1. Presentation Title
Jet Steam Proteomics with Automated Sample Prep for High-Throughput Peptide Quantitation in Clinical Research
11/16/2018
Confidentiality Label
Regulatory statement (if applicable)
Peptide Quantitation
Yanan Yang, PhD
LC/MS Application Scientist
January 2018
For Research Use Only. Not for Use in diagnostic procedures

2. Overview Peptide Quantitation Break Automated sample preparation
Jet Stream proteomics - description and performance
Agilent 6495 Triple Quadrupole MS system – features design to enhance peptide quantitation
Software tools
Application examples
Break
Automated sample preparation
AssayMAP Bravo system – description
Peptide Sample Prep workflow
SISCAPA workflow
Phosphopeptide enrichment
Fractionation workflow
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

3. What is Jet Stream Proteomics
Presentation Title
11/16/2018
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LC sample inlet
ESI
Nebulizing gas
Super-heated sheath gas
MS inlet
AJS
Nozzle voltage
Heated drying gas
The collimated thermal containment zone creates a dramatically “brighter source”
Resistive sampling capillary
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

4. Why is Agilent Jet Stream Technology an Advantage?
Presentation Title
11/16/2018
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Jet Stream source provides 3-5x signal increase compared to ESI
Agilent Jet Stream showed same signal (and LOD) for
2.1 mm ID column at 400 µL/min
0.5 mm ID column at 17 µL/min
Electrospray ionization is concentration- dependent … so what is happening? 2
x10
0.2
0.4
0.6
0.8
1.0
Acquisition Time (min) 3
3.5 4
4.5 5
5.5 6
6.5 7
AJS normalized response
ESI relative response
2.1 mm column
0.5 mm column
575.5937.5
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

5. Why is Agilent Jet Stream Technology an Advantage?
Presentation Title
11/16/2018
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Agilent Jet Stream shows mass-flow dependent behavior
Analytical sensitivity depends on absolute amount (mass) injected
Using smaller i.d. columns with standard sources does NOT increase analytical sensitivity!
This makes standard flow LC/MS practical for proteomics!
2.1 mm column
0.5 mm column
575.5937.5
ESI
AJS
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

6. Journal Article Demonstrates Agilent Jet Stream Performance
Presentation Title
11/16/2018
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Buckenmaier S, Miller CA, van de Goor T, Dittmann MM. J Chromatogr. A 2015, 1377:64-74.
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

7. Comparison of Approaches: Nanoflow vs. Jet Stream Proteomics
Nanoflow LC/MS
Standard Flow LC/MS
Lowest detection limit when sample is limited Longer column re-equilibration times Requires cleaner samples On complex samples, may hit limit of column capacity Requires more skill to maintain and use
Equivalent or better detection limit when sample is not limited Faster analyses with quicker column re- equilibration Higher loading and peak capacity Best run-to-run reproducibility Easiest to use and maintain Greater column selection available
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

8. Standard Flow LC: Excellent Retention Time Reproducibility in Complex Matrices
Zoom view
Depletion has no effect on the LC retention time
Overlay of TICs from for depleted plasma digest (n=3) and non-depleted plasma digest (n=3) samples
In collaboration with Christoph Borchers, University of Victoria
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

9. Agilent 6495 LC/Triple Quadrupole System
Presentation Title
11/16/2018
Ideal for peptide quantitation
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Mass range: 5 – 3,000 Da Maximum MRM rate: 500 MRMs/sec Minimum MRM dwell time: 0.5 ms
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

10. iFunnel Technology Enhanced Analytical Sensitivity Agilent Jet Stream
Presentation Title
11/16/2018
Enhanced Analytical Sensitivity
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Agilent Jet Stream
Hexabore Capillary
Dual Ion Funnel
Thermal gradient focusing
Efficient desolvation
Creates an ion rich zone
Six capillary inlets
Samples x10 times more ion rich gas
Removes the gas but captures the ions
Removes neutral noise
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

11. Increased Mass Range Useful for Peptides
Presentation Title
11/16/2018
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Regulatory statement (if applicable)
QQQ MRM spectrum for glycopeptide: EEQYN[ ]STYR (G1F)
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

12. Improved Detector Design
Presentation Title
11/16/2018
Increased signal (60-120%) with higher HED voltage
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Regulatory statement (if applicable)
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

13. Peptide Quantitation: Outstanding Analytical Sensitivity with Standard Flow Chromatography
Presentation Title
11/16/2018
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blank
3.0 amol
on-column
5.0 amol
7.5 amol
LLOD
LLOQ
Instrumentation: UHPLC + Agilent Jet Stream QQQ
Sample: synthetic peptide standard (LVNEVTEFAK) spiked into enolase tryptic digest
Injection volume: 1 µL
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

