Jet Steam Proteomics with Automated Sample Prep for Superior Peptide Quantitation

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1 Jet Steam Proteomics with Automated Sample Prep for Superior Peptide Quantitation Christine Miller Jason Russell June 2, 205

2 Overview Automated sample preparation Jet Stream Proteomics Peptide quantitation Jet Stream Proteomics Kickoff June 2, 205 2

3 AssayMAP: Automated Sample Prep for Quantitative Proteomics Jason D. Russell LC/MS Scientist AssayMAP ASMS 205

AssayMAP Technology Components Automated workflows designed for analytical chemists Target Customer Microchromatography Cartridges Quantitative binding & elution Simple User Interface Uses customer

Normalization Liquid handling utilities Protein purification PA-W (protein A) PG-W (protein G) SA-W (streptavidin) Reversed-phase cleanup: C8 (peptide) RP-S (peptide) RP-W (reduced mabs) Peptide

4 AssayMAP Technology Components Automated workflows designed for analytical chemists Target Customer Microchromatography Cartridges Quantitative binding & elution Simple User Interface Uses customer language - not automation language Affinity (protein) Purification In-Solution Digestion Peptide Cleanup (desalting) Phosphopeptide Enrichment IMAC Cartridge Customization Fractionation Sample Normalization Liquid handling utilities Protein purification PA-W (protein A) PG-W (protein G) SA-W (streptavidin) Reversed-phase cleanup: C8 (peptide) RP-S (peptide) RP-W (reduced mabs) Peptide Fractionation: SCX RP-S C8 Phosphopeptide enrichment: TiO 2 Positive Displacement Pipetting Fe(III)-NTA Syringes interface directly with cartridges and enable precise, controlled liquid flow through cartridges with no air bubbles to disrupt binding June 2, 205 4

5 AssayMAP Technology Each AssayMAP cartridge is slurry packed, back-pressure tested, and inspected for voids June 2, 205 5

6 AssayMAP Technology June 2, 205 6

7 Protein Sample Prep Workbench June 2, 205 7

8 Workflow Library June 2, 205 8

9 Workflows June 2, 205 9

10 App Library June 2, 205 0

11 Utility Library June 2, 205

12 Syringe Test v.0 June 2, 205 2

13 Harmonized User Interface: Affinity Purification v.0 June 2, 205 3

14 Harmonized User Interface: Peptide Cleanup v2.0 June 2, 205 4

15 Harmonized User Interface: Phosphopeptide Enrichment v2.0 June 2, 205 5

16 Focus On Method Development, Not Automation June 2, 205 6

17 VWorks Driving Assaymap Protocols Behind The User Interface June 2, 205 7

18 Small Elution Volume 5% acetic acid 2 mm HCl/00 mm NaCl Depending on choice of elution buffer, the elution volume can be as low as 0 μl June 2, 205 8

19 Jet Stream Proteomics 6550 ifunnel Q-TOF with Dual Agilent Jet Stream ESI Source AssayMAP Bravo 290 Infinity LC with AdvanceBio Peptide Mapping Column 2. mm x 50 or 250 mm June 2, 205 9

20 AssayMAP Digestion And Cleanup Workflow Each step in the workflow was performed using the AssayMAP Bravo Input: 5 μl 5 μg/μl (225 μg), cleanup of 75 μg of digest June 2,

interday digestion and cleanup for samples prepared

21 AssayMAP Digestion And Cleanup Workflow Day cleanup = C8 Day 2 cleanup = RP-S Low %CVs for both intra- and interday digestion and cleanup for samples prepared using urea or guanidine-based denaturation June 2, 205 2

22 AssayMAP mab Quantification Workflow Each step in the workflow was performed using the AssayMAP Bravo Input = 5 μg mab in 00 μl CCS June 2,

5 EIC overlays of mab peptides from a single row of samples (n

23 AssayMAP mab Quantification Workflow Across 96 sample replicates x EIC overlays of mab peptides from a single row of samples (n = 2) Counts vs. Acquisition Time (min) June 2,

