utilizing an accurate mass and retention time library to ......gradient* in ms-only mode (scan rate...

Utilizing an Accurate Mass and Retention Time Library to Facilitate

Biomarker Discovery

Nichole Reisdorph, PhD and Cole Michel, BSMass Spectrometry Facility

Skaggs School of Pharmacy and Pharmaceutical SciencesUniversity of Colorado Anschutz Medical Campus

6/15/2017

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

Research Program

Core Facility

Informatics

Training

Respiratory disease

International Training Program

Data analysis and tool optimization

Proteomics, lipidomics, metabolomics, targeted analyses (lipid mediators, steroids, amino acids, etc)

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)

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

Nanoflow LC/MS• Improved analytical sensitivity,

especially when sample limited• Longer column re-equilibration

times • Requires cleaner samples-

clogging 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

Nano vs Standard Flow: Pros and Cons

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Sample Information• Pierce Hela protein digest standard (P/N: 88328)• Tryptic digest• Re-suspended @ 1 ug/uL with 3% ACN in 0.1% formic acid• Triplicate injections in each configuration

Column InformationNanoflow LC/MS Standard Flow AJS* LC/MS

• Analytical Column: Thermo 75 µm x 250 mm Acclaim PepMap RSLC Column (P/N: 164941)

• Trapping Column: Thermo 75 µm x 20 mm Acclaim PepMap 100 Trap Column (P/N: 164535)

• Analytical Column: Agilent 2.1 x 25 cm Acclaim PepMap RSLC Column (P/N: 164941)

• Trapping Column: None

*Agilent Jet Stream on 6550 QTOF

Note: AJS parameters were provided by Dr. Vadi Bhat from Agilent.Mr. Cole Michel optimized the nanoflow methods.

Method Information (HPLC)Nanoflow LC/MS Standard Flow AJS LC/MS

• Gradient (~115 min)• Mobile Phase A = 0.1% Formic Acid in H2O• Mobile Phase B = 0.1% formic acid in 90% ACN

• TCC: 65⁰C• Effective Flowrate: 0.2 mL/min• Loading Flowrate: N/A• Sample on Column: 15 µg (15µL)• Date Sample Run: January 17th 2017

• Gradient (~112 min)• Mobile Phase A = 0.1% Formic Acid in H2O• Mobile Phase B = 0.1% formic acid in 90% 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 18th 2017

Method Information (MS/MS)Gas Temp: 175⁰CDrying Gas: 11 L/minVCap: 1300 VFragmentor: 360 VOct 1 RF Vpp: 750 VMode: Auto MS/MSMS Mass Range: 290-1700 m/zMS Acquisition Rate: 10MS/MS Mass Range: 50-1700 m/zMS/MS Acquisition Rate: 3Isolation Width: narrowAdvanced Decision Engine: True (B.08 software)Collision Energy: Use formula

Nanoflow LC/MS Standard Flow AJS LC/MS

Charge Slope Offset

2 3.1 1

3 3.6 ‐4.8

>3 3.6 ‐4.8

Gas Temp | Sheath Gas Temp: 250⁰C | 250⁰C

Drying Gas | Sheath Gas: 14 L/min | 11 L/min

Vcap | Nozzle: 3500 V | 0 V

Fragmentor: 360 V

Oct 1 RF Vpp: 750 V

Mode: Auto MS/MS

MS Mass Range: 290-1700 m/z

MS Acquisition Rate: 10

MS/MS Mass Range: 50-1700 m/z

MS/MS Acquisition Rate: 3

Isolation Width: narrow

Advanced Decision Engine: True (B.08 software)

Collision Energy: Use formula

Nebulizer: 35 psigCharge Slope Offset

2 3.1 1

3 3.6 ‐4.8

>3 3.6 ‐4.8

Method Information (MS/MS)Nanoflow LC/MS Standard Flow AJS LC/MS

Max Precursors Per Cycle: 20Abs. Threshold: 3000 countsRel. Threshold (%): 0.01%Active Exclusion: True

Exclude after: 1 spectraReleased After: 0.4 min

Isotope Model: PeptidesCharge State Preferences: Purity Stringency: 100%Purity Cutoff: 30%ADA Target: true, 40,000 counts/spectrumUse MS/MS accumulation time limit: trueReject Precursors that cannot reach target TIC: false

