Towards Turnkey Targeted Proteomics Solutions …...Towards Turnkey Targeted Proteomics Solutions...
1
Sebastien Gallien 1,2 , Aaron S. Gajadhar 3 , Bhavin Patel 4 , Markus Kellmann 5 , Tabiwang N. Arrey 5 , Christian Thoeing 5 , Alexander Harder 5 , Romain Huguet 3 , Graeme McAlister 3 , Derek Bailey 3 , Shannon Eliuk 3 , Emily L. Chen 2 , Yue Xuan 5 , Andreas Huhmer 3 1 Thermo Fisher Scientific, Paris, France; 2 Thermo Fisher Scientific, Precision Medicine Science Center, Cambridge, MA; 3 Thermo Fisher Scientific, San Jose, CA; 4 Thermo Fisher Scientific, Rockford, IL; 5 Thermo Fisher Scientific, Bremen, Germany SureQuant IS-PRM Methodology The original implementation of the IS-PRM method, using instrument application programming interface, toggled between i) a “watch mode”, in which IS were continuously measured through fast PRM scans over their elution time monitoring windows (dynamically corrected), and ii) a “quantitative mode” that was triggered by the real-time detection of the IS (through dot-product spectral matching), wherein the corresponding pairs of IS and endogenous peptides were measured over their pre-defined elution profiles with optimized PRM acquisition parameters. In order to foster a broader adoption of the approach, we produced a generic implementation of the method (Figure 2), also benefiting from higher usability, in the native instrument control software of latest generation Orbitrap-based instruments. The acquisition scheme has been revised while keeping the same rationale of using IS to drive the acquisition in two modes. More specifically, the watch mode portion has been substantially modified and relies on a two-step process interrogating both MS1 and MS2 data to track the elution of IS. With such set-up, the acquisition fully avoid any dependency on time scheduling. In addition, substituting dot product spectral library matching with fragment matching only for the MS2-based confirmation of the IS improves the robustness and portability of the approach. The structure of the revised acquisition method, detailed in Figure 3, includes different scan events and filters operated with parameters optimized for the highest data quality and the sensitivity of triggering as first priority but also to retain sufficient triggering specificity (Figure 4). ABSTRACT Purpose: A novel internal standard triggered targeted quantification workflow, called ‘SureQuant’ workflow (Figure 1), has been developed and implemented in the native instrument control software of novel Orbitrap mass spectrometers: Thermo Scientific™ Orbitrap Exploris™ 480 mass spectrometer and Thermo Scientific™ Orbitrap Eclipse™ Tribrid™ mass spectrometer. It aims at enabling large-scale target profiling with superior quantitative performance, robustness, and usability. Methods: The SureQuant internal standard triggered-PRM (IS-PRM) acquisition scheme was developed and tested on Orbitrap Exploris 480 and Orbitrap Eclipse Tribrid mass spectrometers. Chromatographic separations were carried out using Thermo Scientific™ UltiMate™ 3000 RSCLC system and Thermo Scientific™ EASY-nLC™ 1200 system. Thermo Scientific™ Pierce™ SureQuant™ AKT Pathway Multiplex Panel kit and PQ500 kit from Biognosys were used to evaluate SureQuant workflows applied to the analyses of cancer cell line models and undepleted plasma samples. Results: The novel SureQuant IS-PRM method significantly enhanced targeted data acquisition efficiency in comparison with conventional PRM method (i.e., 80-90% of the instrument time dedicated to the collection of pertinent data), and exhibited strong robustness against chromatographic variations. Applied to the analyses of a variety of samples supplemented with different sets of stable isotopically labeled (SIL) peptides used as internal standards (IS) in various formats (30-800 IS), the method demonstrated the capability to quantify endogenous peptides in the low amol range at all scales. In addition, Embedding pre-set methods, associated with predefined kits of IS peptides, directly into the instrument control software turned out to be a decisive step towards the provision of turnkey targeted proteomics solutions. INTRODUCTION The advances in HRAM mass spectrometry instrumentation have enabled new approaches for targeted quantitative proteomics, such as the parallel reaction monitoring (PRM) technique. An extension of PRM uses spiked-in internal standards to dynamically