The Most Powerful Instrument DESIGNED TO ADVANCE YOUR SCIENTIFIC PURSUITS. Tribrid Mass Spectrometer

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1 The Most owerful Instrument DESIGNED T ADVANCE YUR SCIENTIFIC URSUITS Tribrid Mass Spectrometer

2 USING NEW FRNTIERS IN SCIENCE Breakthroughs in science rely on great scientists making rapid progress answering challenging questions. ow does lipid metabolism affect cancer? ow do noncancerous cells mutate? What protein modifications are indicative of a disease state? As a leading scientist, you need to be equipped with the latest innovations in analytical technology. With the Thermo Scientific rbitrap Fusion Lumos Tribrid MS, we are committed to keeping pace with your scientific pursuits through continuous innovations.

3 The Most owerful Instrument Designed for igh Impact Discoveries The Thermo Scientific rbitrap Fusion Lumos Tribrid MS delivers superior sensitivity, selectivity and versatility to enable life scientists to obtain the highest quality data. It is designed to pursue the most difficult analyses, including multiplexed quantitation of lowabundance peptides in complex matrices, characterization of positional isoforms of intact proteins, resolution of isobaric metabolites, protein structure characterization using chemical crosslinking and the deepest mining of challenging posttranslational modifications. You trusted us to make this most versatile and powerful mass spectrometer a reality. We now trust you with groundbreaking science and high impact discoveries.

4 Multiplexing Targeted Analysis Accurately Quantifying undreds of roteins in 90 Minutes Multiplexed analyses using isobaric mass tags are widely utilized for high throughput quantitative comparisons of protein abundances. The TMT SS MS workflow available on the Thermo Scientific rbitrap Fusion Lumos Tribrid MS is a proven method which enables the simultaneous analysis of 10 samples together with improved quantitative accuracy. ushing the multiplexing frontiers even further, targeted assays incorporating this technique can now be built to detect and quantify very low levels of target peptides even from undetected precursors. This new TMT SS tms workflow further benefits from the enhanced sensitivity of the rbitrap Fusion Lumos MS, which boosts the number of quantifiable peptides present at low levels. List of eptide Targets Synthetic peptide targets labeled with TMT0 mixed with Native peptides from digested samples labeled with TMT10 MS DETECTIN Synthetic TMT0labeled Target TMT10labeled Native eptide Target Isolation ffset Time (min) Base peak Extracted Ion Chromatogram (XIC). urple peaks are XICs of TMT0labeled peptides. Everley et al. W 66, ASMS 016. The synthetic TMT0labeled peptide is detected in the full MS scan. The native TMT10labeled peptides are isolated by using an offset ( difference between TMT0 and TMT10).

5 The Thermo Scientific rbitrap Fusion Lumos Tribrid MS now supports targeted proteomics assays with multiplexing in two dimensions: targets and samples. These experiments make use of triggeredbyoffsetinmass peptides that allow for quantitation, even from undetected precursors. Using this method, we monitored 11 target peptides corresponding to 69 proteins across the NCI60 cell line in biological triplicate, analyzing 180 samples in only 8 hours (16 min/sample). We found that accurate and reproducible TMT SS tms quantitation elucidated a correlation between expression of key proteins and cellular response to drug treatment. Steven Gygi, rofessor, arvard Medical School CID MS FTMS CD SS tms IMRVED QUANTITATIVE ACCURACY AT LW LEVELS 8 Expected Ratio 6 Fold Change 0 tms SS tms CID MS spectrum of native peptide target contains multiplexed fragments. Gray bars indicate the predicted fragment target ions that are selected for the following SS tms step. Targeted SS MS of the predicted MS/MS fragments generates ten reporter ions used for quantitation. Comparison of results for the TMT tms and TMT SS tms quantification methods shows improved accuracy of the tms method. Each point represents the fold change of a single quantification event for each of the targeted peptides present at 100 amol level in matrix. The expected ratio is 8:1.