14. LVNEVTEFAK: Excellent Precision and Low Amol IDL
Presentation Title
11/16/2018
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Amount measured
Replicates
%RSD
t (99%)
IDL
5.0 amol (LLOQ)
n = 10 injections
14.0
2.821
2.0 amol
MDL = t x (%RSD/100) x Amount = x (14.0/100) x 5.0 amol = 2.0 amol
Inj #
Peak Area 1
30.5 2
28.7 3
30.4 4
33.91 5
35.1 6
31.5 7
34.5 8
20.7 9
26.7 10
26.6
%RSD
14.0
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

15. Peptide Quantitation – 6 Orders of Linear Dynamic Range
LVNEVTEFAK
5 amol – 5 pmol on-column
6 orders of linear dynamic range
R2 = 0.998
Zoom-in 5 – 500 amol
LVNEVTEFAK
Calibration Standards (amount on-column; 1 µL injected)
5.0 amol
7.5 amol
15 amol
30 amol
300 amol
3 fmol
30 fmol
300 fmol
3 pmol
5 pmol
%Accuracy
109.8
108.7
105.0
87.1
85.2
81.4
86.4
87.4
105.6
97.5
Reproducibility (%RSD, n=10)
14.0
16.0
9.4
9.0
1.6
1.2
0.6
0.7
2.1
1.0
RT (%RSD, n=100)
0.12
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

16. Jet Stream Proteomics Provides Better Quantitative Accuracy and Reproducibility Than Nanoflow
Presentation Title
11/16/2018
Confidentiality Label
Jet Stream Proteomics
Nanoflow
MRM Analysis of a peptide INDISHTQSVSSK in mouse plasma
10x more sample injected on Agilent Jet Stream system
Percy, A. J., et al. (2012). "Comparison of standard- and nano-flow liquid chromatography platforms for MRM-based quantitation of putative plasma biomarker proteins." Anal Bioanal Chem 404(4):
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

17. Proven Robustness for Peptide Quantitation
Presentation Title
11/16/2018
Robustness test protocol
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Regulatory statement (if applicable)
Goal: Test the robustness of the 6495 QQQ for peptide quantitation using a complex matrix Strategy: Use MRM Proteomics kits to establish initial performance and monitor performance. (10 µg load, 40 min method) Inject large amounts of plasma digest using short chromatography to accelerate “dirtying” of the system (40 µg load, 15 min method) No cleanup of plasma digest and no flow diversion – maximize dirtying of system
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

18. Standardization of MRM-MS Workflows with Quality Control Kits
Presentation Title
11/16/2018
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Standardization of MRM-MS Workflows
with Quality Control Kits
Regularly and conveniently evaluate instrument performance or total MRM-MS workflow
Identify the source of variability in your human plasma analyses
Includes performance quality guide and Qualis data analysis software
Daily QC
Monthly QC
For Research Use Only. Not for Use in diagnostic procedures

19. Robust Standard Flow Chromatography: 40 Stable Isotope Labeled Peptides Spiked in Plasma
Presentation Title
11/16/2018
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Overlay of 238 transitions from 40 peptides in QC kit from MRM Proteomics
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

20. Proven System Robustness in Complex Matrix: Protein Quantitation in Plasma
Presentation Title
11/16/2018
Confidentiality Label
Regulatory statement (if applicable)
Selected peptides from 42 peptides in the QC sample – normalized to Day 1 response
Peptide QC samples analyzed daily after every ~25 plasma digest injections
No significant signal degradation observed after 853 injections of 40 µg plasma digest per injection and 3.5 weeks of continuous operation
Response %RSD:
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

21. Post-Robustness Test Cleaning of Source
Presentation Title
11/16/2018
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After rinsing with water, a film could be peeled off from the spray shield and endplate. The film was completely removed and the QC sample was re-run.
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

22. 6495 System Robustness Gate-Valve Assembly
Easy capillary maintenance with the gate valve
Gate Valve Assembly
Gate Valve
Capillary Puller Tool
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

23. Other Tools for Peptide Quantitation
Presentation Title
11/16/2018
Source Optimization Wizard
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Regulatory statement (if applicable)
Available for all Agilent QQQ systems
Automates optimizing all source parameters including
Gas flows
Gas temperatures
Voltages
Ion funnel parameters
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

24. Other Tools for Peptide Quantitation
Triggered MRM for confirmation and quantitation
Peptide triggers secondary transitions, collects ‘n’ cycles then returns to primary cycle
Triggered cycle (above threshold)
Threshold
Primary cycle (below threshold)
Advantages of tMRM:
Less cycle time compared to product ion scan
Secondary transitions only collected for peptides found to be present
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

