Christine A. Jelinek, Ph.D. - Johns Hopkins Medicine ... P and Aebersold R Nat Methods 2012...

Selected Reaction Monitoring

Christine A. Jelinek, Ph.D.

Johns Hopkins University School of Medicine

Department of Pharmacology and Molecular Sciences

Middle Atlantic Mass Spectrometry Laboratory

Selected Reaction Monitoring Lecture Agenda

Selected Reaction Monitoring: Technique

Selected Reaction Monitoring: Instrumentation

Quantitation: Absolute vs. Relative

Selected Reaction Monitoring: Method Development

Selected Reaction Monitoring: Application

Selected Reaction Monitoring: Case Studies

Selected Reaction Monitoring:

Technique

Selected reaction monitoring (SRM)

Picotti P and Aebersold R Nat Methods 2012 9:555-566

SRM is a mass spectrometry technique for the detection and quantitation of

specific, pre-determined analytes

SRM selectively monitors specific analyte molecular ion and several fragment

ions generated from the analyte by collisional dissociation

Most effectively used in an LC-coupled MS system

Exploits the unique capabilities of triple quadrupole instruments to act as

mass filters

Originally applied to the measurement of small molecules

SRM allows for the measurement of proteins in low µg or high ng/mL range

November 27, 2012 5 5

Q1 Q2 Q3 Q0

RF only Collision Cell Scanning

RF/DC

Scanning

RF/DC RF only

Selected Reaction Monitoring Mass Spectrometric Assay

Elliott MH et al. J Mass Spectrom 2009 44:1637-1660

6

y11

y9

y7

G

A

G

Q

N

I

I

P

A

S

T

G

A

A

K

X

X

X

Each peptide has a unique mass

and unique sequence fragments Q1 passes only the

molecular mass

Q3 passes three

selected fragments

Selected Reaction Monitoring Mass Spectrometric Assay

Elliott MH et al. J Mass Spectrom 2009 44:1637-1660

November 27, 2012 7

Selected Reaction Monitoring Mass Spectrometric Assay Output

7

Peak Area

November 27, 2012 8

Selected Reaction Monitoring Mass Spectrometric Assay Output

8

Peak Area

Peptide

Sequence

Transition

Ion AUC

HQQQFFQR y6 21.4513

HQQQFFQR y7 6.2568

HQQQFFQR y5 2.0417

HQQQFFQR y4 0.8244

HQQQFFQR y2 0.2809

9

Selected Reaction Monitoring Mass Spectrometric Assay Platform

Selectivity

Specificity

Sensitivity

Mass filters pass only targeted peptides

even when ion signal is below noise

Pre-select unique “transitions”

ions during detection

High duty cycle.

LODs 2-3 orders of magnitude

better than scanned spectra


Instrumentation

Selected Reaction Monitoring Using Triple Quadrapole Mass Spectrometers

Triple Quadrapole Mass Spectrometers

Jim Morrison of LaTrobe University, Australia first developed the QQQ

arrangement for the purpose of studying the photodissociation of gas-

phase ions.

The first triple-quadrupole mass spectrometer was developed at

Michigan State University by Dr. Christie Enke and graduate student

Richard Yost in the late 1970s.

Quantitation:

Absolute vs. Relative

Quantitation Strategies

Picotti P and Aebersold R Nat Methods 2012 9:555-566

Quantitation Labeling Quantitation

strategy

Relative (differential)

quantitation

Label-free

Metabolic stable-isotope

labeling

[15N]ammonium

sulfate

SILAC

Chemical stable-isotope

labeling

ICAT, iTRAQ

mTRAQ

Enzymatic stable-isotope labeling [18O]water

Absolute quantitation

Metabolic stable-isotope labeling QconCAT, PSAQ

Chemical stable-isotope labeling AQUA synthetic

peptides

Method Development Strategy Peptide Standards

Labeled Standards:

PEPTIDE* LAGKPEPTIDEKLAG*

PEPTIDEK* …….KPEPTIDEK………*

Internal Standards:

Non-biomarker peptide present within sample

Used to minimize run-to-run variability from processing and instrumentation

Panel or single peptide can be selected

External Standards:

Heavy isotope labeled standards can be added to each sample

Panel of biomarker peptides, a single biomarker peptide, a panel of biomarker

proteins, or a single protein can be used

Isotope labeling directs absolute quantitation schema

Non-labeled non-biomarker peptide or protein can also be used

16

Relative Quantitation: Replicate Comparison

17

Relative Quantitation: Replicate Comparison

Selected Target Peptide

18

Relative Quantitation: Calibration Curve using External Standard

Conc (fmol/uL) RT/min FWHM Start Time End Time Area Background Height

1 11.5 0.64 10.08 12.97 886 338 19

5 11.28 0.89 10.48 13.93 18741 438 321

10 11.35 0.86 10.16 13.98 58852 322 976

50 11.33 0.86 10.42 14.58 271949 1635 4282

y = 5890.1x - 8475.5 R² = 0.9985

-100000

0

100000

200000

300000

400000

500000

600000

700000

0 20 40 60 80 100 120

Are

a

Concentration of apomyoglobin (fmol/uL)

