New Quadrupole-Ion Trap-Orbitrap Mass Spectrometer Combined with Real Time Search Enhances Proteome Coverage and Quantification Accuracy in Multiplexing Workflows Xiangyun Yang, Xiujie Sun, Yue Zhou, Thermo Fisher Scientific, Building 6, 27 Xinjinqiao Road, Shanghai, China, 210006

Figure 2. Real-time search can elevate the proteome depth of SPS MS3 quantitation. 500ng of TMT11plex For deep human proteome characterization, we also tested RTS-MS3 using fractionated TMT-10plex cell ABSTRACT Yeast Digest Standard was analyzed on a 60min gradient using MS2, SPS MS3, or RTS SPS MS3 methods. line and plasma sample for a total analysis time of 24h or 18h, respectively. We observed more 9728 We evaluated the effect of Real Time Search for TMT identification rates on PSM(2A.), peptide (2B.) and Purpose: Evaluate the performance of Orbitrap Eclipse Tribrid mass spectrometer including modified hardware, protein groups and more than 80000 peptide groups from 12μg TMT labeled 293T samples, and 1411 protein(2C.) levels as well as the SPS mass match distribution at PSM level (2D, 2E). real time search in accuracy, precision, and sensitivity for TMT based quantitation. protein groups and 10000 peptide groups from 12μg top-14 abundant protein depleted plasma samples A (Table 1) . Pierce 6-proteins digest mixtures were spiked in various ratios into the 293T cell or plasma Methods: Thermo Scientific™ Pierce™ TMT11plex yeast digest standard, TMT-10plex cell line and plasma sample 10000 90169002 B 10000 C 2000 8951 8111 8685 1540 1779 8054 peptides (Figure 1). 1ug unlabeled peptides were measured with DIA method. The total analysis time of were analyzed with MS2, SPS-MS3 and Real Time Search based SPS-MS3 (RTS-SPS-MS3). 7590 1535 1552 8000 6564 8000 7211 1600 6548 DIA is equal to TMT analysis time. 6112 1195 Results: RTS-SPS-MS3 method on Orbitrap Eclipse instrument showed deep proteome coverage and enhanced 6000 6000 1200 898 quantitative accuracy performance in multiplexing workflows. 3944 Compared with MS2-based quantification, the measured ratio of the six proteins in RTS-MS3 experiment PSM 83%

4000 4000 Protein 800 Peptides 87% was closer to the expected value. With the same analysis duration, the proteome coverage of TMT- 74% 2000 2000 65% 400 quantification was greater than DIA method (Table 1). Meanwhile, compared to TMT quantification, we INTRODUCTION observed that the quantitative accuracy of DIA is higher (Figure 5). 0 0 0 Multiplexing quantification with isobaric reagents (e.g., TMT) is a widely-used and powerful strategy for proteomics MS2 SPS MS3 RTS SPS MS2 SPS MS3 RTS SPS MS2 SPS MS3 RTS SPS MS3 MS3 MS3 Table 1. Proteins identified from trypsin digest of Figure 5. Evaluation of TMT quantification accuracy studies. A challenge of this technique is the limited quantification accuracy caused by interference from co-isolated 293T cell or top-14 abundant protein depleted and comparison to DIA method. peptides and co-fragmentation in MS2 spectra. To address this challenge, a new technique called Real-time Identified Quantified Identified Quantified Identified Quantified human plasma with TMT or DIA method. Search-MS3 (RTS-MS3) has been developed for the Thermo Scientific™ Orbitrap Eclipse™ quadrupole-ion trap- D E Expected Change is 2X Expected Change is 24X SPS MS3 >70%: 64.11% RTS SPS MS3 >70%: 83.73% Orbitrap instrument, building on the SPS-MS3 method used with previous generations of this platform1. Here, we 1512 7525 Quantified N=6 N=6 1600 1396 8000 Method Proteins evaluate the sensitivity and quantification accuracy of RTS-MS3 on the Orbitrap Eclipse mass spectrometer for 1300 Proteins TMT-based quantitation using TMT11plex yeast digest standard, TMT10plex cell samples and depleted plasma 1200 958 6000 293T_TMT10plex_24h RTS SPS MS3 9728 9347 samples containing a 6-proteins digest. 800 669 4000 Plasma_TMT10plex_18h RTS SPS MS3 1411 1248 400 1611 293T_TMT10plex_25h DIA 7245 682 PSM Count PSM 400 189 Count PSM 2000 39 90 362 11 7 17 16 16 21 55 149 Plasma_TMT10plex_18h DIA 0 0 809 6985 MATERIALS AND METHODS