24 Targeted Peptide Enrichment With Anti-peptide Antibodies Bulk digestion of rat serum performed manually (non-assaymap step) Immobilization input = μg antibody; sample input = 0 μl plasma, digested June 2,

25 Targeted Peptide Enrichment With Anti-peptide Antibodies N = 4, each condition June 2,

26 Targeted Peptide Enrichment With Anti-peptide Antibodies N = 4, each condition June 2,

27 Targeted Peptide Enrichment With Anti-peptide Antibodies The Importance Of An Internal Standard N = 4, each condition June 2,

28 Phosphopeptide Enrichment with Fe(III)-NTA or TiO 2 Cartridges Input: Particulate-free, aqueous solutions of peptides Up to 96 samples ( plate) Up to ml sample load Up to 00x concentration factor Output: Salt-free, solutions of phosphopeptides Elution volume as low as 0 µl June 2,

29 Phosphopeptide Enrichment From α-casein 50 μg load mass June 2,

30 Phosphopeptide Enrichment From α-casein Elution with % aqueous NH 3 (~ ph ) June 2,

31 Phosphopeptide Enrichment From Yeast (S. cerevisiae) Bulk digestion and cleanup (non-assaymap steps) Enrichment input: μg desalted tryptic digests June 2, 205 3

32 Phosphopeptide Enrichment From Yeast 500 μg load mass, n = 3 June 2,

33 Phosphopeptide Enrichment From Yeast 500 μg load mass, n = 3 June 2,

34 Phosphopeptide Enrichment From Yeast June 2,

35 Phosphopeptide Enrichment From Yeast n = 3 June 2,

36 Phosphopeptide Enrichment From Yeast June 2,

$µl sample load Output: Solutions of peptides in various solvents/salts As many as 6 fractions can be collected Can collect small volume fractions (~ 5 µl) June 2, 205$

37 Peptide Fractionation SCX, C8, or RP-S Input: Particulate-free, solutions of peptides Sample plates from Peptide Cleanup protocol Up to 96 samples ( plate) Up to 250 µl sample load Output: Solutions of peptides in various solvents/salts As many as 6 fractions can be collected Can collect small volume fractions (~ 5 µl) June 2,

38 Fractionation User Interface June 2,

39 AssayMAP SCX Fractionation Workflow - E. coli Lysate Each step in the workflow was performed using the AssayMAP Bravo Sample input = 0 μl E. coli 0 μg/μl (00 μg) June 2,

40 AssayMAP SCX Fractionation Workflow - E. coli Lysate Elution by increasing ionic strength (KCl) June 2,

41 AssayMAP SCX fractionation workflow - E. coli lysate Elution by increasing ionic strength (KCl) June 2, 205 4

$AssayMAP SCX fractionation workflow - E.$

42 AssayMAP SCX fractionation workflow - E. coli lysate Elution by increasing ph using volatile buffers Not necessary to do another round of sample cleanup June 2,

43 AssayMAP SCX Fractionation Workflow - E. coli Lysate Elution by increasing ph June 2,

44 AssayMAP Bravo Scalable Sample Preparation for precision proteomics June 2,

45 Rapid Antibody Digestion WP 50 Steve Murphy June 2,

46 Jet Stream Proteomics and Peptide Quantitation Jet Stream Proteomics Kickoff June 2,

47 What is Jet Stream Proteomics? Agilent s unique solution for proteomics that uses the 290+Jet Stream source with our ion funnel MS systems Jet Stream proteomics is Used for both discovery and targeted experiments Enables near nanoflow sensitivity for proteomics Intended for scientists who are not sample limited (plasma, cell cultures, plants, food, etc.) Offers the ease-of-use, robustness and reproducibility associated with standard flow separations Jet Stream Proteomics Kickoff June 2,

ifunnel Technology: Enhancing Sensitivity 5-0x by Transmitting More Ions x0 2.5.4 6460 QQQ 6490 QQQ.6.3.2. 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.5.5 2 2.5 3 3.