LOD of Apomyoglobin in Solvent

LOD In Solvent

Target Peptide

NDIAAK


Concentration (fmol/ L) Average Area Standard Deviation

1 1920 257

5 17634 3050

10 45532 3924

50 287582 51343

100 578626 78607

y = 5884.7x - 9111.7 R² = 0.9998

-100000

0

100000

200000

300000

400000

500000

600000

700000

0 20 40 60 80 100 120

Are

a

Concentration (fmol/ L)

LOD of Apomyoglobin in digested CSF LOD In Matrix

20


Target Peptide

NDIAAK

21

Absolute Quantitation: Calibration Curve using heavy labeled IS peptides

Replicate IEAPQDIK IEAPQDIK*

(heavy)

Standard_1 40 fmol/ul 40 fmol/ul

Standard_2 12.5 fmol/ul 12.5 fmol/ul





Standard_7 .25 fmol/ul .25 fmol/ul

Standard_8 .1 fmol/ul .1 fmol/ul

Stergachis, A. et al. Nature Methods. 2011; 8: 1041–1043.

22


Peak Area Retention Time Representative

Ion Chromatogram


23


Peptide

Sequence

Protein

Name

Replicate Name Ratio to

Standard

IEAIPQIDK GST-tag Standard_1 21.4513








IEAIPQIDK GST-tag FOXN1-GST 0.7079


24


Peptide

Sequence

Protein

Name

Replicate

Name

Ratio to

Standard [fmol

/ul]

IEAIPQIDK GST-tag Standard_1 21.4513 40

IEAIPQIDK GST-tag Standard_2 6.2568 12.5





IEAIPQIDK GST-tag Standard_7 0.0819 .25

IEAIPQIDK GST-tag Standard_8 0.0248 .1

IEAIPQIDK GST-tag FOXN1-GST 0.7079

Stergachis, A. et al. Nature

Methods. 2011; 8: 1041–1043.

Topic:

Assay Development

Selected Reaction Monitoring

SRM-

Assay

November 27, 2012 26

Selected Reaction Monitoring Mass Spectrometric Assay Workflow

Dissertation Defense

April 5, 2012

http://www.hopkinsmedicine.org/mams/

18 Hour Tryptic

Digestion

Solid Phase Extraction

Speedvac and Resuspension

C18 Reverse Phase HPLC

SRM-MS Detection

27

Method Development Strategy “Signature Peptide” selection

Digested

horse

apomyoglobin

ND

IAA

K (

11.3

)

AS

ED

LK

(12.9

)

HG

TV

VLT

ALG

GIL

K

(31.2

)

LF

TG

HP

ET

LE

K

(21.4

)

VE

AD

IAG

HG

QE

VLIR

(23.9

)

ELG

FQ

G (

24.6

)

ALE

LF

R (

28.0

)

YLE

FIS

DA

IIH

VLH

SK

(37.7

)

Tryptic Myoglobin Peptides

28

Method Development Strategy “Signature Peptide” selection

Signature Peptide Selection Criteria: Minimum peptide length

Maximum peptide length

Exclude N-terminal processing peptides

Exclude potential ragged ends

Exclude peptides containing:

o Met

o Cys

o His

o NXT/NXS

o RP/KP

29

Method Development Strategy “Transition Ion” selection

Selected Transitions

y2

y3

y4

y5

y6

y7

y8

y9

y10

y11

Total Ion Chromatogram

TCVADESAENCDK

Transition Selection

y7

y10

y2

30

Iterative Method Development

Method Development Strategy “Transition Ion” selection

Initial 1 Peptide SRM Transition Selection

Poor performing

Transitions

eliminated from

subsequent

experiments


Typical Limit of Detection β-Galactosidase signature peptide in sera matrix

Retention Time (mins)

b4

y3

y4

Inte

nsity (

10

3)

GDFQFNISR

0

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

0 50 100 150 200 250

Sample Concentration (fmol)

y = 4390.1x - 932.73

R² = 0.9982

Experimental Detection Range

Method Development Strategy Scheduling SRM Assay

33

Selected Reaction Monitoring Mass spectrometric validation assay platform

SRM Platform Limitations:

SRM-Assay protocol NOT standardized across instrument manufacturers

Precision of Assay dependent upon technical skill and expertise of technician.

Assay precision varies WIDELY across users, laboratories, test sites.