control the acquisition process and to maximize its efficiency. The original implementation of this internal standard triggered-PRM method on a quadrupole Orbitrap instrument has enabled the systematic monitoring of larger sets of proteins with higher analytical performance. Here, the acquisition scheme of the approach has been revised, while keeping the same rationale, to enable a more generic implementation on latest generation Orbitrap-based instruments. Under this new implementation, the method has exhibited significantly enhanced usability and robustness without sacrificing analytical benefits. MATERIALS AND METHODS Sample Preparation Cell Culture: HCT 116, MCF7, and A549 cells were grown in McCoy’s 5A Media with 10% FBS/1xPenStrep to ~70-80% confluency. HCT116 cells were serum starved in 0.1% charcoal stripped FBS for 24 hours prior to stimulation (15 min hIGF-1 (100ng/mL; Cell Signaling Technology PN#8917SF)). Then cells were lysed with IP-Lysis buffer (Thermo Fisher Scientific PN#87788), and processed by reduction, alkylation and trypsin (Thermo Fisher Scientific PN#90057) digestion overnight at 37 O C. The digested samples were acidified with TFA. A commercial human plasma sample pooled from several individuals purchased from BioreclamationIVT was processed using Thermo Scientific™ EasyPep™ Mini MS Sample Prep Kit. LC-MS sample preparation: A set of 30 SIL peptides from SureQuant AKT Pathway Multiplex Panel (Thermo Fisher Scientific PN#A40080) was spiked at 50 fmol in 500 ng of HeLa digest (Thermo Fisher Scientific PN#88329), HCT116 digest, MCF7 digest, and A549 digest. One µL of the sample was injected for LC-MS/MS analyses. A set of 804 SIL peptides from PQ500 kit (Biognosys PN#Ki-3019-96) was spiked at around 80 fmol (median value) in 1 µg of undepleted plasma sample. One µL of the sample was injected for LC-MS/MS analyses. LC-MS/MS Analysis Chromatographic separations were performed on UltiMate 3000 RSLC system equipped with C 18 trap cartridge (5 µm, 0.3 x 5 mm) and analytical column (2 μm, 0.15 x 150 mm) and EASY-nLC 1200 system equipped with a C 18 analytical column (2 μm, 0.15 x 150 mm) . The LC systems were coupled to Orbitrap Exploris 480 and Orbitrap Eclipse Tribrid mass spectrometers operated in conventional PRM and SureQuant IS-PRM mode. The acquisition parameters are detailed in the pertinent sections of the poster. CONCLUSIONS A novel IS triggered targeted quantification workflow, called ‘SureQuant’ workflow has been successfully implemented in the native instrument control software of Orbitrap Exploris 480 and Orbitrap Eclipse Tribrid MS. The SureQuant method demonstrated exquisite acquisition efficiency and robustness, translating into systematic and highly sensitive quantification of large sets of endogenous peptides ( up to 800) in a variety of samples. Its ‘load-and- play’ execution, especially for embedded application specific kits, significantly improves user experience. For Research Use Only. Not for use in diagnostic procedures. TRADEMARKS/LICENSING © 2019 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. Towards Turnkey Targeted Proteomics Solutions Using Internal Standard Triggered Acquisitions on Modified Orbitrap MS Figure 5. Improved method building usability through ‘Group ID’ feature implementation Figures of Merit of SureQuant Method The native implementation of SureQuant method in the instrument control software facilitates method building, especially for large scale experiments, through the use of the new ‘Group ID’ feature (Figure 5). This feature offers a way to streamline the upload of the necessary information associated with the precursor and fragment ions of the targeted peptides, only requiring a few branches to be included in the method. In order to illustrate the enhanced productivity of SureQuant acquisition, it has been compared with time-scheduled PRM measurement of a pair of IS and endogenous peptides, using a 2.5-min monitoring window (Figure 6). A significantly higher proportion of productive scans was observed in SureQuant analysis (i.e., 80-90% against 10-15% in PRM analysis). In addition, SureQuant acquisition exhibited a strong robustness against chromatographic variations (Figure 7). The introduction of an artificial elution time drift (offset gradient mimicking chormatographic dysfunction) compromised PRM measurements while the capture of the targeted peptides in SureQuant analysis