjoined source ion guide. PROBING THE HIGH MASS TRANSMISSION CHARACTERISTICS OF A CONJOINED ION GUIDE ENABLED QUADRUPOLE / ION MOBILITY / TIME-OF-FLIGHT INSTRUMENT Kevin Giles , Jason Wildgoose, Jonathan Williams and Martin De Cecco Waters Corporation, Manchester, UK The larger diameter (15 mm) ion guide is aligned with the ion/ gas flow exiting the source region, see Figure 3. A DC potential difference between this guide and the second, smaller diameter (5 mm), ion guide extracts and focuses the ions from the main gas stream into the second guide. The ion optical axis of the second ion guide is aligned with the ion optical axes of the subsequent ion guide and the mass analysers. This design simultaneously concentrates the ion beam from the diffuse source and reduces the degree of contamination from neutral material entrained in the gas. The conjoined ion guide operates at a pressure of about 3-4 mb with variable RF amplitudes up to 320 V peak-to-peak at 1 MHz. The DC offset between the conjoined ion guides is typically set at 25 V. In these experiments, the effect of pressure, RF and DC offset on the transmission of three high mass species is investigated. All samples were infused using borosilicate glass nano-electrospray tips and analysed in positive ion mode. Data were acquired in TOF MS or TOF MS/MS sensitivity mode (resolution ~15,000 FWHM) with electrospray voltages of around +1.5 kV used throughout and sample cone voltages of up to 200 V employed for de-clustering. Trap and transfer collision cell pressures of 2-4x10 -2 mb argon were used with trap collision voltages in the 20-40 V range (except as noted for MS/MS). The detector voltage was also increased for best performance with these high mass species. The source backing pressure on the Synapt G2 was optimised at ~7 mb. Samples The samples used in these studies were a mouse monoclonal antibody (MAB) (mw ~150 kDa (Waters, 186006552)), Glutamate Dehydrogenase Hexamer (GDH) (mw 336 kDa (Sigma, G7882)) and GroEL (mw 802 kDa (Sigma, C7688)). All samples were buffer exchanged into 200 mM/100 mM aqueous ammonium acetate (MAB and GDH/GroEL) to a final concentration of 1 mg/mL. RESULTS AND DISCUSSION The mass spectra obtained for the three species using the Synapt G2-S instrument are shown below in Figs 4, 5 and 6. OVERVIEW Investigation of the high mass transmis- sion properties of a conjoined ion guide system Studies undertaken on Synapt G2 and Synapt G2-S quadrupole/ion mobility/ time-of-flight instruments Excellent transmission observed with consistent increases in signal compared with standard ion guide arrangement METHODS Instrumentation The primary instrument used for this work was a Synapt G2-S (Waters Corp.) which is shown schematically in Figure 1, where the dual ion guide arrangement in the source transfer region can be seen. Also shown in Figure 1 is the source region of a Synapt G2, highlighting the presence of a single ion guide between the source and analyser. The conjoined ion guide in the Synapt G2-S consists of two different diameter stacked ring electrode devices which are radially offset, as shown in Figure 2. Figure 1 A schematic diagram of the Synapt G2-S mass spectrometer and the Synapt G2 source region. As can be seen, intense mass spectral peaks are obtained from these samples, even at 1 second acquisition times with essentially equivalent data obtained using analogue- or time-to-digital (ADC or TDC) acquisition modes. Conjoined Guide Transmission Characteristics The transmission characteristics of the conjoined ion guide as a function of applied RF voltage, DC offset between the conjoined ion guides and pressure in the conjoined ion guide are shown in Figures 7, 8 and 9 respectively. The three high mass species show broadly similar transmission characteristics as a function of applied RF and DC. The transmission characteristic of singly charged verapamil (m/z 455) is also plotted for comparison. The RF characteristics of the low and high mass species are as expected but the similarity of response to DC offset over the wide m/z range is perhaps surprising. The pressure in the conjoined ion guide region was increased from the ~4 mb base pressure by throttling the backing pump line using a Speedivalve. It can be seen that there is little or no effect of increased pressure on GDH transmission, unlike that of other systems where some degree of source pressure optimisation is necessary. Synapt G2-S System Performance To compare the relative high mass transmission efficiency of the conjoined ion guide with that of the single ion guide, a series of back-to-back experiments were performed on adjacent Synapt G2 and G2-S instruments using the same sample. An initial