Orbitrap Fusion Lumos: Second-generation "three-in-one" ultra-high resolution mass spectrometry based on Q-OT-qIT platform

Zhang Wei

Thermo Fisher Scientific (China) Co., Ltd. Shanghai 201206

Abstract Orbitrap Fusion Lumos is based on Q-OT-qIT platform second generation "triple play" Mass, improvements by deep ion entrance, ion lens, quadrupole, ETD and vacuum technology, to optimize the overall performance of the instrument Extreme. Combined with the magical power of the “three-in-one” mass spectrometer, Orbitrap Fusion Lumos is able to effectively address the most complex analytical challenges of proteomics and metabolomics. This paper introduces the performance breakthrough and application progress of the second generation "three-in-one" mass spectrometry from the aspects of qualitative quantitative limit sensitivity, anti-pollution performance, post-translation modification and top-down analysis.

Keywords Orbitrap Fusion Lumos, proteomics, metabolomics, top-down

CLC number TH843

Orbitrap Fusion Lumos: Second Generation of “Tribrid” UHR-MS Based Q-OT-qIT

Zhang Wei

ThermoFisher Scientific, Shanghai 201206, China

Abstract Orbitrap Fusion Lumos is the second generation of "Tribrid" MS based on Q-OT-qIT system. The ion inlet, ion optics, quadrupole, ETD and vacuum system are deeply improved, and the instrument performance is extremely optimized. Combined with various Magic functions on "Tribrid", the new system can effectively face the extreme challenge in proteomics and metabolomics. This paper introduces the improvements and advances of the new "Tribrid" MS, including enhanced qual/quan sensitivity, robustness, PTM elucidation and top- Down analysis.

Key words Orbitrap Fusion Lumos, Proteomics, Metabonomics, top-down

In recent years, mass spectrometry has evolved from a single mass analyzer mass spectrometer (such as single quadrupole Q) to a tandem mass spectrometer composed of two mass analyzers (such as quadrupole-time-of-flight Q-TOF) to A combined mass spectrometer with two detectors (such as linear ion trap - electrostatic field orbitrap LTQ-Orbitrap), continuous innovation and breakthrough. In 2013, Thermo developed the world's first three mass analyzers based on a quadrupole-electrostatic field orbitrap-linear ion trap (Q-Orbitrap-LTQ or Q-OT-qIT) structure. "Three-in-one" mass spectrometry Orbitrap Fusion [1]. Utilizing quadrupole's high selectivity and wide linearity, ultra-high resolution and ultra-high quality accuracy of the electrostatic field orbital well, ultra-high sensitivity of linear ion traps and multi-stage MSn analysis, and allowing the mass analyzers to work together, Work in parallel for revolutionary scanning, magical analysis and unparalleled instrument performance. "Three-in-one" mass spectrometry can solve everything from qualitative identification to structural analysis to accurate quantification. Especially for the promotion of proteomics, the identification of all proteins in yeast was completed in an hour, which pioneered the "one-hour proteomics" [2].

At the 2015 American Mass Spectrometry Conference, based on deep optimization of the Q-OT-qIT system, Thermo introduced the second-generation “three-in-one” mass spectrometer – Orbitrap Fusion Lumos. The word "Lumos" is intended to illuminate the wand's fluorescent spell, the second-generation "three-in-one" mass spectro magical function and optimized to the ultimate performance, with intelligent parallel operation technology (ADAPTTM) to fully mobilize all components of the mass spectrometer, Work together and work together to easily address the most complex analytical challenges, just like magic wand fluorescence illuminating an unknown area of ​​analysis.

Identification of very low abundant proteins / compound

The second-generation "three-in-one" mass spectrometer uses a new high-capacity ion transport tube (HCTT) and an electro-ion funnel (EDIF) to allow a larger amount of ions to enter the mass spectrometer through a large-diameter ion-input lens, in conjunction with an active ion beam transport module (AABG). ), effectively focusing and transmitting ions, thus fully breaking through the bottlenecks and limits of instrument sensitivity. Experimental data shows that the new ion inlet and ion lens design increases the detection sensitivity of nanoliter flow rate and normal flow rate by 2 and 5 times, respectively [3]. The increase in sensitivity not only reduces the minimum detection limit and the minimum limit of quantitation, but also improves the signal response of multi-stage analysis. The 1 ng/mL timolol is fragmented to 5 and still obtains high quality spectra. Achieve effective parsing of fine structures.