was not affected. Application to Signaling Pathway Monitoring SureQuant method was applied to the monitoring of AKT/mTOR signaling pathway using SureQuant AKT Pathway Multiplex Panel kit. The kit includes 30 SIL peptides associated to the 12 main protein components of the pathway. After initial assay development, such as described in the upper part of Figure 1, PRM and SureQuant methods have been optimized in order to maximize the sensitivity/selectivity of endogenous peptides measurements in cell line digests while maintaining a chromatographic sampling rate of at least 8 data points over the LC separation. It turned out that with the 30-min gradient used, MS/MS of endogenous peptides can be acquired with 6 times more fill times in SureQuant analyses (Figure 8, upper panel), translating into significantly improved sensitivity of measurements. This higher sensitivity enabled 26 of the 30 targeted endogenous peptides to be detected / quantified by SureQuant, against only 11 by PRM (Figure 8, lower panel). The higher acquisition time (resolution and fill time) dedicated to endogenous peptides measurements in SureQuant analyses significantly improved signal to noise ratio of their extracted fragment ion traces. Applied to other cancer line digests stimulated with IGF-1, SureQuant method delivered near comprehensive quantification of the entire set of targets over a quantification range between 10 amol and 1 fmol, and with exquisite precision, reflected by 60% of the endogenous peptides quantified with CV<5% (Figure 9). LC-MS Analysis Synthetic isotopically labeled peptides (IS) #1 #2 #n …. Sample SureQuant ENDO Peptides Pseudo Spectral Matching ‘QUANT MODE’ High Quality MS2 of Target ‘ WATCH MODE’ Fast, Low Res MS2 of Trigger 1 4 5 2 3 MS1 Survey Scan Mix MS/MS IS IS peptides (trigger) Predefined precursor ions and corresponding intensity threshold of IS are monitored in Full scan MS Candidate precursor ions satisfying filtering criteria subjected to fast MS/MS (low resolution / low fill time) Several “selective” fragment ions of IS are monitored in fast MS/MS The detection of IS triggers the high-quality measurement of corresponding ENDO peptide (high resolution / fill time) 1 2 3 4 5 1 2 4 5 3 Full MS - BPC LC DSGELVAIK (2 + ) MS/MS LC DSGELVAIK (2 + ) - IS MS/MS LC DSGELVAIK (2 + ) - ENDO 100 200 300 400 500 600 700 800 900 1000 1100 1200 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 939.519 131.116 824.492 1099.549 339.245 953.451 276.062 737.462 672.316 246.124 155.125 606.821 887.345 550.276 786.908 438.312 389.135 235.142 1000.439 1066.550 y 9 + y 10 + y 8 + y 7 + y 3 + y 4 + 0 50 100 13.79 13.80 13.81 13.77 13.83 13.76 13.75 13.84 13.74 13.72 13.71 0 50 100 13.79 13.78 13.80 13.76 13.82 13.75 13.83 13.74 13.85 13.73 13.72 13.71 13 78 13.70 13.75 13.80 13.85 0 50 100 13.81 13.78 13.77 13.82 13.76 13.74 13.84 13.73 13.85 13.72 13.71 605.5 606.0 606.5 607.0 607.5 608.0 m/z 0 20 40 60 80 100 Relative Abundance 606.823 606.281 607.297 607.633 607.967 606.325 605.595 NL: 8.85E5 High Resolution (120k); Lock mass Precursor m/z; Tolerance (3-10 ppm); Intensity Threshold (1% Apex) ‘Low’ resolution (7.5k); ‘Low’ Fill time (10 ms) Fragments m/z (6/pep.); Tolerance (10-20 ppm); Min. number frags (5) ‘High’ resolution (e.g., 60k); ‘High’ Fill time (e.g., 116 ms) 1 2 3 4 5 Full MS – Zoom in MS/MS 1 2 3 5 4 1 2 3 4 5 Heavy R, Charge: +2 Heavy K, Charge: +2 Heavy R, Charge: +3 Heavy K, Charge: +3 Heavy R, Charge: +4 Heavy K, Charge: +4 #1 #2 #3 #4 #5 #6 #1 #2 #3 #4 #5 #6 2 4 5 ‘Group ID’ Feature Expected elution window Expected elution window Delayed Elution PRM SureQuant SIL Peptide Detection Original elution profile Delayed elution profile Internal standards in matrix Survey Run SureQuant LC-MS Analysis Internal standards Sample LC-MS Analysis Survey Run Analysis Export reports & Refine analytes definition Targeted Mass Targeted Mass Trigger Update scan filter tables Target Quantification ‘Optimal’ fragments / selected peptide precursor ‘Optimal’ precursor ion / peptide – Intensity response Directed DDA Inclusion list IS precursor ions multiple charge states / peptide Generic template Generic template SureQuant Prepare initial processing method from generic Skyline analysis template Refined processing method Copy/Paste Towards Turnkey Targeted Proteomics Solutions The development of a SureQuant multi-peptides assay requires the preliminary determination