Simplifying Targeted Protein

Quantification with Mass Spec:

The SRM Atlas and Multiplexed
MRM Protein Assays

Simplifying Targeted Protein Quantification with

Mass Spec: The SRM Atlas and Multiplexed MRM
Protein Assays
Broadcast Date: Thursday, December 16, 2010
Time: 1:00 PM EDT
Sponsored by

Simplifying Targeted Protein

Quantification with Mass Spec:
The SRM Atlas and Multiplexed
MRM Protein Assays

Your Moderator

Tamlyn Oliver
Managing Editor
Genetic Engineering & Biotechnology News

Simplifying Targeted Protein

Quantification with Mass Spec:
The SRM Atlas and Multiplexed
MRM Protein Assays

Robert L. Moritz, Ph.D.

Associate Professor and Director of Proteomics
Institute for Systems Biology

RobertMoritz
DevelopmentofaCompleteHuman
Peptide andSRMAtlas

Human SRMAtlas

Duchy of
Luxembourg

HumanSRMAtlas
Designmultiplequantitative
assaysforeveryhumanprotein
usingarobusthighthroughput
platformsuitablefor
clinicalapplications

Understandingtheterrain
Knowingthecomponents

SurveyingtocreateanAtlas

NavigatingtheAtlas

Quantitativeproteomicanalysis
TandemMassSpectrometry(MS/MSorMS2)

SelectedReactionMonitoring(SRM,MRM)
Q1

Q2

Q3

Highsensitivity
targeteddutycycle
Highdynamicrange
7

Selected/MultipleReactionMonitoring(S/MRM)
1 MS analyzer

Fragmentation

SRM peak

3 MS analyzer

m/z

m/z
Q3 / trap mode
or segmented

twolevelsofmassselection: highspecificity
notscanning(Q1/Q3static),highdutycycle: highsensitivity

SRM-triggered
MS2

themostsensitivemassspectrometrymethodknown(lowamole)

....youneedtoknowwhattolookfor!
(themassspectrometristsELISA)

Outline
IntroductiontotheISBPeptideAtlas:Compendiumof
peptidesandinferredproteinsobservedbyMS/MS

DevelopmentofSRMAtlas:Unifiedtransitionresource
Statusofassayproduction
ApplicationofSRMAtlas

PeptideAtlas Workflow
Shotgun
MS/MS
Experiments

NIST

Community

PeptideAtlas
Database &
Web Interface

Tranche
UniPep

PhosphoPep

ProteomExchange

GPMDB
PRIDE

Raw Data
Repository

Sequence
Searching
SpectraST
Searching

www.peptideatlas.org,TPP:TransProteomicPipeline

TPP

PeptideAtlas
Pipeline

Whatisaproteotypic peptide?
Protein
Peptide
region never
observed in
multiple
experiments

Peptide
region
consistently
observed in
multiple
experiments

PeptideAtlas protein view page

Cytoscape view of proteins & peptides

proteins

ambiguously mapped peptide

proteotypic peptides
Nprot = 1 Nobs >121
EPS > 0.3

PeptideAtlas BuildSummary
Build
HumanAll

#MS
Runs

#Exps

Searched
Spectra

IDs
P>0.9

Distinct
Peptides

Distinct
Proteins

Swissprot

424

54k

49M

5.6M

97k

12141

20328

76

48k

16M

1.8M

18k

2486

???

174

1731

7.1M

690K

14K

2771

7426

Yeast

53

2957

6.5M

1.1M

36k

4336

6552

Mouse

59

3097

10M

1.4M

51k

7686

16085

Drosophila

43

1769

7.5M

498k

72k

9124

13,600

Drosophila
PhosphoPep

448

0.9M

170k

10k

4583

???

Halobacterium

88

497

0.5M

76k

12k

1975

2426

S.pyogenes

64

215k

52k

7k

1068

3729

MousePlasma

568

7M

0.8M

9.4k

2075

????

HumanPlasma
Human
Glycosylated

HumanPeptide andPlasmaAtlas
HPPP-II

HumanProteomecoverage

PlasmaProteomecoverage

72,396 distinctpeptidesmappingto
10,297 SwissProtidentifiers,or51%
ofallproteincodinggenes

18,936 distinctpeptidesmappingto
2,238 SwissProtidentifiers

HumanSRMAtlas

20,333proteins(20,2772010version)
32,562proteinsincl.isoforms
658,684tryptic peptides(anylength)
480,284distinctpeptides(730aa)

15

DevelopmentsatISB SRMAtlas
Humanproteins
(fromnaturalsourceor
synthetic)

Synthetic
proteotypic
peptide
(fromcheapsynthesis)

DevelopHuman
PeptideAtlas
(fromtryptic digestsor
syntheticpeptides)

DevelopHuman
SRMAtlas

Develop
optimized
transitionsfrom
PeptideAtlas

(verifiedquantitative
assays)
16

PredictedPeptidesAugmentObserved
Proteincoveragebyobservedpeptides
inHumanPeptideAtlas 201005

Extractedobservedpeptidesfromlarge
internalhumanPeptideAtlasbuild,but
wereunabletouseallobservedpeptides.