6 Advancing eptide Quantitation Large Scale Quantitation with Data Independent Acquisition Data Independent Acquisition (DIA) is widely used today for the global identification and quantification of thousands of peptides in complex mixtures. Stepwise isolation and MS/MS fragmentation of all ions in a defined window cover the targeted mass range and provide ultrahigh resolution MS and MS/MS data for all components in the sample, allowing accurate quantitation of peptides, and the unique opportunity for retrospective analysis of unknowns and new targets of interest in the future. The Thermo Scientific rbitrap Fusion Lumos Tribrid MS provides speed, selectivity and enhanced sensitivity to obtain maximal performance while maintaining high reproducibility of quantitation for low abundance analytes. LCMS 10 min rotein extraction, and digestion Creation of MS Spectral Library ela digest standard was introduced into the rbitrap Fusion Lumos MS using RLC. Data Dependent experiments were used to generate high resolution accurate mass (RAM) MS spectra. The data was searched using SEQUEST TM T in order to generate a spectral library for the following DIA data analysis.. s cycle DATA INDEENDENT RAM CD MS MS scan Sequential 15 amuwide MS/MS scans DIA acquisition was performed using a 10,000 FWM resolution MS scan followed by 15 amuwide MS/MS windows at 0,000 FWM resolution with 60 ms maximum injection time.

7 MATCING DIA SECTRUM T TE SECTRAL LIBRARY QUANTITATIN USING FULL MS 60,000 RTEIN GRUS UNIQUE ETIDES 50,000 50,659,1 0,000 0,000 0,000 10,000 6,501 5,96 0 Spectral Library Quantified by DIA Spectral Library Quantified by DIA DIA MS spectra were searched against the library built from Sequest T search results. Fragment peak detection was performed with Biognosys Spectronaut TM software. eptide quantitation took into account all confidently identified isotopes from consecutive MS full scans, correcting for interference and using crossrun normalization. Nearly 9% of the protein groups and over 85% of the unique peptides were confidently matched to the spectral library and quantified with <6.9% CV when using 500 ng of ela digest. Confident LowAttomole Limit eptide Quantitation Using arallel Reaction Monitoring arallel Reaction Monitoring (RM) is uniquely designed for quantifying hundreds of targeted proteins in complex matrices. Using this approach, precursor ions are isolated and selectively fragmented, with the resulting product ions analyzed in the rbitrap. This approach benefits from the brighter ion source and Advanced Quadrupole Technology of the Thermo Scientific rbitrap Fusion Lumos Tribrid MS, routinely achieving attomolelevel limits of quantification (LQ) in matrix. GILFVGSGVSGGEEGAR LTILEELR GLILVGGYGTR SAAGAFGELSR TASEFDSAIAQDK LSSEAALFQFDLK ELASGLSFVGFK NGFILDGFR SFANQLEVVYSK ELGQSGVDTYLQTK GISNEGQNASIK SSAAR DIVKK IGDYAGIK VLTSIGEK 100 amol 10.6% 5.1%.% 10 amol 5 amol 1 amol 9.8% Fifteen RTC peptides were spiked into 00 ng of ela digest and analyzed by LCMS (0 min run). The rbitrap Fusion Lumos MS provides accurate quantitation of all 15 RTC peptides with some down to 1 attomole levels. Average CV% for each LQ level is shown.

8 New orizons in Intact rotein Analysis ighly Selective Analysis of rotein Isoforms Topdown mass spectrometry is commonly utilized to characterize intact proteins and their modifications. The Advanced Vacuum Technology, unique to the Thermo Scientific rbitrap Fusion Lumos Tribrid MS, provides conditions optimized for improved performance in intact protein analysis. The high selectivity of Advanced Quadrupole Technology allows for isolation of precursors and detection of fragments with very high resolving power in the rbitrap analyzer. Combined, the new system most efficiently delivers the highest quality data for the characterization of protein isoforms and their posttranslational modifications. The Lumos proved capable of high selectivity precursor selection in MS but retained exceptionally high sequence coverage for MS. This enabled the decoding of the most complicated core histone to detect trivalent proteoforms uniquely detected in a cellular model of the Bcell cancer, multiple myeloma. Neil Kelleher, rofessor, Northwestern University INTACT ISTNE SECTRUM, 18+ IG RESLUTIN ISLATIN F METYLATED FRMS F ISTNE FRAGMENTATIN MAS F ISTNE FRM ETD D TM TDWN EXERIMENTS 1 Da (methylation) Center of 0.5 isolation window Advanced Vacuum Technology increases ion transmission, improving the quality of intact protein spectra acquired in the high resolution accurate mass rbitrap analyzer. Zheng, Y et al. Mol Cell roteomics 016, 15, With improved ion transmission provided by the Advanced Quadrupole Technology, it is now possible to efficiently enrich for individual isobaric protein forms for a subsequent topdown analysis. ETD D identification of isomeric forms of histone, differing in the site of trimethylation (K9 vs. K7) from the precursors at 85.. ETD D enhances the dynamic range of ETD spectra by increasing the precursor ion storage capacity. The higher efficiency of ETD D experiments provides greater sequence coverage at faster acquisition rates.