Inactive Active1

unk2 3 >3

Sort by Abundance only

Max Precursors Per Cycle: 20Abs. Threshold: 3000 countsRel. Threshold (%): 0.01%Active Exclusion: True

Exclude after: 1 spectraReleased After: 0.15 min

Isotope Model: PeptidesCharge State Preferences: Purity Stringency: 100%Purity Cutoff: 30%ADA Target: true, 25,000 counts/spectrumUse MS/MS accumulation time limit: trueReject Precursors that cannot reach target TIC: true

Inactive Active1

unk2 3 >3

Sort by Abundance only

Tuning: High analytical sensitivity slicer, low mass range 50-1700, extended 2Ghz922 m/z: Quad narrow isolation efficiency 65%, abundance 10.9 M, Res 22K

Note: AJS parameters were provided by Dr. Vadi Bhat from Agilent.Mr. Cole Michel optimized the nanoflow methods.

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



Protein Summary Results (X% Validation Yield of Filtered Spectra)Software compiles results

*See next slide for settings

Spectrum Mill Parameters (MS/MS Search #1)

Same parameters used for AJS and Nano data files

Spectrum Mill Parameters

Unchanged• SwissProt > Humans• Carbamidomethylation• 2 missed cleavages

Search 1 Search 2 Search 3 Search 4

Enzyme Trypsin Trypsin Trypsin, non-specific N term

Trypsin, non-specific C term

Lysine Carbamylated Acetyl K - -

Ox Meth X X - -

Pyroglutamicacid

X X - -

Deamidated X X - -

Phos S, T, Y X X - -

Search Mode Variable Mod Variable Mod Identity Identity

Spectrum Mill Parameters (Auto-Validation #1)

Spectrum Mill Parameters (Auto-Validation #2)

Nanoflow vs. Standard flow (AJS)Results from HeLA cell digest

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

12492 12181 12573

1725817449 17852 17341

23808

0

5000

10000

15000

20000

25000

Inj 1 Inj 2 inj 3 Total

Dis

tinc

t Pep

tide

s

AJS vs. Nano Source Distinct Peptides

AJS Nano

Protein Comparison

2217 2176 2207

27993170 3217 3029

4115

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4500

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Prot

eins

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ntifi

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AJS vs. Nano Source - Proteins

AJS Nano

Total Ion Chromatograms of Triplicate Injections

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2.4+ESI TIC MS(all) Hela_Jet_Stream_20ug_3.d

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Counts vs. Acquisition Time (min)5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135

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AJS

Nano

Total Ion Chromatograms Overlaid (same scale)

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Nano TIC | AJS TIC

Nano

AJS

Base Peak Chromatograms (Scale Locked)

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AJS BPC | Nano BPC

Nano

AJS

Conclusions

• Triplicate injections of HeLA digest showed good overlay of TIC for both Nano and AJS

• Nanoflow has greater than 20x the sensitivity of AJS– AJS@15 ug: 17,258 peptides and 2799 proteins– Nano@ 1.2 ug: 23,808 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 10x difference in loading.

31593388 3481

4234

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Injection 1 Injection 2 Injection 3 Total

Prot

eins

Ide

ntifi

ed

Nano Source 1.5 ug Injections - Proteins

Nano

1845319157 19002

24412

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30000

Injection 1 Injection 2 Injection 3 Total

Dis

tinc

t Pep

tide

s

Nano Source 1.5 ug Injections - Distinct Peptides

Nano

Triplicate 1.5 ug Injection Hela Standard

Average 3342Std Dev 1665% CV

Average 18870Std Dev 3701% CV

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+ESI TIC Scan Frag=360.0V Hela_05ug_1-r019.d

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Reproducibility studies

• Overlaid TIC of 18 replicate 500 ng Injections with a 120 min gradient* in MS-only mode (Scan rate 1.5 spec/sec)

Pierce Hela protein digest standard (P/N: 88328) re-suspended @ 0.5ug/uL with 3%ACN in 0.1%FA Analytical Column: Thermo 75 µm x 250 mm Acclaim PepMap RSLC Column (P/N: 164941)Trapping Column: Thermo 75 µm x 20 mm Accalim PepMap 100 Trap Column (P/N: 164535)

*68 hour or ~3 days

Overlaid Base Peak Chromatograms7x10

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+ESI BPC Scan Frag=360.0V Hela_05ug_1-r019.d