Depictsnumberofmappingpeptidesforeach
proteininthetargetproteome.Observed
peptidesalonecannotachievefullcoverage.

PeptideTracking&Processing
>150,000peptides

poolsof
96peptides
=

2Dbarcodetube
ISBHJRMXX000026

Laboratory
Information
Management
System

mix&dilutepeptides

DataAquisition
b2

b3

GQEVETSVTYYR,z=2
y4

ISBHJRMXX000026

b4

96peptides

b3-NH3

y7

y6

y3
b5

b6 y5

&
InclusionList

Agilent
6530QTOF

BasedonExpectedm/z
2>2,3
3>2,3,4
4>2,3,4

intensity,cps

Agilent AutoPreferredExcludeMSMSTable
exclusive
5CEstepsperchargestate

converttomzML &
databasesearch
b2
b4

y4

y7

m/z

consensusspectrum
SSR36.49

SSR29.40
SSR21.53
SSR12.92
SSR6.58

SSR41.69

RetentiontimestabilityfromJuneSeptember2010

Agilent
SRMspectrum 6460QQQ

Q1

Q3

RT

691.9

659.4

25.2

691.9

730.4

25.2

691.9

900.2

25.2

691.9

303.1

25.2

transitionlist

Transitioniontransportability
QTof

ILPLTGIPVLFLPGNAGSYK[M+2H]2+
int.

int.

2
3
6

QQQ

1
2
3
4
5
6
7
8
9
10

8
5
4
3
2
7
6
9
10
1

9
10

HumanProteomeSRMAssayCoverage
99%oftheHumanProteome
(SwissProtCorecoverage)

Results SyntheticpeptideadditionstoPeptideAtlas

99%withSRMAtlas

51%fromshotgun
experimentsalone

HumanProteomeSRMCoverage(subsets)
Singleaminoacidmutations
2726MajorSNPs (>30%frequency=1363)resultinginnon
synonymousmutations

Glycosylated proteins
5199Membraneproteinswith11,269 Nglycosites
1748secretedproteins

Splicedisoforms notpresentinSwissProt
11,309additionalproteotypic peptidesforuniqueidentification

SRMAtlasinterface
FragmentSpectrum(QQQ)

www.srmatlas.org

Suggested transitions,
Collision Energy plots,
Calculated hydrophobicity
Fragment relative intensities

Conclusions
Created comprehensive highresolution MS/MS (fragment ion)
spectra for >99% of the human proteome via a standardized
pipeline
GoldStandard fragmentiondatabasethatcanbeusedbyall
asareferenceforcreatingSRMassays
First pass creation of the human proteome database allowing
communityparticipationtofurtheradddataonposttranslation
modificationsetc
Referencefragmentationdatabasehasmanyotherusessuchas
spectralmatchingetc.

www.srmatlas.org

Acknowledgements
KenMiller
ChristineMiller
UlrikeKusebauch
EricDeutsch
DavidCampbell
MiYounBrusniak
DouglasSpicer
CarolineChu
JeffreyStevens
Zhi Sun
JoeSlagel
LuisMendoza
TerryFarrah
SungTatKwok
HectorRamos
DavidShteynberg

Weiwu Heandcolleagues
OpenBiosystems
RuediAebersold
ChristineCarapito
PaolaPicotti
OliverRinner
BerndWollscheid
LeroyHood
Ilya Shmulevich
SarahKillcoyne
JohnBoyle

JoelLouette
Theracode
HolgerWenschuh

ARRA1RC2HG00580501

Duchyof
Luxembourg

www.systemsbiology.org

Simplifying Targeted Protein

Quantification with Mass Spec:
The SRM Atlas and Multiplexed
MRM Protein Assays

Leigh Anderson, Ph.D.