25. Other Tools for Peptide Quantitation
Presentation Title
11/16/2018
Agilent Automation Tool for Skyline
Confidentiality Label
Regulatory statement (if applicable) CE
Replicate 10 20 30 40 50 60 P e a k A r ( 1 ^ 3 )
Opt
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

26. Examples of Peptide Quantitation Analysis Using Jet Stream Proteomics
Presentation Title
11/16/2018
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Regulatory statement (if applicable)
mAb digest
Simple analysis of a biopharma mAb
Fast analysis – 6 minute gradient and 2 minute post-time
Used triggered MRM (tMRM) for confirmation plus quantitation
Synthetic peptide (MMSETAPLAPTIPAPAEK)
Analysis of neat standard
Analysis of peptide spiked into rat serum
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

27. tMRM Results for LLIYDTSK From mAb
Presentation Title
11/16/2018
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Regulatory statement (if applicable)
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

28. Calibration Curve for LLIYDTSK from mAb
Presentation Title
11/16/2018
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Regulatory statement (if applicable)
Peptide LLIYDTSK
External Calibration Concentration (amount on-column)
1 amol
2 amol
5 amol
10 amol
20 amol
50 amol
100 amol
1 fmol
10 fmol
100 fmol
Accuracy (%, n=6)
104.6
96.4
98.7
93.9
94.0
99.1
105.8
103.4
98.0
Cal. Conc. %RSD (n=6)
18.0
10.2
7.2
6.0
5.8
4.0
2.0
0.9
1.3
Retention Time %RSD (n=60)
0.12 %
Range: 1 amol to 100 fmol
R2>0.999
Type: Linear, Origin: Ignore, Weight:1/x
Zoom in: amol/µl
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

29. LOD/LOQ for LLIYDTSK: 1 amol (0.95 fg) On-column
Presentation Title
11/16/2018
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Regulatory statement (if applicable)
Blank, 5 runs
1 amol, 5 runs
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

30. Calibration Curve for Neat Peptide Standard
Presentation Title
11/16/2018
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Regulatory statement (if applicable)
Peptide Standard
External Calibration Concentration
20 fg/µl
50 fg/µl
100 fg/µl
200 fg/µl
500 fg/µl
1 pg/µl
2 pg/µl
5 pg/µl
10 pg/µl
Accuracy (%, n=4)
105.18
88.18
100.08
98.90
88.33
103.90
102.68
101.35
111.43
Cal. Conc. %RSD (n=4)
9.83
10.95
8.09
11.14
7.03
2.94
2.98
0.96
0.86
Retention Time %RSD (n=36)
0.15%
Range: 20 fg/µl to 10 pg/µl
R2>0.996
Type: Linear, Origin: Ignore, Weight:1/x
Zoom in: fg/µl
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

31. LOQ for Neat Peptide Standard: <20 fg (<10 amol) On-column
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

32. Calibration Curve for Peptide Spiked into Rat Serum
Presentation Title
11/16/2018
Confidentiality Label
Regulatory statement (if applicable)
Peptide
External Calibration Concentration
0.2 ng/ml
0.5 ng/ml
1 ng/ml
2 ng/ml
5 ng/ml
10 ng/ml
20 ng/ml
100 ng/ml
Accuracy (%, n=6)
90.70
106.43
92.83
105.93
96.68
103.63
103.07
99.15
Cal. Conc. %RSD (n=6)
14.38
10.22
16.89
4.36
4.92
3.93
3.09
2.73
Retention Time %RSD (n=48)
0.17%
Range: 0.2 ng/ml to 100 ng/ml
R2>0.998
Type: Linear, Origin: Ignore, Weight:1/x
Zoom in: ng/ml
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

33. LOD and LOQ for Peptide Spiked Into Rat Serum
LOD: 0.2 ng/ml (9 amol)
LOQ: 0.5 ng/ml (22.7 amol)
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

34. tMRM Results for Peptide Spiked Into Rat Serum
Presentation Title
11/16/2018
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Regulatory statement (if applicable)
Triggered transitions
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures

35. Summary
Agilent Jet Stream technology provides enhanced analytical sensitivity and mass-flow dependent ionization The Agilent 6495 Triple Quadrupole mass spectrometer with iFunnel technology has the analytical sensitivity, mass range and robustness to facilitate routine peptide quantitation Additional tools such as triggered MRM (tMRM) mode and Agilent Automation for Skyline support peptide quantitation workflows
Peptide Quant For Research Use Only. Not for Use in diagnostic procedures