of the optimal precursor ion of each peptide target together with their intensity response and optimal associated fragment ions (Figure 1). This information is typically obtained from a directed DDA analysis of the corresponding IS peptide mixture. The target list of multiple theoretical precursor ions of each IS peptide included in such survey runs enables comprehensive data collection, and therefore the refinement of analytes definition for subsequent SureQuant analyses. The process is supported by Skyline software and facilitated by the generic data acquisition and processing templates provided. The transfer of the generated information can be achieved through simple copy/paste from Skyline reports to ‘Targeted Mass’ and ‘Targeted Mass Trigger’ tables of SureQuant method. The survey analysis of a SureQuant assay needs to be run one time as the onset of the project without further adjustment over time, unless a major change in the experimental design is applied (e.g., LC set up, spiked amount of SIL peptides). In the latter case, only the intensity response of predefined IS precursor ions needs to be re-adjusted though an additional survey run performed under the modified experimental conditions (Figure 10). Therefore, the ability of embedding pre-set methods associated with predefined kits of IS peptides expedites SureQuant assay development at the user sites. Several validated method templates of this type have already been included in the instrument control software, such as the PQ500 related methods (Figure 11), prepared for the analyses of plasma samples supplemented with PQ500 kit (which includes 804 SIL peptides associated with more than 500 plasma proteins). The applications of the SureQuant PQ500 method to the analyses of undepleted plasma samples enabled the robust and precise quantification of around 560 endogenous peptides, corresponding to around 400 proteins in a 70-min LC gradient. 0 0.4 0.8 1.2 1.6 6 8 10 12 14 16 18 20 22 Cycle time range enabling optimal sampling rate: ≥ 8 data points (15-s peak width) Cycle Time [s] IT: 20 ms IT: 116 ms AENGPGPGVLVLR 1 15 AQEALDFYGEVR 10 11 DGFYPAPDFR 0 1 ETSFNQAYGR 2 6 FEISETSVNR 0 9 GALAEAAR 5 13 GNNLQDTLR 0 6 GQPEGPLPSSSPR 0 8 GYTISDSAPSR 0 5 IDIHSC[Carbamidomethyl]NHEAEK 1 12 IYNLC[Carbamidomethyl]AER 0 15 LC[Carbamidomethyl]DSGELVAIK 5 15 LFDAPEAPLPSR 11 11 LLEYTPTAR 0 15 LPFYNQDHER 0 17 LVPPFKPQVTSETDTR 0 0 NDGTFIGYK 0 13 NNIDDVVR 0 4 SDGSFIGYK 0 13 SNPTDIYPSK 0 9 SQEVAYTDIK 1 9 SSDEENGPPSSPDLDR 0 8 SVSAPQQIINPIR 0 0 TFHVDTPEER 0 0 TGIAAEEVSLPR 0 0 TLDQSPELR 2 10 TPPEAIALC[Carbamidomethyl]SR 1 20 TTINNEYNYR 0 7 VTTVVATLGQGPER 6 15 YSFQTHDR 0 10 Peptide PRM 15K-20ms SQ IS-PRM_60k/116ms Number of spectra including ≥ 5 ref. fragments 0 1-2 ≥ 3 ≤LOD Detection >>> Quantification Number of ENDO pept. detected: 11 26 SSDEENGPPSSPDLDR SQEVAYTDIK 16.6 16.7 16.8 16.2 16.35 16.5 16.2 16.35 16.5 16.2 16.35 16.5 16.2 16.35 16.5 6 e 6 6 e 6 6 e 3 6 e 3 PRM SureQuant 16.6 16.7 16.8 16.6 16.7 16.8 16.6 16.7 16.8 SIL ENDO SIL ENDO ≈ 1.2 e 7 1.2 e 7 4 e 4 4 e 4 0 0.2 0.4 0.6 0.8 1 1.2 HCT116 (+IGF1) MCF7 (+IGF1) A549 (+IGF1) [fmol] 0 0.5 1 1.5 2 2.5 3 200 600 1000 1400 0 10 20 30 40 50 60 70 80 90 [min] m/z [s] Endo. Pept. non-quantified (238) Endo. Pept. Quantified (564) Cycle time Figure 2. SureQuant acquisition scheme. Figure 3. SureQuant method structure. Figure 4. SureQuant acquisition process and critical parameters. Figure 6. Acquisition efficiency of conventional PRM and SureQuant methods Figure 7. Robustness of SureQuant acquisition against chromatographic variations Figure 8. Comparison of the analytical performance of PRM and SureQuant analyses Figure 9. Sensitive and precise quantification of AKT/mTOR peptides by SureQuant analyses. Figure 10. Overall SureQuant acquisition worklow of embedded application specific kits. Figure 11. SureQuant analysis of plasma enabled by embedded preset methods Figure 1. Overall SureQuant acquisition workflow 19 20 21 22 23 24 19 20 21 22 23 24 19 20 21 22 23 24 19 20 21 22 23 24 LCDSGELVAIK [min] [min] SIL ENDO PRM SureQuant Non-productive MS2 scans Productive MS2 scans TIC TIC TIC TIC Proportion productive MS2 scans (% Acq. Time) PRM: 10 – 15 % SureQuant: 80 – 90 % 2.5-min window 2.5-min window >6 data points / peak (20-s peak width) PO65533-EN0519S
Transcript of Towards Turnkey Targeted Proteomics Solutions …...Towards Turnkey Targeted Proteomics Solutions...