48 ifunnel Technology: Enhancing Sensitivity 5-0x by Transmitting More Ions x QQQ 6490 QQQ Counts vs. Acquisition Time (min) Samples more ions Removes neutrals Gives higher ion yield LVNEVTEFAK June 2, 205

49 Agilent Jet Stream Ion Generation LC sample inlet ESI Nebulizing gas Super-heated sheath gas MS inlet Nozzle voltage Heated drying gas AJS The collimated thermal containment zone creates a dramatically brighter source Resistive sampling capillary

50 Why is Agilent Jet Stream Technology an Advantage? Agilent Jet Stream shows massflow dependent behavior Sensitivity depends on absolute amount (mass) injected Using smaller i.d. columns with standard sources does NOT increase sensitivity! Jet Stream source provides 3-5x signal increase compared to ESI Combined with sensitivity of ion funnel systems, makes standard flow feasible! x x mm column 0.5 mm column Counts vs. Acquisition Time (min) AJS normalized response ESI relative response Acquisition Time (min) Jet Stream Proteomics Kickoff June 2,

51 Journal Article Demonstrates Agilent Jet Stream Performance Buckenmaier S, Miller CA, van de Goor T, Dittmann MM. J Chromatogr. A 205, 377: Jet Stream Proteomics Kickoff June 2, 205 5

52 MRM Analysis of INDISHTQSVSSK in Mouse Plasma Jet Stream Proteomics Nanoflow x0 3 x Counts vs. Acquisition Time (min) Counts vs. Acquisition Time (min) 0x more injected on Agilent Jet Stream system

53 Why Standard Flow? Superior chromatography Sub-2 micron columns with narrow peaks Greater loading and peak capacity Stable retention time Faster analysis Easier operation.not everyone is sample limited!! Jet Stream Proteomics Kickoff June 2,

54 Comparison of Standard Flow and Nanoflow Chromatography: Sharper Peaks with UHPLC x0 4 x0 4 x0 4 Jet Stream Proteomics x0 5 x0 5 x NanoLC Counts vs. Acquisition Time (min) Counts vs. Acquisition Time (min) Counts vs. Acquisition Time (min) Jet Stream Proteomics Kickoff June 2,

55 Superior Standard Flow Chromatography with the AdvanceBio Peptide Mapping Column Packing has 20Å pore size with superficially porous 2.7 μm particles Specially tested with a challenging peptides mix to ensure reliable peptide mapping performance Exceptional resolution and speed for UHPLC, and excellent results for conventional HPLC too Available in a range of column lengths including 250 mm! Jet Stream Proteomics Kickoff June 2,

56 Peptide Quantitation: Outstanding Sensitivity with Standard Flow Chromatography x blank x LLOD amol on-column x LLOQ amol on-column x amol on-column Counts vs. Acquisition Time (min) Instrumentation: 290 UHPLC + Agilent JetStream QQQ Sample: synthetic peptide standard (LVNEVTEFAK) spiked into enolase tryptic digest Injection volume: µl Jet Stream Proteomics Kickoff June 2,

57 LVNEVTEFAK: Excellent Precision and Low Amol IDL Amount measured Replicates %RSD t (99%) IDL 5.0 amol (LLOQ) n = 0 injections amol MDL = t x (%RSD/00) x Amount = 2.82 x (4.0/00) x 5.0 amol = 2.0 amol x x Inj # Peak Area x0 x x x x0 x x x Counts vs. Acquisition Time (min) Counts vs. Acquisition Time (min) %RSD QQQ LC/MS for Peptide Quantitation 6/2/205 57

58 Robust Standard Flow Chromatography: 40 SIS Peptides in Plasma x Counts vs. Acquisition Time (min) Overlay of 238 transitions from 40 peptides in QC kit from MRM Proteomics