Counts vs. Acquisition Time (min)20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130

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Counts vs. Acquisition Time (min)55 55.5 56 56.5 57 57.5 58 58.5 59 59.5 60 60.5 61 61.5 62 62.5 63 63.5 64 64.5 65 65.5 66 66.5 67 67.5 68 68.5 69 69.5 70 70.5 71 71.5 72 72.5 73 73.5 74 74.5 75

RT drift was

CV Analysis

CV cut‐off Compounds Total compounds Percentage5% 4944 15325 32.3%10% 11789 15325 76.9%15% 13040 15325 85.1%20% 13590 15325 88.7%30% 14297 15325 93.3%40% 14616 15325 95.4%50% 14813 15325 96.7%

Filtered on Raw Data

CV cut‐off Compounds Total compounds Percentage5% 14484 15325 94.5%10% 14704 15325 95.9%15% 14717 15325 96.0%20% 14718 15325 96.0%30% 14942 15325 97.5%40% 15029 15325 98.1%50% 15077 15325 98.4%

Filtered on Normalized data

• Data extracted using Profinder (v 8.0)• CV analysis performed in MPP on

features that were found in 100% of all samples (to remove artifacts due to extraction)

• Since these are all the same sample, we expect to approach 100% of all compounds having CVs < 20%; this would indicate excellent analytical platform reproducibility

Typical Peak Shape Characteristics for Most Peptides in a 120 min Run

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Counts vs. Acquisition Time (min)50.6 50.8 51 51.2 51.4 51.6 51.8 52 52.2 52.4 52.6 52.8 53 53.2 53.4 53.6

Height: ~7.7 x 105FWHM: 0.30 minPeak width at 5%: 0.66 min (51.83 – 51.17 min)Peak Mid-Point: 51.50 minTailing Factor: 1.0

AAYLQETGKPLDETLK; 1776.938 MH+; Annexin A1

Summary- Nanoflow method development


• Good proteome coverage compared to AJS• 18 replicate injections showed excellent reproducibility

– Close to 90% of peptides had CVs < 20% (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


• 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

CSF/Plasma

MS/MS AMRT library

Immunodepletion(MARS Hu 14)

Protein Digestion

High pH RPLC

Low abundance proteins

Protein Digestion

Low abundanceproteins

MS QuantMS/MS Quant

MS/MS Quant

MS only Quant

Strategy

Sample Preparation Workflow > AMRT library development

Pooled CSF from control and MS patientsConcentrated using 3kDa molecular weight cut off filter > Immunodepletion using MARS Hu-14 column Tryptic digest using FASP methodHigh 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 MS + immunodepletion: 171 proteins in 2007 http://pubs.acs.org/doi/pdfplus/10.1021/pr070293w

LTQ + immunodepletion: 2800-3500 proteins in various sampleshttps://www.ncbi.nlm.nih.gov/pubmed/24039694 (2013)

AMRT Library LC/MS/MS Method

• The 15 concatenated high pH-RPLC fractions were analyzed in MS/MS mode on the Agilent 6550 QTOF with the Nano-adapter and nanosource.

• Column = 0.075 x 250 mm Acclaim PepMap RSLC• Trap = 0.075 x 20 mm PepMap 100, nanoviper• Mobile Phase A = 0.1% Formic Acid in H2O• Mobile Phase B = 0.1% formic acid in 90% aq. ACN• Primary Flow = 0.120 mL/min; Effective Flow = 330

nL/min; Loading Flow = 3.2 µL/min• Column Temperature = 42⁰C• Injection Volume = 2.0 uL or 480 ng on column• 1290 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 50%. – 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 3rd injection• AMRT library built using in-house scripts (subsequently used

MPP)

Sample Preparation and LCMS Workflow > Sample analysis

Individual CSF samples from control and MS patientsConcentrated using 3kDa molecular weight cut off filter > Immunodepletion using MARS Hu-14 column Tryptic digest using FASP methodSpike in retention time standardsAnalyze using LC/MS and identify peptides/proteins using AMRT library

MS-Only Quant Method

Digested CSF samples were loaded onto a 2cm PepMAP 100, nanoviper trapping column and chromatographically resolved on-line using a 0.075 x 250 mm, 2.0µ Acclaim PepMap reverse phase nano column (Thermo Scientific) using a 1290 Infinity II LC system equipped with a nanoadapter(Agilent).