Sebastien Gallien1,2, Aaron S. Gajadhar3, Bhavin Patel4, Markus Kellmann5, Tabiwang N. Arrey5, Christian Thoeing5, Alexander Harder5, Romain Huguet3, Graeme McAlister3, Derek Bailey3, Shannon Eliuk3, Emily L. Chen2, Yue Xuan5, Andreas Huhmer3
1Thermo Fisher Scientific, Paris, France; 2Thermo Fisher Scientific, Precision Medicine Science Center, Cambridge, MA; 3Thermo Fisher Scientific, San Jose, CA; 4Thermo Fisher Scientific, Rockford, IL; 5Thermo Fisher Scientific, Bremen, Germany
SureQuant IS-PRM MethodologyThe original implementation of the IS-PRM method, using instrument application programming interface, toggled betweeni) a “watch mode”, in which IS were continuously measured through fast PRM scans over their elution time monitoringwindows (dynamically corrected), and ii) a “quantitative mode” that was triggered by the real-time detection of the IS(through dot-product spectral matching), wherein the corresponding pairs of IS and endogenous peptides were measuredover their pre-defined elution profiles with optimized PRM acquisition parameters. In order to foster a broader adoption ofthe approach, we produced a generic implementation of the method (Figure 2), also benefiting from higher usability, in thenative instrument control software of latest generation Orbitrap-based instruments. The acquisition scheme has beenrevised while keeping the same rationale of using IS to drive the acquisition in two modes. More specifically, the watchmode portion has been substantially modified and relies on a two-step process interrogating both MS1 and MS2 data totrack the elution of IS. With such set-up, the acquisition fully avoid any dependency on time scheduling. In addition,substituting dot product spectral library matching with fragment matching only for the MS2-based confirmation of the ISimproves the robustness and portability of the approach. The structure of the revised acquisition method, detailed inFigure 3, includes different scan events and filters operated with parameters optimized for the highest data quality andthe sensitivity of triggering as first priority but also to retain sufficient triggering specificity (Figure 4).
ABSTRACTPurpose: A novel internal standard triggered targeted quantification workflow, called ‘SureQuant’ workflow (Figure 1),has been developed and implemented in the native instrument control software of novel Orbitrap mass spectrometers:Thermo Scientific™ Orbitrap Exploris™ 480 mass spectrometer and Thermo Scientific™ Orbitrap Eclipse™ Tribrid™mass spectrometer. It aims at enabling large-scale target profiling with superior quantitative performance, robustness, andusability.
Methods: The SureQuant internal standard triggered-PRM (IS-PRM) acquisition scheme was developed and tested onOrbitrap Exploris 480 and Orbitrap Eclipse Tribrid mass spectrometers. Chromatographic separations were carried outusing Thermo Scientific™ UltiMate™ 3000 RSCLC system and Thermo Scientific™ EASY-nLC™ 1200 system. ThermoScientific™ Pierce™ SureQuant™ AKT Pathway Multiplex Panel kit and PQ500 kit from Biognosys were used to evaluateSureQuant workflows applied to the analyses of cancer cell line models and undepleted plasma samples.
Results: The novel SureQuant IS-PRM method significantly enhanced targeted data acquisition efficiency incomparison with conventional PRM method (i.e., 80-90% of the instrument time dedicated to the collection of pertinentdata), and exhibited strong robustness against chromatographic variations. Applied to the analyses of a variety of samplessupplemented with different sets of stable isotopically labeled (SIL) peptides used as internal standards (IS) in variousformats (30-800 IS), the method demonstrated the capability to quantify endogenous peptides in the low amol range at allscales. In addition, Embedding pre-set methods, associated with predefined kits of IS peptides, directly into the instrumentcontrol software turned out to be a decisive step towards the provision of turnkey targeted proteomics solutions.
INTRODUCTIONThe advances in HRAM mass spectrometry instrumentation have enabled new approaches for targeted quantitativeproteomics, such as the parallel reaction monitoring (PRM) technique. An extension of PRM uses spiked-in internalstandards to dynamically control the acquisition process and to maximize its efficiency. The original implementation of thisinternal standard triggered-PRM method on a quadrupole Orbitrap instrument has enabled the systematic monitoring oflarger sets of proteins with higher analytical performance. Here, the acquisition scheme of the approach has been revised,while keeping the same rationale, to enable a more generic implementation on latest generation Orbitrap-basedinstruments. Under this new implementation, the method has exhibited significantly enhanced usability and robustnesswithout sacrificing analytical benefits.