9 Comprehensive Characterization of Intact Monoclonal Antibodies Recent advances in protein and antibodybased therapeutics have led to a demand for mass spectrometers capable of comprehensive characterization of antibody heterogeneity in addition to providing accurate mass measurements. The Thermo Scientific rbitrap Fusion Lumos Tribrid MS allows high accuracy mass analysis of the intact monoclonal antibody with isotopic resolution of the heavy and light chains. The combination of the various fragmentation methods available on the rbitrap Fusion Lumos MS and the improved efficiency provided by ETD D enables users to obtain high sequence coverage for the light and heavy chains. INTACT mab GLYCFRMS ISTIC RESLUTIN F mab LIGT AND EAVY CAINS TDWN SEQUENCE CVERAGE FR mab LIGT AND EAVY CAINS Light Chain, Da G1F/GF Glycosylated eavy Chain, Da Da G1F/G1F G0F/G1F G0F/G0F G0/G0F Average Mass Matched Matched Delta Glycoforms Mass (ppm) Relative Abundance Raw spectrum showing baseline resolution of major intact mab glycoforms. Deconvolution results are shown in the table above. Isotopic resolution of light chain (top panel), and heavy chain (bottom panel), showing the detection of glycosylated forms. Combination of ETD D, CID and CD fragmentation modes provided 91% sequence coverage for the light chain (top panel) and 6% sequence coverage for the heavy chain (bottom panel).

10 Revolutionizing Glycoproteomics Simplified Glycopeptide Analysis Analysis of posttranslational modifications (TMs) is often central to biological research because of their roles in cellular function and disease states. Some TMs, such as glycosylation, are particularly challenging to analyze because they require information to both accurately determine the composition of the modification as well as correctly identify the modification site. The multiple fragmentation capabilities (CID, CD, ETD and EThcD) on the Thermo Scientific rbitrap Fusion Lumos Tribrid MS are essential for highthroughput, comprehensive glycopeptides analysis. The high dynamic range of ETD and EThcD D yields high quality MS/MS spectra of glycopeptide backbone fragmentation, resulting in higher sequence coverage and accurate localization of the modification site. Accompanying CD spectra provide complementary information about glycan composition. Combination of fragmentation techniques, in addition to intelligent instrument control workflows for glycopeptide analysis unique to the rbitrap Fusion Lumos MS, enables deeper mining of glycosylation modifications in a variety of biological samples. EThcD MS/MS spectrum of linked glycopeptide RBITRA FUSIN LUMS CAN RDUCE: exnac(1)ex(1)neuac() Large scale LCMS intact glycopeptide identification M+eNeuAc M+e LAGT ESVREEGEDFAAR Intelligent acquisition strategies for complete characterization of intact glycopeptide structure using CDproduct ion dependentethcd/cid Increased peptide sequence coverage and confidence in glycosylation site localization with EThcD Quantitation of intact glycopeptides using isobaric tags via SS MS y1 z1 c1 c exnac NeuAc18 NeuAc c z exnacex y z5 ep + z1 z y5 z6 y6 y1 ep+exnac_+ ep+exnacex_+ exnac(1)ex(1)neuac() z15 c9 z16 y15 y8 y7 M+eAcetyl M+e17 c10 y9 ep+exnacex_+ c15 c1 c1 z10 c z11 y10 y11 c c19 y17 c5 c18 c17 M+eNeuAc M+e M+e17 M+eAcetyl c7 c

