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1 Utilizing an Accurate Mass and Retention Time Library to Facilitate Biomarker Discovery Nichole Reisdorph, PhD and Cole Michel, BS Mass Spectrometry Facility Skaggs School of Pharmacy and Pharmaceutical Sciences University of Colorado Anschutz Medical Campus 6/15/217 Conflict of Interest Statement The SSPPS mass spectrometry facility is a Center of Excellence for Agilent Technologies Dr. Richard Reisdorph consults for Agilent Technologies Cole Michel Richard Reisdorph, PhD

2 Respiratory disease Research Program Proteomics, lipidomics, metabolomics, targeted analyses (lipid mediators, steroids, amino acids, etc) Training Core Facility International Training Program Informatics Data analysis and tool optimization Overview Background: Quantitative proteomics Addressing challenges with choice of methodology Nano vs Standard flow Comparison using commercially available cell extract Application to clinical research project Analyzing Cerebrospinal Fluid (CSF) to understand Multiple Sclerosis (MS) phenotypes Accurate mass retention time library vs spectral counting (MS-only quantitation vs MS/MS quantitation)

3 Background- Quantitative proteomics Objective was to develop robust and reproducible method for quantitation of complex protein samples Cells > tissues > biofluids Pros/Cons of labeling vs non-labeling methods Pros/Cons of MS vs MS/MS Accurate Mass Retention Time Library Spectral Counting Quadrupole Time of Flight What strategy do we choose? Use of biofluids in research limits options sample limited, challenging to multiplex Compare standard flow versus nanoflow using the same sample and assess: Reproducibility Coverage (# of peptides and proteins identified) Compare MS-based quantitation to MS/MS quantitation Not conducted as part of standard vs nano flow experiments

4 Nano vs Standard Flow: Pros and Cons Nanoflow LC/MS Improved analytical sensitivity, especially when sample limited Longer column re-equilibration times Requires cleaner samplesclogging can be an issue Column capacity may be reached when loading complex samples Requires more skill to maintain and use HPLC Chip vs Nanoflow columns Standard Flow LC/MS Equivalent or better detection limit when sample is not limited Faster analyses with quicker column re-equilibration More forgiving of matrix Higher loading and peak capacity Easiest to use and maintain Greater commercial column selection available Best run-to-run reproducibility Sample Information Pierce Hela protein digest standard (P/N: 88328) Tryptic digest 1 ug/ul with 3% ACN in.1% formic acid Triplicate injections in each configuration Column Information Nanoflow LC/MS Analytical Column: Thermo 75 µm x 25 mm Acclaim PepMap RSLC Column (P/N: ) Trapping Column: Thermo 75 µm x 2 mm Acclaim PepMap 1 Trap Column (P/N: ) Standard Flow AJS* LC/MS Analytical Column: Agilent 2.1 x 25 cm Acclaim PepMap RSLC Column (P/N: ) Trapping Column: None Note: AJS parameters were provided by Dr. Vadi Bhat from Agilent. Mr. Cole Michel optimized the nanoflow methods. *Agilent Jet Stream on 655 QTOF

5 Method Information (HPLC) Nanoflow LC/MS Gradient (~112 min) Mobile Phase A =.1% Formic Acid in H2O Mobile Phase B =.1% formic acid in 9% ACN Standard Flow AJS LC/MS Gradient (~115 min) Mobile Phase A =.1% Formic Acid in H2O Mobile Phase B =.1% formic acid in 9% ACN TCC: 42⁰C Effective Flowrate: 335 nl/min Loading Flowrate: 3.2 µl/min Sample on Column: 1.2 µg (1.2µL) Date Sample Run: January 18 th 217 TCC: 65⁰C Effective Flowrate:.2 ml/min Loading Flowrate: N/A Sample on Column: 15 µg (15µL) Date Sample Run: January 17 th 217 Method Information (MS/MS) Nanoflow LC/MS Gas Temp: 175⁰C Drying Gas: 11 L/min VCap: 13 V Fragmentor: 36 V Oct 1 RF Vpp: 75 V Mode: Auto MS/MS MS Mass Range: m/z MS Acquisition Rate: 1 MS/MS Mass Range: 5-17 m/z MS/MS Acquisition Rate: 3 Isolation Width: narrow Advanced Decision Engine: True (B.8 software) Collision Energy: Use formula Charge Slope Offset > Standard Flow AJS LC/MS Gas Temp Sheath Gas Temp: 25⁰C 25⁰C Drying Gas Sheath Gas: 14 L/min 11 L/min Vcap Nozzle: 35 V V Fragmentor: 36 V Oct 1 RF Vpp: 75 V Mode: Auto MS/MS MS Mass Range: m/z MS Acquisition Rate: 1 MS/MS Mass Range: 5-17 m/z MS/MS Acquisition Rate: 3 Isolation Width: narrow Advanced Decision Engine: True (B.8 software) Collision Energy: Use formula Charge Slope Offset Nebulizer: 35 psig >