MATERIALS AND METHODSSample PreparationCell Culture: HCT 116, MCF7, and A549 cells were grown in McCoy’s 5A Media with 10% FBS/1xPenStrep to ~70-80%confluency. HCT116 cells were serum starved in 0.1% charcoal stripped FBS for 24 hours prior to stimulation (15 minhIGF-1 (100ng/mL; Cell Signaling Technology PN#8917SF)). Then cells were lysed with IP-Lysis buffer (Thermo FisherScientific PN#87788), and processed by reduction, alkylation and trypsin (Thermo Fisher Scientific PN#90057) digestionovernight at 37OC. The digested samples were acidified with TFA.A commercial human plasma sample pooled from several individuals purchased from BioreclamationIVT was processedusing Thermo Scientific™ EasyPep™ Mini MS Sample Prep Kit.LC-MS sample preparation: A set of 30 SIL peptides from SureQuant AKT Pathway Multiplex Panel (Thermo FisherScientific PN#A40080) was spiked at 50 fmol in 500 ng of HeLa digest (Thermo Fisher Scientific PN#88329), HCT116digest, MCF7 digest, and A549 digest. One µL of the sample was injected for LC-MS/MS analyses.A set of 804 SIL peptides from PQ500 kit (Biognosys PN#Ki-3019-96) was spiked at around 80 fmol (median value) in 1µg of undepleted plasma sample. One µL of the sample was injected for LC-MS/MS analyses.
LC-MS/MS AnalysisChromatographic separations were performed on UltiMate 3000 RSLC system equipped with C18 trap cartridge (5 µm, 0.3x 5 mm) and analytical column (2 μm, 0.15 x 150 mm) and EASY-nLC 1200 system equipped with a C18 analytical column(2 μm, 0.15 x 150 mm) . The LC systems were coupled to Orbitrap Exploris 480 and Orbitrap Eclipse Tribrid massspectrometers operated in conventional PRM and SureQuant IS-PRM mode. The acquisition parameters are detailed inthe pertinent sections of the poster.
CONCLUSIONS A novel IS triggered targeted quantification workflow, called ‘SureQuant’ workflow has been successfully implemented
in the native instrument control software of Orbitrap Exploris 480 and Orbitrap Eclipse Tribrid MS.
The SureQuant method demonstrated exquisite acquisition efficiency and robustness, translating into systematic andhighly sensitive quantification of large sets of endogenous peptides ( up to 800) in a variety of samples. Its ‘load-and-play’ execution, especially for embedded application specific kits, significantly improves user experience.
For Research Use Only. Not for use in diagnostic procedures.
TRADEMARKS/LICENSING© 2019 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific andits subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe theintellectual property rights of others.
Towards Turnkey Targeted Proteomics Solutions Using Internal Standard Triggered Acquisitions on Modified Orbitrap MS
Figure 5. Improved method building usability through ‘Group ID’ feature implementation
Figures of Merit of SureQuant MethodThe native implementation of SureQuant method in the instrument control software facilitates method building, especiallyfor large scale experiments, through the use of the new ‘Group ID’ feature (Figure 5). This feature offers a way tostreamline the upload of the necessary information associated with the precursor and fragment ions of the targetedpeptides, only requiring a few branches to be included in the method. In order to illustrate the enhanced productivity ofSureQuant acquisition, it has been compared with time-scheduled PRM measurement of a pair of IS and endogenouspeptides, using a 2.5-min monitoring window (Figure 6). A significantly higher proportion of productive scans wasobserved in SureQuant analysis (i.e., 80-90% against 10-15% in PRM analysis). In addition, SureQuant acquisitionexhibited a strong robustness against chromatographic variations (Figure 7). The introduction of an artificial elution timedrift (offset gradient mimicking chormatographic dysfunction) compromised PRM measurements while the capture of thetargeted peptides in SureQuant analysis was not affected.
Application to Signaling Pathway MonitoringSureQuant method was applied to the monitoring of AKT/mTOR signaling pathway using SureQuant AKT PathwayMultiplex Panel kit. The kit includes 30 SIL peptides associated to the 12 main protein components of the pathway. Afterinitial assay development, such as described in the upper part of Figure 1, PRM and SureQuant methods have beenoptimized in order to maximize the sensitivity/selectivity of endogenous peptides measurements in cell line digests whilemaintaining a chromatographic sampling rate of at least 8 data points over the LC separation. It turned out that with the30-min gradient used, MS/MS of endogenous peptides can be acquired with 6 times more fill times in SureQuant analyses(Figure 8, upper panel), translating into significantly improved sensitivity of measurements. This higher sensitivity enabled26 of the 30 targeted endogenous peptides to be detected / quantified by SureQuant, against only 11 by PRM (Figure 8,lower panel). The higher acquisition time (resolution and fill time) dedicated to endogenous peptides measurements inSureQuant analyses significantly improved signal to noise ratio of their extracted fragment ion traces. Applied to othercancer line digests stimulated with IGF-1, SureQuant method delivered near comprehensive quantification of the entire setof targets over a quantification range between 10 amol and 1 fmol, and with exquisite precision, reflected by 60% of theendogenous peptides quantified with CV<5% (Figure 9).