Successful target detection in biological fluids is dependent upon front-end

sample preparation

Laborious sample preparation limits total throughput capability

Skyline is an open-source tool available at https://brendanx-

uw1.gs.washington.edu/labkey/project/home/software/Skyline/begin.view

Skyline is a windows-based application that allows researchers to build

methods and analyze data results from Selected Reaction Monitoring

experiments.

Originally developed in conjunction with NCI's Clinical Proteomic

Technology Assessment for Cancer network (CPTAC) program.

Skyline users can directly access raw mass spectrometry data from data

files generated by almost all instrument manufactures.

Selected Reaction Monitoring Bioinformatics Solutions

Selected Reaction Monitoring Bioinformatics Solutions


Application

Biomarker Assay Workflow Role of Selected Reaction Monitoring in Biomarker Research

Picotti P and Aebersold R Nat Methods. 2012; 9:555-566.

Biomarker - a biological molecule found in blood, other body fluids,

or tissues that is a sign of a normal or abnormal process, or of a

condition or disease (National Cancer Institute)

Predictive biomarker – gives an indication of the probable effect of a

treatment on a patient; helps to assess the most likely response to a

particular treatment type

Diagnostic biomarker – determines whether disease already exists

Prognostic biomarker – shows disease progression with or without

treatment

Clinical Biomarkers Definition


Clinical Biomarkers Working Hypothesis

Localized Tissue

Damage

Disease

Process

Molecular

Cues

Mol Oncol. 2009; 3(1): 33-44.

Biomarker Assays Clinical Sample Types

Tissue

Tissue-derived proteins become highly diluted in the systemic circulation

Proximal fluids o Urine – prostate, bladder or kidney diseases

o CSF – intracranial processes

o Nipple aspirate – breast cancer

o Local drainage sites for disease-derived proteins

Serum

Primary component of biobank archives

Clotting process may lead to neo-generation of peptides

Plasma

>90% of plasma proteome is comprised of ~10 proteins

41

Sandwich ELISA Validation Assay Platform of Choice

ELISA Platform Advantages:

• Quantitative measurements

• High sensitivity for desired target

• High selectivity for target antigen

• Rapid diagnostic

• High-throughput platform

• Easily performed diagnostic

• Easily interpretable results

• Platform requirements are compatible

with clinical testing facilities

42

Sandwich ELISA Validation Assay Platform of Choice

ELISA Platform Limitations:

• Target constrained by antibody

availability, epitope recognition and

binding specificity

• Quality of available antibodies often

varies across manufacturers.

• Constrained to single-plex analysis

• Assay development costs are

prohibitively high for most biomarker

target development projects

• When initiated, assay development

proceeds for multiple years

Biomarker Assay Workflow Role of Selected Reaction Monitoring in Biomarker Research

Picotti P and Aebersold R Nat Methods. 2012; 9:555-566.

Case Study #1:

A. Hoofnagle, HDL Assay

Hoofnagle, A. et al. “Multiple-Reaction Monitoring–Mass Spectrometric

Assays Can Accurately Measure the Relative Protein Abundance in Complex

Mixtures.” Clinical Chemistry. April 2012; 58(4): 777-78.

November 27, 2012 45 November 27, 2012 45

HDL Assay SRM-Assay measurements

Hoofnagle, A. et al. “Clinical Chemistry. April 2012; 58(4): 777-78.


HDL Assay: Study Methodology SRM-Assay measurements

6-plex MRM-Assays

1-plex Immuno-Assays

Label-free shotgun Mass

Spectrometric Assay

All isotope-labeled

peptides incorporated

Single isotope-labeled

peptides incorporated

Comparing Assay Platforms

for Apolipoproteins

HDL purified from

plasma from 30 donors



HDL Assay: Study Results SRM-Assay measurements



HDL Assay: Study Results SRM-Assay measurements

Comparing ApoA1 Assay Results

Isotope dilution MRM-MS Assay vs Immunoassay


49

HDL Assay: Study Conclusions SRM-Assay measurements

Relative quantification by shotgun proteomics approaches correlated

poorly with the 6 protein immunoassays.

Peak area ratios measured in multiplexed MRM-MS assays correlate

well with immunochemical measurements.

o The isotope dilution MRM-MS approaches showed correlations with

immunoassays of r 0.61– 0.96.

o The MRM-MS approaches had high reproducibility and linearity--

13% CV and linearity (r 0.99)

A single protein internal standard applied to all proteins performed as

well as multiple protein-specific peptide internal standards.

The ISprot approach was capable of correcting for digestion variability

but the ISpep approach was not.