comparison was undertaken using the fragments of the doubly charged Glu-fibrinopeptide B (GFP) ion, as shown in Figure 10. The observed factor of ~25 increase in transmission using the conjoined ion guide is as expected from previous studies. The results for the high mass species are shown in Figure 11. The signals on the conjoined ion guide system (G2-S) are consistently higher than those on the single ion guide system (G2). Absolute comparisons between instruments was challenging for these samples, the magnitude of the difference is CONCLUSIONS The high mass transmission characteristics of a conjoined ion guide in the source region of a Q-IM- TOF has been investigated Excellent transmission is observed for species ranging from 150 kDa to 802 kDa with notable increases in performance over a single source ion guide design The optimum conjoined guide operating parameters are broadly similar to those for lower molecular weight species allowing relatively generic settings Further studies are needed to qualify transmission differences between the single and conjoined guides Figure 2 Diagrams of the conjoined stacked ring ion guides. Opposite phases of RF voltage ap- plied to adjacent electrodes Conjoined Ion Guides Electrodes ~120mm 15mm 5mm 11mm Ions + Gas From API Source Rough Pump Gas Ions Differential Aperture Conjoined Ion Guides Ion Trajectories Figure 3 Schematic diagrams illustrating the operating principle of the conjoined ion guide. X Y Diffuse Ion Cloud Compact Ion Cloud Electric Field Potential Contours X Y m/z 7500 10000 % 0 0 4mb 5mb 6mb 7mb 8mb 9mb 10mb Conjoined Ion Guide Pressure Intensity Figure 9 NanoESI mass spectra of GDH as a function of con- joined ion guide pressure. Figure 4 NanoESI mass spectra of MAB: Synapt G2-S. 24+ charge state peak FWHM = 28 m/z, ~350 counts/sec peak top m/z 5000 5250 5500 5750 6000 6250 6500 6750 7000 7250 7500 7750 8000 8250 8500 8750 % 0 100 m/z 5000 5250 5500 5750 6000 6250 6500 6750 7000 7250 7500 7750 8000 8250 8500 8750 % 0 100 m/z 5000 5250 5500 5750 6000 6250 6500 6750 7000 7250 7500 7750 8000 8250 8500 8750 % 0 100 m/z 5000 5250 5500 5750 6000 6250 6500 6750 7000 7250 7500 7750 8000 8250 8500 8750 % 0 100 24+ 24+ ADC 60 secs ADC 1 sec TDC 60 secs TDC 1 sec Figure 5 NanoESI mass spectra of GDH: Synapt G2-S. 38+ charge state peak FWHM = 53 m/z, ~170 counts/sec peak top m/z 7000 7500 8000 8500 9000 9500 10000 10500 % 0 100 m/z 7000 7500 8000 8500 9000 9500 10000 10500 % 0 100 m/z 7000 7500 8000 8500 9000 9500 10000 10500 % 0 100 m/z 7000 7500 8000 8500 9000 9500 10000 10500 % 0 100 38+ 38+ ADC 60 secs ADC 1 sec TDC 60 secs TDC 1 sec Figure 6 NanoESI mass spectra of GroEL: Synapt G2-S. 67+ charge state peak FWHM = 30 m/z, ~70 counts/sec peak top m/z 10000 10500 11000 11500 12000 12500 13000 13500 % 0 100 m/z 10000 10500 11000 11500 12000 12500 13000 13500 % 0 100 m/z 10000 10500 11000 11500 12000 12500 13000 13500 % 0 100 m/z 10000 10500 11000 11500 12000 12500 13000 13500 % 0 100 67+ 67+ ADC 60 secs ADC 1 sec TDC 60 secs TDC 1 sec Figure 8 Transmission plots through the conjoined ion guide as a function of DC offset voltage (at 1 MHz and 320 V pk-pk RF). 0 20 40 60 80 100 0 20 40 60 80 Normalised Transmission (%) DC Offset (V) MAB GDH GroEL m/z 455 Figure 7 Transmission plots through the conjoined ion guide as a func- tion of RF pk-pk voltage (at 1 MHz and 25V DC offset). 0 20 40 60 80 100 0 50 100 150 200 250 300 350 Normalised Transmission (%) RF Voltage (V) MAB GDH GroEL m/z 455 m/z 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 % 0 100 m/z 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 % 0 100 x25 G2 G2-S Figure 10 ESI mass spectra of GFP fragment ions obtained using the Synapt G2 and Synapt G2-S systems. being investigated further. Figure 12 highlights the data quality obtainable using the Synapt G2-S system with the improved transmission of the conjoined guide. Figure 12 MS/MS spectra obtained using the Synapt G2-S sys- tem. With and without quadrupole mass isolation. m/z 2500 5000 7500 10000 12500 15000 17500 20000 22500 25000 27500 30000 % 0 100 m/z 2500 5000 7500 10000 12500 15000 17500 20000 22500 25000 27500 30000 % 0 100 11801 1735 20701 x10 11630 1790 20700 20137 21291 21915 22585 Collision Voltage = 75 V x10 Figure 11 NanoESI mass spectra obtained using the Synapt G2 and G2-S systems. m/z 10000 10500 11000 11500 12000 12500 13000 13500 % 0 100 m/z 10000 10500 11000 11500 12000 12500 13000 13500 % 0 100 x20 x20 G2 G2-S m/z 7000 7500 8000 8500 9000 9500 10000 10500 % 0 100 m/z 7000 7500 8000 8500 9000 9500 10000 10500 % 0 100 x5 G2 G2-S x5 m/z 5000 5500 6000 6500 7000 7500 8000 8500 % 0 100 m/z 5000 5500 6000 6500 7000 7500 8000 8500 % 0 100 x10 x10 G2 G2-S