LC-MS Analysis
Synthetic isotopically labeled peptides (IS)
#1#2
#n….
SampleSureQuant
ENDO Peptides Pseudo Spectral Matching
‘QUANT MODE’High Quality MS2 of Target
‘WATCH MODE’
Fast, Low Res MS2 of Trigger
1
4
5
2 3
MS1 Survey Scan
Mix
MS/MS IS
IS peptides (trigger)
Predefined precursor ions and corresponding intensity threshold of IS are monitored in Full scan MS
Candidate precursor ions satisfying filtering criteria subjected to fast MS/MS (low resolution / low fill time)
Several “selective” fragment ions of IS are monitored in fast MS/MS
The detection of IS triggers the high-quality measurement of corresponding ENDO peptide (high resolution / fill time)
1
2
3
4
5
1 2
4
5
3
Full MS - BPC LCDSGELVAIK (2+)
MS/MS LCDSGELVAIK (2+) - IS
MS/MS LCDSGELVAIK (2+) - ENDO
p @ [ ]
100 200 300 400 500 600 700 800 900 1000 1100 1200m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive
Abun
danc
e
939.519
131.116
824.492 1099.549339.245
953.451276.062737.462
672.316246.124155.125
606.821 887.345550.276 786.908438.312389.135
235.142 1000.4391066.550
y9+
y10+y8
+
y7+y3
+
y4+
0
50
10013.79 13.80
13.8113.77
13.8313.7613.75 13.84
13.7413.7213.71
13 79
0
50
100
013.7913.78 13.8013.76
13.8213.75 13.8313.74 13.8513.73
13.7213.71
13 8113 78
13.70 13.75 13.80 13.85
0
50
1000
13.8113.7813.77 13.8213.76
13.7413.8413.73 13.8513.7213.71
p [ ]
605.5 606.0 606.5 607.0 607.5 608.0m/z
0
20
40
60
80
100
Rela
tive
Abun
danc
e
606.823606.281
607.297
607.633607.967606.325
605.595
NL: 8.85E5
High Resolution (120k); Lock mass
Precursor m/z; Tolerance (3-10 ppm); Intensity Threshold (1% Apex) ‘Low’ resolution (7.5k); ‘Low’ Fill time (10 ms)
Fragments m/z (6/pep.); Tolerance (10-20 ppm); Min. number frags (5)
‘High’ resolution (e.g., 60k); ‘High’ Fill time (e.g., 116 ms)
12345
Full MS – Zoom in
MS/MS
1 2
3
54
1
2345
Heavy R, Charge: +2
Heavy K, Charge: +2
Heavy R, Charge: +3
Heavy K, Charge: +3
Heavy R, Charge: +4
Heavy K, Charge: +4
#1 #2 #3 #4 #5 #6
#1#2#3#4#5#6
2 4 5
‘Group ID’ Feature
Expected elutionwindow
Expected elutionwindow
Delayed Elution
PRM SureQuantSIL Peptide Detection
Original elution profile
Delayed elution profile
Internal standards in matrix
Survey Run
SureQuant
LC-MS Analysis
Internal standards Sample LC-MS Analysis
Survey Run Analysis
Export reports & Refine analytes definition
Targeted Mass
Targeted Mass Trigger
Update scan filter tables Target Quantification
‘Optimal’ fragments / selected peptide precursor
‘Optimal’ precursor ion / peptide – Intensity response
Directed DDAInclusion list IS precursor ions multiple charge states / peptide
Generic template
Generic templateSureQuant
Prepare initial processing method from generic Skyline analysis template
Refined processing method
Copy/Paste
Towards Turnkey Targeted Proteomics SolutionsThe development of a SureQuant multi-peptides assay requires the preliminary determination of the optimal precursor ionof each peptide target together with their intensity response and optimal associated fragment ions (Figure 1). Thisinformation is typically obtained from a directed DDA analysis of the corresponding IS peptide mixture. The target list ofmultiple theoretical precursor ions of each IS peptide included in such survey runs enables comprehensive data collection,and therefore the refinement of analytes definition for subsequent SureQuant analyses. The process is supported bySkyline software and facilitated by the generic data acquisition and processing templates provided. The transfer of thegenerated information can be achieved through simple copy/paste from Skyline reports to ‘Targeted Mass’ and ‘TargetedMass Trigger’ tables of SureQuant method. The survey analysis of a SureQuant assay needs to be run one time as theonset of the project without further adjustment over time, unless a major change in the experimental design is applied(e.g., LC set up, spiked amount of SIL peptides). In the latter case, only the intensity response of predefined IS precursorions needs to be re-adjusted though an additional survey run performed under the modified experimental conditions(Figure 10). Therefore, the ability of embedding pre-set methods associated with predefined kits of IS peptides expeditesSureQuant assay development at the user sites. Several validated method templates of this type have already beenincluded in the instrument control software, such as the PQ500 related methods (Figure 11), prepared for the analyses ofplasma samples supplemented with PQ500 kit (which includes 804 SIL peptides associated with more than 500 plasmaproteins). The applications of the SureQuant PQ500 method to the analyses of undepleted plasma samples enabled therobust and precise quantification of around 560 endogenous peptides, corresponding to around 400 proteins in a 70-minLC gradient.