Case Study #2:

CPTAC Program Clinical Proteomic Tumor Analysis Consortium

Addona, T. et al. “Multi-site assessment of the precision and

reproducibility of multiple reaction monitoring–based measurements of

proteins in plasma.” Nature Biotechnology. June 2009; 27(7): 633-64.


CPTAC Program Program Mission and Objectives

The Clinical Proteomic Tumor Analysis Consortium (CPTAC) is a comprehensive and

coordinated effort to accelerate the understanding of the molecular basis of cancer

through the application of robust, quantitative, proteomic technologies and workflows.

Mission:

Objective 1: Identify and characterize the protein inventory from tumor and normal tissue

biospecimens

Objective 2: Integrate genomic and proteomic data from analysis of common cancer

biospecimens

Objective 3: Develop assays against proteins prioritized in the discovery stage as potential

biomarker candidates

Objective 4: Perform testing of verification assays in relevant cohorts of biospecimens

Objectives:

http://proteomics.cancer.gov/


CPTAC Study: Study Objectives Assessing SRM Assay Reproducibility and Precision

Study 1

synthetic peptides

Study 3

undigested synthetic proteins

Study 2

digested synthetic proteins

Comparing SRM-MS Assay Protocols

within and between laboratories:

Location Nano-LC MS QQQ

8 Sites

Eksignet

nanoLC-1D

Plus

ABI-Sciex

4000 QTRAP

Eksigent

nano LC-2D

Thermo TSQ

Quantum

Ultra Agilent 1100

Nanosystem

Addona, T. et al. “Nature Biotechnology. June 2009; 27(7): 633-64.


CPTAC Target Proteins and Signature Peptides

Protein Abbrev Species Signature peptide MH+

(mono)

MRM transitions (m/z)

Q1 Q3

Aprotinin APR-AGL Bovine AGLCQTFVYGGCR 1493.7 747.3 863.4 964.5 1092.5

Leptin LEP-IND Mouse INDISHTQSVSAK 1407.3 469.9 590.8 647.8 728.4

Myoglobin MYO-LFT Horse LFTGHPETLEK 1279.7 427.2 510.3 583.8 724.4

Myelin basic

protein

MBP-HGF Bovine HGFLPR 732.4 366.7 391.3 538.3 595.4

MBP-YLA Bovine YLASASTMDHAR 1328.6 443.5 491.2 526.8 823.4

Prostate-specific

antigen

PSA-IVG Human IVGGWECEK 1082.5 541.7 808.3 865.4 969.4

PSA-LSE Human LSEPAELTDAVK 1280.7 640.8 783.4 854.5 951.2

Peroxidase HRP-SSD Horseradish SSDLVALSGGHTFGK 1483.8 495.3 711.4 798.4 982.5

C-reactive

protein

CRP-ESD Human ESDTSYVSLK 1136.6 568.8 617.4 704.4 805.4

CRP-GYS Human GYSIFSYATK 1144.6 572.8 724.4 837.5 924.5

CRP-YEV Human YEVQGEVFTKPQLWP 1826.9 914.0 1053.5 1181.6 1525.8


CPTAC Study: SRM-MS Assay Targets Assessing SRM Assay Reproducibility and Precision


CPTAC Study: Study Workflow Assessing SRM Assay Reproducibility and Precision





CPTAC Study: Study Workflow Assessing SRM Assay Reproducibility and Precision





CPTAC Study: Study Results Assessing SRM Assay Reproducibility and Precision

Comparing SRM Assay Results

Intralaboratory Assay CVs





Intralaboratory Assay CVs





Intralaboratory Assay LOQs


59


Reproducibility and Precision of the quantitative measurements for 9 of 10 peptides ranged from 4–14%, 4–13%, and 10–23%.

Intralaboratory CVs were predominantly <15% and <25% at the identical concentration for studies I/II and III, respectively.

Interlaboratory and intralaboratory CVs improved with increasing analyte concentration.

Under real plasma biomarker conditions (study III): o Performance was below that expected for clinical assays (<10–15%).

o Sensitivity was sufficiently low for verifying candidate biomarkers present in plasma with a concentration higher than 2–6 g/ml in plasma

o Assay demonstrated a linear dynamic range spanning three orders of magnitude


60

CPTAC Study: Study Conclusions Assessing SRM Assay Reproducibility and Precision

The most frequent cause of poor peptide performance was the presence of interference from the background plasma digest matrix.

Monitoring a minimum of three transitions per analyte is critical in maintaining assay selectivity and recognizing signal interference.

SRM Assay method development and optimization must be completed independently for all instrument platforms included within a multi-site validation effort.

SRM Assay design impacts assay measurements, assay LOQ, and reproducibility.

.

SOP compliance is critical for assay reproducibility.


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Christine A. Jelinek, Ph.D. - Johns Hopkins Medicine ... P and Aebersold R Nat Methods 2012...

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