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Increased Analytical Performance on a Hybrid Linear Ion Trap-FTMS

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Increased Analytical Performance on a Hybrid Linear Ion Trap-FTMS Increased Analytical Performance on a Hybrid Linear Ion Trap-FTMS Mass Spectrometer with a High-Field Orbitrap Mass Analyzer Martin Zeller, Catharina Crone, Mathias Mueller, Eugen Damoc, Eduard Denisov, Alexander Makarov, Dirk Nolting, and Thomas Moehring Thermo Fisher Scientific, Bremen, Germany Intact Protein Analysis and Top-Down Proteomics Figure 3 shows the HCD and ETD spectra of the Histone H4 protein, a 12.3 kDa The same E.coli sample was analyzed with the same LC setup on the Orbitrap Elite Overview Results protein. Top-down proteomics is done on-line on complex protein mixtures and usually and the LTQ Orbitrap Velos hybrid mass spectrometers with a top 15 HCD method Figure 2 shows on the example of the intact yeast enolase (47 kDa) the increase in employs serveral microscans to enhance the quality of the MS/MS spectrum. Figure 4 and with different gradient lengths. Due to the higher scan rate and sensitivity, Purpose: Implementation of a next-generation high-field OrbitrapTM mass analyzer High-Field Orbitrap Mass Analyzer resolving power using both the next generation Orbitrap mass analyzer and advanced shows the benefit of higher resolution and higher sensitivity of the Orbitrap Elite hybrid substantially more peptide spectrum matches (PSM) were identified on the Orbitrap and advanced signal processing algorithm on a hybrid ion trap-Orbitrap mass data signal processing. Elite. The performance increase also allows shorter LC gradients and increases One of the most important changes in the Orbitrap Elite™ mass spectrometer is the mass spectrometer for top-down proteomics. More ETD fragment ions can be spectrometer. analysis throughput. use of a new high-field Orbitrap mass analyzer. The new Orbitrap is 1.5 times smaller identified with higher resolution with fewer microscans, making the Orbitrap Elite Methods: Various experiments (direct infusion, online LC-MS/MS) have been carried than the standard Orbitrap while the central electrode is relatively thicker. However, The same performance increase can also be observed for TMT-labeled proteins with FIGURE 2. Intact yeast enolase infusion on the Orbitrap Elite (top trace) and suitable for on-line top-down proteomics. out to characterize the performance of the high-field Orbitrap mass analyzer on the this down-scaling means that the entrance aperture is also reduced more than twice in different protein amounts loaded on column. The Orbitrap Elite hybrid mass TM Orbitrap Elite hybrid mass spectrometer. cross-section. To avoid a loss of sensitivity, a miniature lens system was developed to Orbitrap Velos Pro (bottom trace) hybrid mass spectrometers showing the spectrometer identifies more proteins than the LTQ Orbitrap Velos mass spectrometer focus ions into a much smaller spot. The increase in resolution on the high-field increased resolution of the Orbitrap Elite for both very low amounts (20 ng) and high amounts (200 ng). (Figure 6) Results: The new high-field Orbitrap mass analyzer, in combination with advanced Figure 4. Influence of the number of microscans for an ETD top-down spectrum Orbitrap is about a factor of 1.8 compared to the standard Orbitrap. Figure 1 shows the TM data processing, provides increased performance for the characterization of intact Yeast Enolase, 47+ on the Orbitrap Elite and the LTQ Orbitrap XL hybrid mass spectrometers on standard Orbitrap and the new high-field Orbitrap mass analyzers true-to-scale. 993.9770 NL: 1.00E6 R=110221 Enolase_994_transients_1 the example of Ubiquitin proteins and complex mixtures. 100 FIGURE 6. Number of identified TMT-labeled E.coli proteins with different 993.8706 e5_v2#1-15 RT: R=107582 994.1036 0.00-0.34 AV: 15 T: protein amounts loaded on column on the Orbitrap Elite and the LTQ Orbitrap e Orbitrap Elite 80 R=108475 FTMS + p ESI Full ms [150.00-2000.00] Velos hybrid mass spectrometers FIGURE 1. Standard Orbitrap mass analyzer (left) and new high-field Orbitrap 60 Orbitrap Elite c-type fragment ions (120k) Orbitrap Elite z-type fragment ions (120k) Introduction 994.1885 mass analyzer (right), true-to-scale R=109018 40 993.6367 Orbitrap Elite c-type fragment ions (240k) Orbitrap Elite z-type fragment ions (240k) ve Abundanc One of the biggest challenges in shot-gun proteomics of complex samples remains i t 993.0619 R=108853 a 993.4244 994.4433 l R=109083 e 994.7200 undersampling. Besides the ion accumulation time to reach the target value in ion trap 20 R=99350 R=105946 994.9536 R LTQ Orbitrap XL c-type fragment ions (60k) LTQ Orbitrap XL z-type fragment ions (60k) R=109283 R=98949 hybrid mass spectrometer, the detect time in the Orbitrap detector has the biggest 0 70 portion in the overall scan process. Improvements resulting in higher resolution or in 994.0309 NL: 5.48E3 R=44604 enolase_otvelospro_1850 100 higher scan speed at the same resolution significantly decrease the overall cycle time. 993.9030 #1 RT: 92.40 AV: 1 T: 65 R=42104 FTMS + p ESI SIM ms 80 In this work we present the advances in analytical performance using modifications to 993.7112 994.1802 LTQ Or[992.bi00-tra997p .V00]e los R=35704 R=42804 60 hardware and software on an ion trap-hybrid MS based instrument platform. 