Sample Preparation SPS Mass Match [%] SPS Mass Match [%] TMT11-labeled triple knockout (TKO) yeast standards (P/N A40938), Thermo Scientific™ Pierce™ Top14 Abundant Protein Depletion Mini Spin Columns (P/N A36369) and Pierce™ Intact Protein Standard Mix (P/N A33527) were Figure 3. Intelligent acquisition strategies of real-time search can improve the TMT quantification accuracy. CONCLUSIONS obtained from Thermo Fisher Scientific. Pierce TMT11plex Yeast Digest Standard including three triplicated knock-out strains (Met6Δ, His4Δ, or . We use a set of complex models including Pierce™ TMT11plex yeast digest standard and, Ura2Δ) (3A.). These three knock out proteins can be used to evaluate the interference free index4 (IFI) at TMT-10plex cell line and plasma sample to evaluate. High abundant proteins of human plasma were depleted using Top 14 Abundant Protein Depletion Spin Columns. the protein level (3B.) or peptide level (3C.). Trypsin digested human 293T cell, depleted plasma and Pierce intact protein standard mix were prepared in lab follow common digestion workflow2. 0.5 to 48 pmol trypsin digested 6 protein standard were added to 20 μg trypsin A . 6 proteins digest with known relative abundances were spike into cell and plasma proteome. digested human 293T cell and plasma, and then 15μg peptides were labeled with TMT 10plex regent. As needed, met6Δ his4Δ ura2Δ BY4741 This allowed us to study the accuracy and precision of TMT based quantification. TMT labeled 293T cell peptides and plasma were separated via reversed phase chromatography (Figure 1). . RTS-MS3 method on Orbitrap Eclipse instrument is the new gold standard for highest DDA Test Method quantitative accuracy and proteome depth. TMT11PLEX YEAST DIGEST STANDARD Thermo Scientific™ Pierce™ TMT11plex yeast digest standard is used for optimizing method and assessing the Interference free index at the protein level C 1.0 . DIA quantification can be used to further verify the results from TMT quantification. With these sensitivity of RTS-MS3 quantitation. In order to evaluate the accuracy, precision, and sensitivity, 1μg of TMT B 86.18% 93.35% 1.0 84.50% techniques, researchers will be able to find more reliable quantifications, which will finally be labeled peptides containing a 6-proteins digest mixtures were analyzed with Orbitrap Eclipse using MS2, SPS-MS3 83.72% 89.04% 84.29% and RTS-SPS-MS3. For comprehensive proteome characterization, each unlabeled samples was analyzed with 0.8 70.86% translated into meaningful biological findings. 64.25% 60.99% DIA method. Meanwhile, each fraction of TMT labeled samples was analyzed using RTS-MS3 method on an 0.6 0.5 Orbitrap Eclipse, with Xcorr value set to 1 and ∆Cn set as 0.1. 0.4 25%~75% Interference Free Index REFERENCES 0.2 Range within 1.5IQR Data Analysis Median Line Outliers 0.0 0.0 1. Erickson, B.K., et al., Active instrument engagement combined with a real-time database search All TMT data analysis was performed using Thermo Scientific™ Proteome Discoverer™ 2.4 software with a 10ppm MS2 RTS MS3 SPS MS3 MS2 RTS MS3 SPS MS3 MS2 RTS MS3 SPS MS3 Interference Free Index HIS4 MET6 URA2 HIS4 MET6 URA2 for improved performance of sample multiplexing workflows. J Proteome Res., 2019. 18(3): MS2 SPS MS3 RTS SPS MS3 MS1 and 0.6 Da MS2 mass tolerance, TMT6plex (229.163 Da) set as a static modification, co-isolation threshold p. 1299-1306. set to 75. False-discovery rate of PSM, peptide and protein was set to 1%. DIA data was analyzed with DIA-NN software3. Figure 4. Evaluate the performance of real time search with single-phased TMT labeled peptides 2. Wiśniewski, J.R., et al., Universal sample preparation method for proteome analysis. Nat Methods. , 2009. 6(5): p. 359-362. Figure 1. TMT-labeling strategy for the preparation of 293T cell and depleted plasma peptides containing a using HRMS2, SPS-MS3 and RTS-MS3. 1ug of 293T or plasma peptides was analyzed on a 120min and 90min gradient separately. 6-proteins digest mixtures. 0.5 to 48 pmol trypsin digested 6 protein standard were added to 20 μg trypsin 3. Demichev, V., et al., DIA-NN: neural networks and interference correction enable deep proteome digested human 293T cell and plasma peptide. Peptide mixtures were analyzed using MS2, SPS-MS3, RTS- A TMT Labeled 293T Protein B TMT Labeled 293T Peptide C TMT Labeled 293T PSM coverage in high throughput. Nat Methods. , 2020. 17(1): p. 41-44. SPS-MS3 and DIA method. 4000 20000 16314 20000 16797 2876 16783 2741 2561 14918 ， 3000 13538 13838 Pierce 6 Protein Digest Equimolar 0 pmol 0.5pmol 1 pmol 2 pmol 4 pmol 8 pmol 16 pmol 24 pmol 32 pmol 48 pmol 1789 2307 15000 10824 15000 8910 11620 4. Paulo, J.A ., et al, A triple knockout (TKO) proteomics standard for diagnosing ion interference in DIA 8788 5 μg 1728 8348 8797 293T or Plasma Peptide 20ug 20ug 20ug 20ug 20ug 20ug 20ug 20ug 20ug 20ug Analysis 2000 10000 10000 isobaric labeling experiments. J. Am. Soc. Mass Spectrom., 2016. 27(10): p. 1620-1625. PSMs