11 Comprehensive Lipid rofiling Enhancing roductivity with Fast olarity Switching For specific lipid subclasses such as phosphatidylcholine (C), both positive and negative CD MS/MS data are required for full characterization of the individual molecular species. The Thermo Scientific rbitrap Fusion Lumos Tribrid MS allows fast polarity switching and intelligent acquisition of CD and CID MS n. This provides complementary fragmentation information for identifying C molecular species and elucidating coeluting triacylglycerol (TG) isomers within a single LCMS run, yielding higher throughput, increased sensitivity and more confident analysis of lipid molecular species. SITIVE MDE 018 MIN CD MS/MS of ddms ddms T T CD CD 15,000 15,000 ddms T CD ddms 15,000 ddms T CD T 15,000 CD 15,000 ddms T CD 15,000 ddms ddms T CD 15,000 NEGATIVE MDE 018 MIN T CD 15,000 SITIVE MDE 188 MIN NL[FA(16:0)]+N NL[FA(16:0)]+N NL[FA(16:)]+N NL[FA(16:)]+N NL[FA(16:0)]+N 5.7 NL[FA(16:0)]+N NL[FA(16:)]+N NL[FA(16:)]+N NL[FA(16:)]+N NL[FA(16:0)]+N 5.7 CD MS/MS CD MS/MS FA(0:) FA(0:) FA(0:) (_Ch)+ FA(18:0) 0.9 FA(0:) 0.9 FA(0:) NL[FA(18:)] NL[FA(1:0)]+N (_Ch)+ of FA(18:0) 0.9 (_Ch)+ FA(18:0) NL[FA(18:)] of NL[FA(1:0)]+N NL[FA(18:)] NL[FA(1:0)]+N (_Ch)+ FA(18:0) NL[FA(18:)] NL[FA(16:0)]+N NL[FA(16:)]+N NL[FA(1:0)]+N (_Ch)+ FA(18:0) NL[FA(18:)] NL[FA(16:)]+N NL[FA(1:0)]+N NL[FA(16:0)]+N NL[FA(16:)]+N NL[FA(16:0)]+N FA(1:0) FA(1:0) FA(1:0) FA(1:0) FA(1:0) FA(0:)C FA(0:)C FA(0:) FA(0:)C FA(0:)C FA(0:)FA(0:)C FA(0:) (_Ch)+ FA(18:0) FA(18:) NL[FA(18:)] NL[FA(1:0)]+N FA(18:) (_Ch)+ FA(18:0) FA(18:) MFA1FA+C MFA1FA+C FA(1:0) 6.7 NL[FA(1:0)]+N FA(18:) NL[FA(18:)] NL[FA(1:0)]+N (_Ch)+ FA(18:0) NL[FA(18:)] NL[FA(1:0)]+N FA(1:0) 6.7 NL[FA(1:0)]+N FA(18:) MFA1FA+C MFA1FA+C 8.6 FA(1:0) 6.7 FA(1:0) FA(1:0) 6.7 NL[FA(1:0)]+N NL[FA(1:0)]+N MFA1FA+C FA(1:0) 6.7 NL[FA(1:0)]+N.0691 FA(1:0) FA(0:)C FA(1:0) MG(1:0) FA(0:)C FA(0:)C MG(1:0) (Cho)C MG(1:0) MG(1:0) (Cho)C (Cho)C MG(1:0) (Cho)C 85.9 LC(18:0)C 59.0 (Cho)C FA(18:) MFA1FA+C LC(18:0)C FA(18:) LC(18:0)C MC MC LC(18:0)C FA(18:) FA(1:0) 6.7 NL[FA(1:0)]+N MC LC(18:0)C MFA1FA+C MC MFA1FA+C FA(1:0) 6.7 NL[FA(1:0)]+N MC FA(1:0) 6.7 NL[FA(1:0)]+N MG(1:0) MG(1:0) MG(1:0) (Cho)C (Cho)C LC(18:0)C (Cho)C MC LC(18:0)C MC LC(18:0)C MC CID MS/MS of C (18:0_0:) C (18:0_0:) of LC(0:) LC(0:) FA(18:) LC(0:) FA(18:) NL[FA(1:0)] FA(18:) M+ LC(0:) 8.6 FA(18:) NL[FA(1:0)] NL[FA(1:0)] 56.9 M+ M+ LC(0:) M+ 8.6 FA(18:) NL[FA(1:0)] NL[FA(1:0)] :0 FA, C :0 FA, 18 C 5 M :0 FA, C : FA, C :0 5 FA, C LC(18:0) LC(0:) LC(18:0) LC(0:) LC(18:0) LC(0:) LC(18:0) LC(0:) LC(18:0) LC(0:) LC(0:) N >57.7 FA(18:) NL[FA(1:0)]+N 08.9 NL[FA(1:0)] NL[FA(1:0)]+N 08.9 NL[FA(1:0)]+N N M+ + N + N LC(0:) FA(18:) NL[FA(1:0)]+N LC(0:) FA(18:) 57.7 NL[FA(1:0)] 08.9 NL[FA(1:0)] 8.6 N M NL[FA(1:0)]+N C C :0 FA, C 18 5 C LC(18:0) M+ 8.6 C C C C 18:0 FA, C C LC(18:0) LC(18:0) LC(18:0) C 18:0 FA, C C LC(18:0) LC(18:0) LC(0:) LC(18:0) LC(0:) LC(18:0) LC(0:) NL[FA(1:0)]+N N [MC [MC ] : FA, C C ] C LC(18:0) 0: FA, 0 C 1 0 [MC 1 ] NL[FA(1:0)]+N N [MC ] 08.9 NL[FA(1:0)]+N N : FA, C : FA, C 0 1 [MC C C ] LC(18:0) 0: C FA, C LC(18:0) C [MC ] [MC [MC 0: FA, C 0 1 ] 0: FA, C ] 0: FA, C TG (1:0_1:0_18:) :0 FA, C :0 FA, 1 C 8 1 1:0 FA, C :0 FA, C 18:0 FA, C 18:0 FA, 18 C :0 8 FA, 18 18:0 FA, C C :0 FA, C :0 6 FA, C N N :0 N N + FA, C 1 8 N N 1:0 FA, C 1 8 N 18:0 FA, + N C N 1:0 FA, C N 18:0 FA, C :0 FA, C C N C N 5 15 N C + C 5 15 N N N C N N + + N N + [M+N [M+N ] + N + N ] + [M+N N [M+N :0 FA, C :0 FA, 1 C 1 N ] + N ] + N [M+N ] + N : FA, C 1 1:0 FA, C 1 N 0: FA, 0 C 1:0 FA, C C N 1 1 N 0: FA, C :0 1 N 0: FA, C FA, C 1 1 N 0: FA, C C 5 15 N 0 + C 5 15 N [M+N ] + N [M+N [M+N ] + N NEGATIVE ] + N :0 FA, C 1 1 N 0: FA, C 1:0 FA, C 1:0 FA, C N 0: FA, C N 0: FA, C 0 CID MS SITIVE 0 MIN Fast polarity switching during LC/MS analysis. A cycle with 18 MIN one positive and one negative rbitrap MS scan at 10,000 resolving power takes less than seconds to complete. 8 MIN