59 Robustness Test Protocol 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. (0 µg load, 40 min method) Inject large amounts of plasma digest using short chromatography to accelerate dirtying of the system (40 µg load, 5 min method) No cleanup of plasma digest and no flow diversion maximize dirtying of system 6495 QQQ LC/MS for Peptide Quantitation 6/2/205 59

60 Proven System Robustness in Complex Matrix: Protein Quantitation in Plasma 40.00% 20.00% 00.00% 80.00% 60.00% 40.00% 20.00% 0.00% 0/06/4 0//4 0/6/4 0/2/4 0/26/4 0/3/4 02/05/4 Apolipoprotein E L-selectin Plasminogen Albumin_serum Kininogen Transthyretin Hemopexin Selected peptides from 42 peptides in the QC sample normalized to Day 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: QQQ LC/MS for Peptide Quantitation 6/2/205 60

61 Examples of Peptide Quantitation Analysis Using Jet Stream Proteomics 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 Fast analysis 6 minute gradient and 2 minute post-time Jet Stream Proteomics Kickoff June 2, 205 6

Triggered MRM for Confirmation and Quantitation Triggered cycle (above threshold) Peptide triggers secondary transitions, collects n cycles then returns to primary cycle x0 4 2.8.6.4.2 0.8 0.

62 Triggered MRM for Confirmation and Quantitation Triggered cycle (above threshold) Peptide triggers secondary transitions, collects n cycles then returns to primary cycle x Cpd : peptide 3: +ESI MRM Frag=20.0V CID@20.0 ( > ) HSA-tDMRM-pep-02.d Primary 0 cycle (below threshold) Threshold Counts vs. Acquisition Time (min) Advantages of tmrm: Less cycle time compared to product ion scan Secondary transitions only collected for peptides found to be present 62

63 tmrm Results for LLIYDTSK From mab x0 5 Cpd 3: LLIYDTSK.light: + tmrm CF=0.000 DF=0.000 ( > ) mab digest 0f-r004.d Counts vs. Acquisition Time (min) x0 4 Cpd 3: LLIYDTSK.light: +ESI MRM ( min, 9 Scans) Frag=380.0V CF=0.000 DF=0.000 ( > **) mab digest 0f-r004.d Counts vs. Mass-to-Charge (m/z)

64 Calibration Curve for LLIYDTSK from mab Peptide LLIYDTSK amol 2 amol External Calibration Concentration (amount on-column) 5 amol 0 amol 20 amol 50 amol 00 amol fmol 0 fmol 00 fmol Accuracy (%, n=6) Cal. Conc. %RSD (n=6) Retention Time %RSD (n=60) 0.2 % LLIYDTSK.light - 0 Levels, 0 Levels Used, 58 Points, 58 Points Used, 0 QCs x0 6 y = * x R^2 = Type:Linear, Origin:Ignore, Weight:/x Range: amol to 00 fmol R 2 >0.999 Type: Linear, Origin: Ignore, Weight:/x Zoom in: -00 amol/µl Concentration (amol) /2/205

65 LOD/LOQ for LLIYDTSK: amol (0.95 fg) On-column Blank, 5 runs amol, 5 runs x0 + MRM CF=0.000 DF=0.000 ( > ) Blank-r006.d + MRM CF=0.000 DF=0.000 ( > ) mab digest a-r002.d Noise (RMS) = 0.4; SNR (2.323min) = 9.4 x x0 + MRM CF=0.000 DF=0.000 ( > ) Blank-r007.d + MRM CF=0.000 DF=0.000 ( > ) mab digest a-r003.d Noise (RMS) = 0.7; SNR (2.333min) = 52.7 x0 5 5 * x0 + MRM CF=0.000 DF=0.000 ( > ) Blank-r008.d + MRM CF=0.000 DF=0.000 ( > ) mab digest a-r004.d Noise (RMS) = 0.36; SNR (2.323min) = 2.2 x0 5 5 * x0 + MRM CF=0.000 DF=0.000 ( > ) Blank-r009.d + MRM CF=0.000 DF=0.000 ( > ) mab digest a-r005.d Noise (RMS) = 0.38; SNR (2.323min) = 8.5 x0 5 * x0 + MRM CF=0.000 DF=0.000 ( > ) Blank-r00.d + MRM CF=0.000 DF=0.000 ( > ) mab digest a-r006.d Noise (RMS) = 0.42; SNR (2.328min) = 9. x Counts vs. Acquisition Time (min) Counts vs. Acquisition Time (min)