6 Method Information (MS/MS) Nanoflow LC/MS Max Precursors Per Cycle: 2 Abs. Threshold: 3 counts Rel. Threshold (%):.1% Active Exclusion: True Exclude after: 1 spectra Released After:.4 min Isotope Model: Peptides Inactive Active 1 2 Charge State Preferences: unk 3 >3 Purity Stringency: 1% Sort by Abundance only Purity Cutoff: 3% ADA Target: true, 4, counts/spectrum Use MS/MS accumulation time limit: true Reject Precursors that cannot reach target TIC: false Standard Flow AJS LC/MS Max Precursors Per Cycle: 2 Abs. Threshold: 3 counts Rel. Threshold (%):.1% Active Exclusion: True Exclude after: 1 spectra Released After:.15 min Isotope Model: Peptides Inactive Active 1 2 Charge State Preferences: unk 3 >3 Purity Stringency: 1% Sort by Abundance only Purity Cutoff: 3% ADA Target: true, 25, counts/spectrum Use MS/MS accumulation time limit: true Reject Precursors that cannot reach target TIC: true Note: AJS parameters were provided by Dr. Vadi Bhat from Agilent. Mr. Cole Michel optimized the nanoflow methods. Tuning: High analytical sensitivity slicer, low mass range 5-17, extended 2Ghz 922 m/z: Quad narrow isolation efficiency 65%, abundance 1.9 M, Res 22K Spectrum Mill Parameters (General Workflow for Iterative Searching) Extraction- Default settings MS/MS Search #1* Auto-Validation #1 Auto-Validation #2 MS/MS Search #2 Auto-Validation #1 Auto-Validation #2 MS/MS Search #3 Auto-Validation #1 Auto-Validation #2 MS/MS Search #4 Auto-Validation #1 Auto-Validation #2 Protein Summary Results (X% Validation Yield of Filtered Spectra) Software compiles results *See next slide for settings

7 Spectrum Mill Parameters (MS/MS Search #1) Same parameters used for AJS and Nano data files Spectrum Mill Parameters Search 1 Search 2 Search 3 Search 4 Enzyme Trypsin Trypsin Trypsin, nonspecific N term Trypsin, nonspecific C term Lysine Carbamylated Acetyl K - - Ox Meth X X - - Pyroglutamic X X - - acid Deamidated X X - - Phos S, T, Y X X - - Search Mode Variable Mod Variable Mod Identity Identity Unchanged SwissProt > Humans Carbamidomethylation 2 missed cleavages

8 Spectrum Mill Parameters (Auto-Validation #1) Spectrum Mill Parameters (Auto-Validation #2) Nanoflow vs. Standard flow (AJS) Results from HeLA cell digest

9 SpectrumMill Summary AJS 15 ug on column Nano 1.2 ug on column Results could be different due to MS/MS parameters, eg multiple peptide hits. Peptide Comparison 25 AJS vs. Nano Source Distinct Peptides 2388 Distinct Peptides Inj 1 Inj 2 inj 3 Total AJS Nano

10 Protein Comparison Proteins Identified AJS vs. Nano Source - Proteins Inj 1 Inj 2 inj 3 Total AJS Nano x Total Ion Chromatograms of Triplicate Injections +ESI TIC MS(all) Hela_Jet_Stream_2ug_3.d AJS Counts vs. Acquisition Time (min) x1 7 +ESI TIC MS(all) HELA_1ug_condition_4.d Nano Counts vs. Acquisition Time (min)

