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