66 Skyline Software: Agilent Automation Tool for Automatic CE Optimization

CE Optimization Using Skyline SW Opt CE 60 25 Peak Area

0 0 CE Replicate Intensity (0^3) 5 0 5 0 Skyline SW will

67 CE Optimization Using Skyline SW Opt CE Peak Area (0^3) CE Replicate Intensity (0^3) Skyline SW will export MRM list or acquisition method to run on Agilent QQQ with optimal CE for each transition

68 Calibration Curve for Neat Peptide Standard Peptide Standard External Calibration Concentration 20 fg/µl 50 fg/µl 00 fg/µl 200 fg/µl 500 fg/µl pg/µl 2 pg/µl 5 pg/µl 0 pg/µl Accuracy (%, n=4) Cal. Conc. %RSD (n=4) Retention Time %RSD (n=36) 0.5% MMSETAPLAPTIPAPAEK.light - 9 Levels, 9 Levels Used, 36 Points, 36 Points Used, 0 QCs x0 4 y = * x R^2 = Type:Linear, Origin:Ignore, Weight:/x Range: 20 fg/µl to 0 pg/µl R 2 >0.996 Type: Linear, Origin: Ignore, Weight:/x Zoom in: fg/µl ncentration (fg/ul) /2/205

69 LOQ for Neat Peptide Standard: <20 fg (<0 amol) On-column +ESI MRM Frag=380.0V CF=0.000 DF=0.000 ( > ) 20fg-r00-r00-r002.d Noise (RMS) = 0.84; SNR (2.290min) = 2.3 x0 5.5 * ESI MRM Frag=380.0V CF=0.000 DF=0.000 CID@5.0 ( > ) 20fg-r00-r00-r003.d Noise (RMS) = 0.83; SNR (2.274min) = 6.3 x0 6 * ESI MRM Frag=380.0V CF=0.000 DF=0.000 CID@5.0 ( > ) 20fg-r00-r00-r004.d Noise (RMS) =.05; SNR (2.279min) = 3. x0 6 * ESI MRM Frag=380.0V CF=0.000 DF=0.000 CID@5.0 ( > ) 20fg-r00-r00-r005.d Noise (RMS) = 0.60; SNR (2.274min) = 23.4 x0 6 * Counts vs. Acquisition Time (min)

70 Calibration Curve for Peptide Spiked into Rat Serum Peptide External Calibration Concentration 0.2 ng/ml 0.5 ng/ml ng/ml 2 ng/ml 5 ng/ml 0 ng/ml 20 ng/ml 00 ng/ml Accuracy (%, n=6) Cal. Conc. %RSD (n=6) Retention Time %RSD (n=48) 0.7% MMSETAPLAPTIPAPAEK.light - 8 Levels, 8 Levels Used, 47 Points, 47 Points Used, 0 QCs x0 4 y = * x R^2 = Type:Linear, Origin:Ignore, Weight:/x Range: 0.2 ng/ml to 00 ng/ml R 2 >0.998 Type: Linear, Origin: Ignore, Weight:/x Zoom in: ng/ml ncentration (ng/ml) /2/205