11 x Total Ion Chromatograms Overlaid (same scale) Nano TIC AJS TIC +ESI TIC MS(all) Hela_Jet_Stream_2ug_1.d Nano AJS Counts vs. Acquisition Time (min) x x Base Peak Chromatograms (Scale Locked) +ESI BPC Scan Frag=36.V Hela_Jet_Stream_2ug_1.d 1 1 AJS AJS BPC Nano BPC +ESI BPC Scan Frag=36.V HELA_1ug_condition_3.d Nano Counts vs. Acquisition Time (min)

12 Conclusions Triplicate injections of HeLA digest showed good overlay of TIC for both Nano and AJS Nanoflow has greater than 2x the sensitivity of AJS ug: 17,258 peptides and 2799 proteins 1.2 ug: 23,88 peptides and 4115 proteins 1.37 fold more peptides and 1.47 fold more proteins While AJS settings could be re-optimized, Nanoflow clearly provides good proteome coverage Next step is to evaluate reproducibility over time Note: 25 ug is considered optimal for the AJS system; our goal was to evaluate a 1x difference in loading. Triplicate 1.5 ug Injection Hela Standard 3 25 Nano Source 1.5 ug Injections - Distinct Peptides Average 1887 Std Dev 37 1% CV Nano Source 1.5 ug Injections - Proteins Average 3342 Std Dev 166 5% CV Distinct Peptides Proteins Identified Injection 1 Injection 2 Injection 3 Total Injection 1 Injection 2 Injection 3 Total Nano Nano

13 ESI BPC Scan Frag=36.V Hela_5ug_1-r19.d Counts vs. Acquisition Time (min) x1 8 +ESI TIC Scan Frag=36.V Hela_5ug_1-r19.d Reproducibility studies Overlaid TIC of 18 replicate 5 ng Injections with a 12 min gradient* in MS-only mode (Scan rate 1.5 spec/sec).2.1 Pierce Hela protein digest standard (P/N: 88328) with 3%ACN in.1%fa Analytical Column: Thermo 75 µm x 25 mm Acclaim PepMap RSLC Column (P/N: ) Trapping Column: Thermo 75 µm x 2 mm Accalim PepMap 1 Trap Column (P/N: ) Counts vs. Acquisition Time (min) *68 hour or ~3 days Overlaid Base Peak Chromatograms x replicate injections x1 7 +ESI BPC Scan Frag=36.V Hela_5ug_1-r19.d 1.6 (55-75 min) RT drift was <.4.5 minutes, which is acceptable for data analysis Counts vs. Acquisition Time (min)

14 CV Analysis Data extracted using Profinder (v 8.) CV analysis performed in MPP on features that were found in 1% of all samples (to remove artifacts due to extraction) Since these are all the same sample, we expect to approach 1% of all compounds having CVs < 2%; this would indicate excellent analytical platform reproducibility Filtered on Raw Data CV cut off Compounds Total compounds Percentage 5% % 1% % 15% % 2% % 3% % 4% % 5% % Filtered on Normalized data CV cut off Compounds Total compounds Percentage 5% % 1% % 15% % 2% % 3% % 4% % 5% % Typical Peak Shape Characteristics for Most Peptides in a 12 min Run AAYLQETGKPLDETLK; MH + ; Annexin A1 x1 5 +ESI EIC( ) Scan Frag=36.V Hela_5ug_1-r19.d Counts vs. Acquisition Time (min) Height: ~7.7 x 1 5 FWHM:.3 min Peak width at 5%:.66 min ( min) Peak Mid-Point: 51.5 min Tailing Factor: 1.