• Column = 0.075 x 250 mm Acclaim PepMap RSLC • Trap = 0.075 x 20 mm PepMap 100, nanoviper• Mobile Phase A = 0.1% Formic Acid in H2O• Mobile Phase B = 0.1% formic acid in 90% aq. ACN• Primary Flow = 0.120 mL/min; Effective Flow = 330

nL/min; Loading Flow = 3.2 µL/min• Column Temperature = 42⁰C• Injection Volume = 3.2uL or 480 ng on column• 1290 Infinity II LC system from Agilent: Binary Pump,

Multisampler, MCT, Capillary Pump and Nanoadapter

Import into MPPImport into MPP

• Mass Profiler Professional

• Normalize

FilterFilter

• Present in 60-90%

• CV RT• CV MH+• CV

Intensity

Compare intensityCompare intensity

• PCA• ANOVA• Flag

differentially abundant ions

Export mass list Export mass list

• Search MS/MS library to identify peptides and proteins of interest

1) Extract peptides MH+, RT, Peak Height (and AUC), z for each molecule

2) Data Analysis

MS-only quant data analysis workflow

MS Quant Data Extraction

• Data Extracted with Profinder V.8.0– Recursive workflow (MFE MPP FBIon)– Parameters

• MFE = Input Range 10-72.5 min; Height: > 8000 counts; + ions: H+, Na+, K+; charge state: 2-6; two or more ions required; RT window 5% + 0.8 min; mass window 20 ppm + 0.2mDa; Absolute Height >13,000; MFE score > 80

• FBIon = + ions: H+ Na+ K+; charge state 2-6; mass and RT required for match; mass tolerance 20 ppm; RT tolerance 0.6 min; Height 10000 counts; Agile 2 Algorithm used for integration

• Converted CEF Files area to height w/perl script

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 < 0.05 and FC > 2.0 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 30% 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

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 0.4 – 0.5

minutes.

* Experimental samples, not library

QC of RT Mix: Library Building

Angiotensin Frag. 1-7 [300.4960 m/z

(Z=3)]

Bradykinin [354.1944 m/z

(Z=3)]

Angiotensin II [349.5188 m/z

(Z=3)]

Angiotensin I [432.9022 m/z

(Z=3)]

Renin Substrate [440.4886 m/z

(Z=4)]

Enolase T35 [624.9956 m/z

(Z=3)]

• 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

QC: Retention time reproducibility over 2 weeks

Black = MS-only Quant: 21 Sample injections [scan rate 1.5]run 02/09/17 Red = Library Building: 42 injections of concatenated fractions [scan rate 10] run 01/25/2017

Angiotensin I [432.9022 m/z

(Z=3)]

Bradykinin [354.1944 m/z

(Z=3)]

Angiotensin Frag. 1-7 [300.4960 m/z

(Z=3)]

Renin Substrate [440.4886 m/z

(Z=4)]

Enolase T35 [624.9956 m/z

(Z=3)]

Angiotensin II [349.5188 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

50044 4651 5005 7614 7968 9600 fdr < 1%, Peptide score > 0 & SPI > 0%, Protein Score > 0








Summary

• Overall strategy proved effective for quantitating proteins from complex biofluid (CSF)

• Immunodepletion > high pH RPLC > MS/MS on Nano 6550 system resulted in almost 40,000 peptides and over 5,000 proteins in our AMRT library

• Nano system is robust and reproducible• Initial pilot study showed good coverage compared to

previous studies (~3500 vs 5000 proteins)• Next step is to increase sample sizes in all categories

Analytical Position

Binary Pump

NanoSplitter (1:400)

To wasteTrap Column

Analytical Column

Auto sampler

Cap Pump

Detector

Loading Position

Binary Pump

NanoSplitter (1:400)

To wasteTrap Column

Analytical Column

Auto sampler

Cap Pump

Detector Switching Valve in TCC at 42oC

Switching Valve in TCC at

42oC

Nano Configuration with Binary and Cap Pumps

http://www.ucdenver.edu/academics/colleges/pharmacy/Research/CoreFacilities/Spectrometry/Pages/default.aspx

utilizing an accurate mass and retention time library to ......gradient* in ms-only mode (scan rate...

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