71 LOD and LOQ for Peptide Spiked Into Rat Serum LOD: 0.2 ng/ml (9 amol) +ESI MRM Frag=380.0V CF=0.000 DF=0.000 ( > ) p2ngml-nomatrix-r00.d Noise (RMS) = 2.68; SNR ( LOQ: 0.5 ng/ml (22.7 amol) +ESI MRM Frag=380.0V CF=0.000 DF=0.000 CID@5.0 ( > ) p5ngml-nomatrix-r00.d Noise (RMS) = 2.42; SNR ( x0 x0 5.5 * * ESI MRM Frag=380.0V CF=0.000 DF=0.000 CID@5.0 ( > ) p2ngml-nomatrix-r003.d Noise (RMS) = 3.92; SNR ( +ESI MRM Frag=380.0V CF=0.000 DF=0.000 CID@5.0 ( > ) p5ngml-nomatrix-r002.d Noise (RMS) = 3.90; SNR ( x0 x * * ESI MRM Frag=380.0V CF=0.000 DF=0.000 CID@5.0 ( > ) p2ngml-nomatrix-r004.d Noise (RMS) = 2.60; SNR ( +ESI MRM Frag=380.0V CF=0.000 DF=0.000 CID@5.0 ( > ) p5ngml-nomatrix-r003.d Noise (RMS) = 2.85; SNR ( x0 x0 6 * * ESI MRM Frag=380.0V CF=0.000 DF=0.000 CID@5.0 ( > ) p2ngml-nomatrix-r005.d Noise (RMS) = 2.90; SNR ( +ESI MRM Frag=380.0V CF=0.000 DF=0.000 CID@5.0 ( > ) p5ngml-nomatrix-r004.d Noise (RMS) = 2.8; SNR ( x0 x * * ESI MRM Frag=380.0V CF=0.000 DF=0.000 CID@5.0 ( > ) p2ngml-nomatrix-r006.d Noise (RMS) = 2.35; SNR ( +ESI MRM Frag=380.0V CF=0.000 DF=0.000 CID@5.0 ( > ) p5ngml-nomatrix-r006.d Noise (RMS) = 3.5; SNR ( x0 x0 5.5 * * Counts vs. Acquisition Time (min) Counts vs. Acquisition Time (min)

72 tmrm Results for Peptide Spiked Into Rat Serum x Cpd : MMSETAPLAPTIPAPAEK.light: +ESI MRM Frag=380.0V CF=0.000 DF=0.000 CID@5.0 ( > ) tmrm-0pg pep-04.d x Counts vs. Acquisition Time (min) +ESI MRM:2 (rt: min) Frag=380.0V CF=0.000 DF=0.000 CID@5.0 ( > ) tmrm-0pg pep-04.d Counts vs. Mass-to-Charge (m/z)

73 Q-TOF Targeted Peptide Quantitation Using All Ions Data Independent Analysis Jet Stream Proteomics Kickoff June 2,

Reproducibility of Jet Stream Proteomics: Proteins Identified at % FDR from MDA-MB-23 Unique Peptides 35000 30000 25000 20000 5000 0000 5000 Unique Peptides Unique Proteins 5,905 4,875 4,824 4,946

74 Reproducibility of Jet Stream Proteomics: Proteins Identified at % FDR from MDA-MB-23 Unique Peptides Unique Peptides Unique Proteins 5,905 4,875 4,824 4, ,000 6,000 5,000 4,000 3,000 2,000,000 Unique Proteins 0 0 Unique peptides: 32,446 Unique Proteins: 5,905 Jet Stream Proteomics for Sensitive and Robust Standard Flow LC/MS ( EN) US HUPO 205 March 8, 205

75 Conclusions Jet Stream Proteomics provides: Faster analysis times for complex samples (superior chromatography) Faster re-equilibration times (modern columns and the 290) For targeted proteomics, superior retention-time reproducibility narrower MRM retention-time windows and more robust methods For discovery proteomics - almost as many protein identifications as nanoflow (with µg sample loaded) Jet Stream Proteomics Kickoff June 2,

Sample Preparation Automation and QQQ Workflows for Peptide Quantitation in DMPK

Sample Preparation Automation and QQQ Workflows for Peptide Quantitation in DMPK Alex Zhu & Yanan Yang LCMS Applications Scientist Agilent Technologies Why is Agilent Workflow a Good Choice for DMPK? AssayMAP