15 Summary- Nanoflow method development Objective was to develop robust and reproducible method for quantitation of complex protein samples Good proteome coverage compared to AJS 18 replicate injections showed excellent reproducibility Close to 9% of peptides had CVs < 2% (96% when using normalized data) System is robust- can switch from AJS to nano in under an hour with no loss of signal. Application: CSF proteomics Objective was to develop robust and reproducible method for quantitation of complex protein samples Cerebrospinal fluid (CSF) and plasma are complex mixtures High abundant proteins make up >95% of CSF proteome Clinical research studies can result in hundreds of samples over time Can be some differences in processing and storing AMRT library approach vs MS/MS quantitation

16 Strategy MS/MS Quant CSF/Plasma MS Quant Low abundance proteins Immunodepletion (MARS Hu 14) Low abundance proteins Protein Digestion MS only Quant Protein Digestion MS/MS Quant MS/MS AMRT library High ph RPLC Sample Preparation Workflow > AMRT library development Pooled CSF from control and MS patients Concentrated using 3kDa molecular weight cut off filter > Immunodepletion using MARS Hu-14 column Tryptic digest using FASP method High ph reverse phase liquid chromatography (hp-rplc) > 45 fractions were collected Equal volumes of non-adjacent fractions were concatenated for a final total of 15 fractions. Spike in retention time standards > Analyze using MS/MS to build library LTQ + immunodepletion: proteins in various samples (213) LTQ MS + immunodepletion: 171 proteins in 27

17 AMRT Library LC/MS/MS Method The 15 concatenated high ph-rplc fractions were analyzed in MS/MS mode on the Agilent 655 QTOF with the Nano-adapter and nanosource. Column =.75 x 25 mm Acclaim PepMap RSLC Trap =.75 x 2 mm PepMap 1, nanoviper Mobile Phase A =.1% Formic Acid in H2O Mobile Phase B =.1% formic acid in 9% aq. ACN Primary Flow =.12 ml/min; Effective Flow = 33 nl/min; Loading Flow = 3.2 µl/min Column Temperature = 42⁰C Injection Volume = 2. ul or 48 ng on column 129 Infinity II LC system from Agilent: Binary Pump, Multisampler, MCT, Capillary Pump and Nanoadapter AMRT Library Database Searching and Exclusion List Method Exclusion lists were utilized to achieve deeper coverage of the CSF Proteome. Each of the 15 fractions was injected 3 times. The first run was searched in SpectrumMill and summarized using the peptide summary with a cut-off score of 9 and SPI of 5%. This summary was used to generate an exclusion list, this was imported into QTOF software and peptides on the list did not have MS/MS performed for subsequent runs This was repeated prior to the 3 rd injection AMRT library built using in-house scripts (subsequently used MPP)

18 Sample Preparation and LCMS Workflow > Sample analysis Individual CSF samples from control and MS patients Concentrated using 3kDa molecular weight cut off filter > Immunodepletion using MARS Hu-14 column Tryptic digest using FASP method Spike in retention time standards Analyze using LC/MS and identify peptides/proteins using AMRT library MS-Only Quant Method Digested CSF samples were loaded onto a 2cm PepMAP 1, nanoviper trapping column and chromatographically resolved on-line using a.75 x 25 mm, 2.µ Acclaim PepMap reverse phase nano column (Thermo Scientific) using a 129 Infinity II LC system equipped with a nanoadapter (Agilent). Column =.75 x 25 mm Acclaim PepMap RSLC Trap =.75 x 2 mm PepMap 1, nanoviper Mobile Phase A =.1% Formic Acid in H2O Mobile Phase B =.1% formic acid in 9% aq. ACN Primary Flow =.12 ml/min; Effective Flow = 33 nl/min; Loading Flow = 3.2 µl/min Column Temperature = 42⁰C Injection Volume = 3.2uL or 48 ng on column 129 Infinity II LC system from Agilent: Binary Pump, Multisampler, MCT, Capillary Pump and Nanoadapter

19 MS-only quant data analysis workflow 1) Extract peptides MH+, RT, Peak Height (and AUC), z for each molecule 2) Data Analysis Import into MPP Mass Profiler Professional Normalize Filter Present in 6-9% CV RT CV MH+ CV Intensity Compare intensity PCA ANOVA Flag differentially abundant ions Export mass list Search MS/MS library to identify peptides and proteins of interest MS Quant Data Extraction Data Extracted with Profinder V.8. Recursive workflow (MFE MPP FBIon) Parameters MFE = Input Range min; Height: > 8 counts; + ions: H+, Na+, K+; charge state: 2-6; two or more ions required; RT window 5% +.8 min; mass window 2 ppm +.2mDa; Absolute Height >13,; MFE score > 8 FBIon = + ions: H+ Na+ K+; charge state 2-6; mass and RT required for match; mass tolerance 2 ppm; RT tolerance.6 min; Height 1 counts; Agile 2 Algorithm used for integration Converted CEF Files area to height w/perl script