12 Small Molecule Analysis Ultimate Confidence in Identification and Quantitation The high sensitivity and high resolution of the Thermo Scientific rbitrap Fusion Lumos Tribrid MS makes it a powerful instrument for the identification and quantitation of small molecules. Due to the instrument s very high resolution capabilities, fine isotopic structure can be observed, enabling the determination of highly accurate molecular formulae. This direct measurement removes the ambiguity of pattern matching estimations and is critical in cases where monoisotopic elements like fluorine or phosphorous may be present in the compound, as shown in the example of norfloxacin below. Furthermore, the enhanced sensitivity of the rbitrap Fusion Lumos MS enables far lower levels of quantitation. Improved Low Limit f Quantitation S N Resolving Isobaric Interferences N N F N Relative Abundance Relative Abundance Relative Abundance C1 C X X C 1 C Time (min) LQ 500 fg 100 fg Standard Ion Source New Ion Source on rbitrap Fusion Lumos MS 15 N15 N N15 1 NC 1 C 1 C C.19 Extracted ion chromatogram of Irganox 105 (M+N ) + ion at (100 fg on column). Irganox is a plasticizer known to leach into foods stored in plastic and must be quantified at very low levels. The rbitrap Fusion Lumos MS operated in SIM mode was able to quantify Irganox 105 in food simulant matrix, achieving an LQ of 100 fg with linear dynamic range of 5 orders and <10% CV for all levels. This LQ is 5x lower than previously achieved on an rbitrap instrument equipped with a standard ion source igh resolution MS spectra of norfloxacin. The direct observations of fine isotopes of the drug are essential for determining elemental composition.