0
0.4
0.8
1.2
1.6
6 8 10 12 14 16 18 20 22
PRM 15K MS2 SureQuant 60K MS2
Cycle time range enabling optimal sampling rate: ≥ 8 data points (15-s peak width)Cycle Time [s]
IT: 20 ms IT: 116 ms
AENGPGPGVLVLR 1 15AQEALDFYGEVR 10 11DGFYPAPDFR 0 1ETSFNQAYGR 2 6FEISETSVNR 0 9GALAEAAR 5 13GNNLQDTLR 0 6GQPEGPLPSSSPR 0 8GYTISDSAPSR 0 5IDIHSC[Carbamidomethyl]NHEAEK 1 12IYNLC[Carbamidomethyl]AER 0 15LC[Carbamidomethyl]DSGELVAIK 5 15LFDAPEAPLPSR 11 11LLEYTPTAR 0 15LPFYNQDHER 0 17LVPPFKPQVTSETDTR 0 0NDGTFIGYK 0 13NNIDDVVR 0 4SDGSFIGYK 0 13SNPTDIYPSK 0 9SQEVAYTDIK 1 9SSDEENGPPSSPDLDR 0 8SVSAPQQIINPIR 0 0TFHVDTPEER 0 0TGIAAEEVSLPR 0 0TLDQSPELR 2 10TPPEAIALC[Carbamidomethyl]SR 1 20TTINNEYNYR 0 7VTTVVATLGQGPER 6 15YSFQTHDR 0 10
Peptide PRM 15K-20ms SQ IS-PRM_60k/116ms Number of spectra including ≥ 5 ref. fragments
0 1-2 ≥ 3
≤LOD Detection >>> Quantification
Number of ENDO pept. detected: 11 26 SSDEENGPPSSPDLDR
SQEVAYTDIK
16.6 16.7 16.8
16.2 16.35 16.516.2 16.35 16.5 16.2 16.35 16.516.2 16.35 16.5
6e6 6e66e3 6e3
PRM SureQuant
16.6 16.7 16.8 16.6 16.7 16.816.6 16.7 16.8
SIL ENDO SIL ENDO
≈
1.2e7 1.2e74e4 4e4
0
0.2
0.4
0.6
0.8
1
1.2
1.4 HCT116 (+IGF1) MCF7 (+IGF1) A549 (+IGF1)[fmol]
0
0.5
1
1.5
2
2.5
3
200
600
1000
1400
0 10 20 30 40 50 60 70 80 90 [min]
m/z [s]Endo. Pept. non-quantified (238) Endo. Pept. Quantified (564) Cycle time
Figure 2. SureQuant acquisition scheme.
Figure 3. SureQuant method structure.
Figure 4. SureQuant acquisition process and critical parameters.
Figure 6. Acquisition efficiency of conventional PRM and SureQuant methods
Figure 7. Robustness of SureQuant acquisition against chromatographic variations
Figure 8. Comparison of the analytical performance of PRM and SureQuant analyses
Figure 9. Sensitive and precise quantification of AKT/mTOR peptides by SureQuant analyses.
Figure 10. Overall SureQuant acquisition worklow of embedded application specific kits.
Figure 11. SureQuant analysis of plasma enabled by embedded preset methods
Figure 1. Overall SureQuant acquisition workflow
19 20 21 22 23 24
19 20 21 22 23 24
19 20 21 22 23 24
19 20 21 22 23 24
LCDSGELVAIK
[min]
[min]
SIL
ENDO
PRM SureQuant
Non-productive MS2 scans Productive MS2 scans
TIC TIC
TIC TIC
Proportion productive MS2 scans (% Acq. Time)PRM: 10 – 15 % SureQuant: 80 – 90 %
2.5-min window
2.5-min window
>6 data points / peak(20-s peak width)
PO65533-EN0519S