In addition, the new three-stage quadrupole (AQT) is designed so that the analyte is always in the stable region of the quadrupole field, thus stably passing the quadrupole, significantly improving the efficiency of ion selection, avoiding the selection of window edges and high quality. Discrimination effect. The improvement of the ion lens coupled with the new three-stage quadrupole enables new high-throughput identification of complex samples to reach new heights: 1 μg of HeLa cell total protein, 140-minute gradient, 2 replicates, and a total of 5,211 non-redundant proteins were identified. Using the selection windows of 0.4, 0.7, and 1.6 m/z to identify 5211, 5184, and 5187 non-redundant proteins, the window reduction did not reduce the sensitivity, but further eliminated the co-eluting peptide interference, resulting in very low abundance protein. The identification is more sensitive and accurate. Total protein analysis of cancer cell lines, fractionation of 12 components, identification of 109578 non-redundant peptides, corresponding to 10210 non-redundant proteins, easy to achieve deep coverage of proteome. In addition, the further upgraded “Universal Method” does not require optimization of the optimal parameters for the sample size. One method is suitable for all unknown concentration sample analysis, and the best results are obtained for either very low or high sample loads.

2 　 Adapt to 7x24-hour testing of large-scale clinical samples

The 7x24 hour continuous analysis of complex samples is a challenge to the anti-pollution ability of mass spectrometry, especially the large molecular weight ions such as peptide/protein, which will cause serious pollution to the mass analyzer and cause the sensitivity to decrease. The new pre-quadrupole (Q00) of the second-generation "three-in-one" mass spectrometer is similar in structure to the main quadrupole and consists of four round rods with selective capabilities. This design allows the new pre-quadrupole to have a more efficient ion pre-screening capability than conventional pre-quadrupoles, and the pre-quadrupole will efficiently target outside the target selection range regardless of the scan mode. Molecular weight ions (usually m/z > 1300) are excluded, preventing high molecular weight ions from entering the mass analyzer to form contamination [4]. The effective elimination of high molecular weight ions greatly improves the anti-pollution ability of the instrument. Even in highly complex clinical samples such as plasma and tissue, it can maintain the high sensitivity of the instrument under large-scale, high-throughput high-intensity analysis. And low impurity noise.

3 　 The most accurate quantitative analysis

Increased sensitivity and selectivity make quantification of very low-abundance proteins/compounds in complex matrices more sensitive and accurate [5, 6]. Parallel reaction monitoring (PRM) quantification, quantification of 15 synthetic peptides (PRTC re-peptide) in 200 ng HeLa matrix samples, 10 of which had LOQs of 5 amol (10-18 mol) and below, and CVs were less than 10 %. Data-independent acquisition (DIA), with Q ≤ 0.01 and CV ≤ 20% as standard, 155-minute effective gradient identified and quantified 44,506 peptides and 5,277 non-redundant proteins from total HeLa cell proteins. % of the protein has accurate and reliable quantitative information. Based on the stable isotope-labeled Shotgun quantification, using 1 μg of TMT-labeled HeLa cell total protein for quantitative parental ion selection (SPS) MS3 quantitative analysis, the number of quantified peptides was increased by 37% compared to the past, if the sample was diluted 100 times, ie 10 With ng loading, the number of quantitative peptides increased by 300%. The second-generation "three-in-one" mass spectrometer is a tool for quantification of very low-abundance proteins/compounds, from large-scale quantification of shotguns to quantitative quantification of DIA to quantitative quantification of PRM targets.

4 　 The clearest modification structure analysis

Obtaining precise site and structural information by ETD fragmentation is an effective means of post-translational modification studies. When traditional ETD is applied in clinical research, due to the complexity of clinical samples, the sensitivity of ETD fragmentation is low and the fragmentation effect is poor. It is still difficult to use as a routine lysis method for clinical sample research. The development of HD ETD (ETD HD) has completely changed the many shortcomings of ETD. ETD HD uses the middle trap of the linear ion trap to stabilize and store the parent ion, and then reacts with the fluoranthene to generate ETD, which significantly improves the sensitivity, dynamic range and secondary spectrum quality [7]. ETD HD works with Easy-ETD source, intelligent parent ion sorting, real-time product ion triggering, EthcD/ETciD and other innovative modes to enable post-translational modification analysis of very low-abundance proteins in complex matrices, especially glycosylation structures. may. ETD HD was used to analyze human serum-enriched glycoproteins, with a 2-hour gradient, 2 replicates, and a total of more than 1000 intact glycopeptides with different glycoform structures, most of which were glycopeptides with molecular weights greater than 5 kDa. The second generation of "three-in-one" mass spectrometry ETD has become a simple and efficient fragmentation mode that can be effectively applied to the analysis of low-abundance proteins in complex samples.