60 993.5618 994.4540 R=52804 1.2 x R=47304 1.5 x 55 40 993.1357 994.6257 R=42304 R=37604 994.8610 Methods 20 R=32404 50 Sample Preparation 0 993.0 993.5 994.0 994.5 995.0 45 m/z E. coli cell lysate (BioRad) was reduced, alkylated and enzymatically degraded. For the TMT experiments, the enzymatic digest was divided into six aliquots and labeled with 40 Standard Orbitrap H igh-Field Orbitrap m/z 126, 127, 128, 129, 130 and 131 TMT tags according to the manufacturer’s 35 protocol. Samples were subsequently mixed in various ratios, concentrated by a FIGURE 3. Histone H4 (12.3 kDa) HCD (top trace) and ETD (bottom trace) spectra 30 Thermo Scientific SpeedVac system and analyzed by LC-MS/MS. Intact proteins (all and identification and annotation using Thermo Scientific ProSightPC software from Sigma Aldrich, St. Louis, MO, USA) were dissolved in 50% methanol containing 0 5 10 15 20 25 30 35 40 45 50 Advanced Signal Processing 0.1% formic acid. number of µscans The Orbitrap Elite instrument employs advanced signal processing based on Liquid Chromatography enhanced Fourier transformation. This produces a high degree of synchronization of ETD HCD Peptides were separated on a Michrom Bioresources spray tip (75 µm inner diameter, the ion motion in the Orbitrap mass analyzer. The resolution increase with this 200 mm length) Magic™ C18 column or on a Thermo Scientific BioBasic peptide trap advanced signal processing is a factor of about 1.7. S G R G K G GK G L G K G GA K R H R K V L R D N I Bottom-Up Proteomics (100 μm inner diameter, 2 cm length) and a BioBasic™ C18 analytical column (75 μm The improvements in resolution permit a faster scan rate. The resolution settings and Figure 5 shows the benefits of the higher scan speed and higher sensitivity for typical inner diameter, 10 cm length) using a Thermo Scientific EASY-nLC with a flow rate of the resulting scan speed are summaried in Table 1. Conclusion 300 nL/min. Mobile phase A was water containing 0.1% formic acid and phase B was Q G I T K P A I R RL A R RG GV K R I S G L I Y E ET bottom-up proteomics experiments. The new high-field Orbitrap mass analyzer and advanced signal processing acetonitrile containing 0.1% formic acid. Different LC gradients were used for the enhance resolution, scan speed and sensitivity on the Orbitrap Elite hybrid mass bottom-up proteomics experiments. Table 1. Summary of resolution and scan speed on the Orbitrap Elite hybrid R G V L K V F L E N V I R D A V T Y T E H A K R K T V Figure 5. Number of peptide spectrum matches for different LC gradients on the spectrometer. Mass Spectrometry mass spectrometer T A M D V VY A L K R Q G R T L Y G F G G Orbitrap Elite and the LTQ Orbitrap Velos hybrid mass spectrometers for a top . Higher MW intact proteins can be isotopically resolved for accurate molecular mass Data were acquired using Thermo Scientific Orbitrap Elite, LTQ Orbitrap Velos, and 15 HCD method determination. Resolution settings Resolution LTQ Orbitrap XL hybrid mass spectrometers, all equipped with electron transfer Scan speed [Hz] Orbitrap Elite at m/z 400 . More fragment ions can be identified in top-down MS/MS spectra. dissociation (ETD). 843.9602 NL: 4.46E4 R=158115 Histone2_808_HCD_110415 z=2 000415-qb#1 RT: 2.10 AV: . More peptides and proteins can be identified in bottom-up proteomics experiments. 15.000 7.7 15.700 100 Data Analysis HCD spectrum1 T: FTM ofS His+ p ESItone Ful lH4 ms2 [email protected] Thermo Scientific Proteome Discoverer software version 1.2 with Mascot 2.3 search ce 80 [120.00-2000.00] 30.000 6.9 31.700 ndan 762.4287 Acknowledgements engine was used for protein identification. Carbamidomethyl modification on cysteine u 60 b R=158642 and TMTsixplex on lysine and N-terminal were set as fixed modifications for the TMT- 40 z=2 ve A 1001.0440 We would like to thank Rosa Viner (Thermo Fisher Scientific, San Jose, CA, USA) for the i 385.7177 R=115639 labeled samples. 60.000 4.0 60.000 lat e 20 R=197410 z=2 TMT analysis.
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