13 The list of the viewable parameters in the scan and filter properties can now be customized MS T TMT SS MS Run advanced experiments effortlessly with validated template methods for a variety of applications ddms T CD Edit parameters in easily accessible properties panels, featuring recommended defaults for each experiment Focus on Your Science, Not on Instrument Set Up The highly intuitive method editor features a userfriendly interface that includes optimized predesigned templates for a wide range of applications.

14 Building on Revolutionary ADVANCED ACTIVE IN BEAM GUIDE revents neutrals and high velocity clusters from entering mass resolving quadrupole Tribrid Architecture The Thermo Scientific rbitrap Fusion Lumos Tribrid mass spectrometer further amplifies the power and versatility of the innovative Tribrid design, first pioneered on the rbitrap Fusion MS. The rbitrap Fusion Lumos mass spectrometer incorporates the latest technologies and groundbreaking innovations in ion transmission, dissociation and detection. The combination of these improvements makes it the most sensitive, most selective and most versatile mass spectrometer to date. With the rbitrap Fusion Lumos MS, we are true partners, committed to innovations to advance your scientific pursuits. EASY ETD SURCE Based on Townsend discharge; reliable and easy to use ELECTRDYNAMIC IN FUNNEL Focuses ions after igh Capacity Transfer Tube; broad tuning curves

15 DUALRESSURE LINEAR IN TRA ETD D Improved dynamic range and detection limits for ETD/EThcD events MS n and sensitive mass analysis of fragments resulting from CID, CD, ETD and EThcD ULTRAIG FIELD RBITRA ANALYZER ffers resolution >500,000 FWM and scan rates up to 0 z at 15,000 FWM IN RUTING MULTILE Enables parallel analysis; allows CD at any MS n stage ADVANCED VACUUM TECNLGY Reduces pressure in UV region, improving transmission to the rbitrap analyzer ADVANCED QUADRULE TECNLGY Segmented design improves transmission at higher resolution; symmetric transmission across the isolation window IG CAACITY TRANSFER TUBE Increases ion flux into the mass spectrometer

16 ushing New Frontiers in Science The Industry s Leading ortfolio of Mass Spectrometry Solutions Metabolomics roteomics Bioanalysis Lipidomics Exactive Series MS Nontargeted Analysis RAM Tribrid Series MS Biomarker Discovery roteomics Metabolism Metabolomics Lipidomics Quantitative Applied Markets Transform Your Science Research Markets Qualitative Food Safety Environmental Clinical Research/ Forensic Toxicology Triple Quadrupole MS MS, MS n Ion Trap MS TM Analysis Lipidomics roteomics Targeted Analysis FR RESEARC USE NLY. NT FR USE IN DIAGNSTIC RCEDURES thermofisher.com/lumos 016 Thermo Fisher Scientific Inc. All rights reserved. SEQUEST T is a registered trademark of the University of Washington. Biognosys Spectronaut is a registered trademark of BiognoSYS AG. TMT is a trademark of roteome Sciences plc. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. lease consult your local sales representative for details. Africa Australia Austria Belgium Canada China (free call domestic) Denmark Europether Finland France Germany India Italy Japan Korea Latin America Middle East Netherlands New Zealand Norway Russia/CIS Singapore Spain Sweden Switzerland UK USA BR69EN 0716M