1000 Peptides 5000 5000 TMT 10plex Labeling Proteins 0 0 0 MS2 SPS MS3 RTS SPS MS2 SPS MS3RTS SPS MS2 SPS MS3RTS SPS ACKNOWLEDGEMENTS MS3 MS3 MS3 We thank our colleagues from Thermo Fisher Scientific China and all over the world. Proteins Quantified Proteins Peptide Quantified Peptides PSM Quantified PSM

D TMT Labeled Plasma Protein E TMT Labeled Plasma Peptide F TMT Labeled Plasma PSM 4708 4586 Sigle-phase Analysis: Reversed-Phase Fractionation 400 339 333 333 5000 4403 8000 5827 TRADEMARKS/LICENSING 296 3812 MS2, SPS MS3, RTS SPS MS3 285277 4000 3625 3487 5818 4571 5757 300 6000 4559 4500 3000 © 2020 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo 200 4000 RTS SPS MS3 Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these RESULTS 2000 PSMs Proteins 100 Peptides 1000 2000 products in any manner that might infringe the intellectual property rights of others. SPS-MS3 based methods provide higher accuracy than HR-MS2 methods for TMT quantitation. However, 0 0 0 conducting an MS3 scan takes time, and not every MS3 scan is useful. Besides, quantitative accuracy depends on MS2 SPS MS3 RTS SPS MS2 SPS MS3 RTS SPS MS2 SPS MS3 RTS SPS MS3 MS3 which fragments are selected for MS3 fragmentation. The real-time Search capability enables instruments to make MS3 an intelligent decision on when to perform an MS3 scan and speeds up this workflow. Using the optimized method, Proteins Quantified Proteins Peptide Quantified Peptides PSM Quantified PSM we could quantify more than 1500 proteins using RTS-MS3 or MS2 based method, while just about 900 proteins could be quantified using SPS-MS3 method from TMT11plex yeast digest standard (Figure 2). What’s more, Real- time search can determine which fragment should be chosen for MS3 fragmentation. The TMT-11plex yeast result showed that the interference could be reduced by 20% compared to MS2-quantification (Figure 3). PO65753-EN 0422S