5 　 A new era of Top-Down research

Top-Down research still faces many bottlenecks after years of development, and the key to breaking through the bottleneck is to reduce the loss of intact protein during transport and to improve the fragmentation efficiency of protein intermediate sequences. Based on the second-generation "three-in-one" mass spectrometer advanced vacuum technology, the overall vacuum of the instrument is further improved by a combination of various techniques, especially the vacuum between C-trap and Orbitrap. Minimize the loss of intact proteins from collisions with residual gases from C-trap ejection to Orbitrap [8]. The experimental results show that the deep high vacuum technology can increase the signal-to-noise ratio of intact carbonic anhydrase detection by 3 times, and then cooperate with ETD HD fragmentation to increase the carbonic anhydrase fragment coverage from 38% to 65%. The same method was used for IgG Top-Down analysis, with light chain fragment coverage of 91% and heavy chain fragmentation of 63% [9]. In addition, using the high selectivity of the three-stage quadrupole, a 0.5m/z window was used to screen the histone H3 intact protein (18+) with different degrees of methylation, achieving similar molecular weights (interval of only 0.78 m/z). Effective isolation and differentiation of different protein variants (Proteoform). The development of innovative technologies such as deep high vacuum technology, ETD HD, and three-stage quadrupole has completely broken through the bottleneck of Top-Down analysis, making online LC-MS Top-Down analysis of complex samples possible, and Top-Down research has entered A new era.

In summary, the second-generation "three-in-one" mass spectrometer - Orbitrap Fusion Lumos solves the qualitative and quantitative determination of very low-abundance proteins/compounds, structural analysis of complex glycopeptides, and intact protein Top-- through many technological innovations and deep optimization of performance. The most challenging problems, such as Down analysis, can be effectively applied to 7x24-hour high-intensity testing of large-scale clinical samples.

references

[1] Senko MW, Remes PM, Canterbury JD, et al. (2013) Novel Parallelized Quadrupole/Linear Ion Trap/Orbitrap Tribrid Mass Spectrometer Improving Proteome Coverage and Peptide Identification Rates. Anal. Chem. 85: 11710-11714

[2] Hebert AS, Richards AL, Bailey DJ, et al. (2014) The One Hour Yeast Proteome. Mol. Cell. Proteomics 13: 339-347

[3] Wouters ER, Prasad S, Dunyach JJ. Advancements in Atmospheric-Vacuum Interfaces of Mass Spectrometers with Increased Gas Throughput and Enhanced Sensitivity. ASMS 2015: PN 64478

[4] McAlister GC, Senko M. Pre-filtering Ions in the Source Region to Improve Instrument Robustness and Dynamic Range. ASMS 2015: PN 64475

[5] Blank M, Huguet R, Bomgarden R, et al. Revolutionizing Data Independent Acquisition on q-OT-IT Tribrid Mass Spectrometers. ASMS 2015: PN 64461

[6] Huguet R, Blank M, Soltero N, et al. Low Attomole Limit of Quantification on an Orbitrap Fusion Lumos Tribrid Mass Spectrometer. ASMS 2015: PN 64492

[7] Mullen C, Earley L, Weisbrod C, et al. Considerations for Attaining Improved ETD Performance for Top Down Applications. ASMS 2015: 64463

[8] Canterbury J, Izgarian N, Senko M, et al. Improvements for High Resolution Analysis on a Modified Tribrid Mass Spectrometer. ASMS 2015: PN 64468

[9] Sharma S, Mallick P, Stoyanova T, et al. Optimizing Top Down Analysis of Proteins on Orbitrap Fusion TM Lumos TM Tribrid TM Mass Spectrometer. ASMS 2015: PN 64439

Received date: 2015-07-17

About the author: Zhang Wei (1982-), male, application engineer of Thermo Fisher Scientific Life Science and Mass Spectrometry, has long been engaged in the research of bio-mass spectrometry, proteomics and post-translational modification of proteins.