20 MS Quant Data Analysis Data analyzed using Mass Profiler Professional V.14.8 Used AMRT Library and ID Browser to annotate peptides ANOVA w/o multiple testing correcting (MTC) was performed on all groups to filter results to those most relevant = ANOVA list P <.5 and FC > 2. were used as thresholds Only proteins with > 2 peptides were included Using ANOVA list, a series of moderated T-test or ANOVA were performed. If more than 3% of peptides were significant with similar trends, a protein was considered a valid hit. Fold Changes were calculated by transforming the normalized transformed data back to raw data and summing all peptides that were identified and linked to the protein regardless of the significance of the peptide. RESULTS

21 Quality Control QC is used to evaluate sensitivity, variability in retention time, and percent CV (%CV) Pooled QC of all CSF samples* for MS and MS/MS quant A total of 7 QC samples were injected over 4 days of analysis. Waters MassPrep Peptide Retention Time Mix (RT Mix) was spiked in to control for retention time shifts. For library building, MS quant, and MS/MS quant Maximum shift in retention time at any given point is.4.5 minutes. * Experimental samples, not library QC of RT Mix: Library Building 42 injections of 15 concatenated fractions 6 MassPrep Peptides were identified in each fraction Some variability due to different amounts being spiked (to assess fold-change differences) and possible degradation of peptides Angiotensin Frag. 1-7 [3.496 m/z (Z=3)] Angiotensin I [ m/z (Z=3)] Bradykinin [ m/z (Z=3)] Renin Substrate [ m/z (Z=4)] Angiotensin II [ m/z (Z=3)] Enolase T35 [ m/z (Z=3)]

22 QC: Retention time reproducibility over 2 weeks Black = MS-only Quant: 21 Sample injections [scan rate 1.5]run 2/9/17 Red = Library Building: 42 injections of concatenated fractions [scan rate 1] run 1/25/217 Angiotensin Frag. 1-7 [3.496 m/z (Z=3)] Bradykinin [ m/z (Z=3)] Angiotensin II [ m/z (Z=3)] Angiotensin I [ m/z (Z=3)] Renin Substrate [ m/z (Z=4)] Enolase T35 [ m/z (Z=3)] Results: Spectrum Mill Summary of CSF MS/MS Library Total Distinct Peptides Total protein groups with > 2 Distinct Peps Total possible proteins with > 2 Distinct Peps Total protein groups Total possible proteins Total Proteins Spectrum Mill Cut-offs fdr < 1%, Peptide score > & SPI > %, Protein Score > fdr < 1%, Peptide score > 8 & SPI > 5%, Protein Score > fdr < 1%, Peptide score > 9 & SPI > 5%, Protein Score > fdr < 1%, Peptide score > 1 & SPI > 5%, Protein Score > fdr < 1%, Peptide score > 8 & SPI > 5%, Protein Score > fdr < 1%, Peptide score > 9 & SPI > 5%, Protein Score > fdr < 1%, Peptide score > 1 & SPI > 5%, Protein Score > fdr < 1%, Peptide score > 1 & SPI > 7%, Protein Score > 13

23 Summary Overall strategy proved effective for quantitating proteins from complex biofluid (CSF) Immunodepletion > high ph RPLC > MS/MS on Nano 655 system resulted in almost 4, peptides and over 5, proteins in our AMRT library Nano system is robust and reproducible Initial pilot study showed good coverage compared to previous studies (~35 vs 5 proteins) Next step is to increase sample sizes in all categories Cap Pump Nano Configuration with Binary and Cap Pumps Analytical Column Detector Auto sampler Loading Position Trap Column Switching Valve in TCC at 42 o C Nano Splitter (1:4) To waste Binary Pump Cap Pump Auto sampler Analytical Column Detector Analytical Position Trap Column Switching Valve in TCC at 42 o C Nano Splitter (1:4) To waste Binary Pump

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