Founder and CEO
Plasma Proteome Institute

Improving Measurement of Protein

Biomarkers by Mass Spectrometry:
Methods and Workflows

Leigh Anderson PhD

Plasma Proteome Institute

GEN Webinar
16 December 2010

The Plasma Proteome Exhibits Wide

Dynamic Range

The human plasma proteome: History, character, and diagnostic prospects. Anderson,
N.L. and Anderson, N.G., Molecular and Cellular Proteomics, 1.11, 845-867 (2002)

The Clinical Plasma Proteome

Proteins measured in clinically-available tests in the US
109 proteins via FDA-cleared or approved tests
Clinical test costs range from $9 (albumin) to $122 (Her2)
90% of those ever approved are still in use

96 additional proteins via laboratory-developed tests (not FDA

cleared or approved)
Total 205 proteins ( products of 211genes, excluding Igs)

Clinically applied proteins thus account for

About 1% of the baseline human proteome (1 gene :1 protein)
About 10% of the 2,000+ proteins observed in deep discovery
plasma proteome datasets

The Clinical Plasma Proteome: A Survey of Clinical Assays for Proteins in

Plasma and Serum. Anderson, L. Clin Chem, 56:177185 (2010)

New Protein Diagnostics Are FDA-Cleared at a Rate of ~1.5/yr:

Insufficient to Meet Dx or Rx Development Needs

*
* 87 tests prior to 1993 at a rate of >5/yr
The Clinical Plasma Proteome: A Survey of Clinical Assays for Proteins in
Plasma and Serum. Anderson, L. Clin Chem, 56:177185 (2010)

Immunoassays Do Not See the

Analytes Chemical Structure
What actually forms the
immunoassay sandwich
- or prevents its
formation - is not directly
visualized:
Immunoassays do not
provide a molecular
microscope

Sandwich Immunoassays Depend on Highly

Specific 3-D Structural Interactions
False Negatives

Blocking Complex

Autoantibody

False Positive

Ideal Sandwich
HAMA

Cleaved Ag

MRM of Proteotypic Tryptic Peptides Provides Highly

Specific Assays for Proteins > 1ug/ml in Plasma
Afamin
Albumin
-1-acid glycoprotein 1
-1-antichymotrypsin
-1B-glycoprotein
-2-antiplasmin
-2-macroglobulin
Angiotensinogen
Antithrombin-III
Apolipoprotein A-I
Apolipoprotein A-II
Apolipoprotein A-IV
Apolipoprotein B-100
Apolipoprotein C-I
Apolipoprotein C-III
Apolipoprotein E
-2-glycoprotein I
Ceruloplasmin
Clusterin
Coagulation factor XIIa HC
Complement C3
Complement C4
Complement C4
Complement C9

DADPDTFFAK
LVNEVTEFAK
NWGLSVYADKPETTK
EIGELYLPK
LETPDFQLFK
LGNQEPGGQTALK
LLIYAVLPTGDVIGDSAK
PKDPTFIPAPIQAK
DDLYVSDAFHK
ATEHLSTLSEK
SPELQAEAK
SLAPYAQDTQEK
FPEVDVLTK
TPDVSSALDK
DALSSVQESQVAQQAR
LGPLVEQGR
ATVVYQGER
EYTDASFTNR
LFDSDPITVTVPVEVSR
VVGGLVALR
TGLQEVEVK
ITQVLHFTK
VGDTLNLNLR
AIEDYINEFSVR

Quantitative Mass Spectrometric MRM Assays for Major Plasma Proteins, Leigh
Anderson and Christie Hunter, Mol Cell Proteomics, 5.4: 573-588 (2006).

Complement factor B
Complement factor H
Fibrinogen
Fibrinogen
Fibrinogen
Fibronectin
Gelsolin, isoform 1
Haptoglobin
Hemopexin
Heparin cofactor II
Histidine-rich glycoprotein
Inter--trypsin inhibitor HC
Kininogen
L-selectin
Plasma retinol-binding protein
Plasminogen
Prothrombin
Serum amyloid P
Transferrin
Transthyretin
Vitamin D-binding protein
Vitronectin
Zinc--2-glycoprotein

EELLPAQDIK
SPDVINGSPISQK
GSESGIFTNTK
QGFGNVATNTDGK
DTVQIHDITGK
DLQFVEVTDVK
TGAQELLR
VGYVSGWGR
NFPSPVDAAFR
TLEAQLTPR
DSPVLIDFFEDTER
AAISGENAGLVR
TVGSDTFYSFK
AEIEYLEK
YWGVASFLQK
LFLEPTR
ETAASLLQAGYK
VGEYSLYIGR
EDPQTFYYAVAVVK
AADDTWEPFASGK
THLPEVFLSK
FEDGVLDPDYPR
EIPAWVPFDPAAQITK

MRM Limits of Quantitation in Unfractionated

Plasma Digests Average 1g/ml Protein

A Multi-site Assessment of Precision and Reproducibility of Multiple Reaction Monitoring-based

Measurements by the NCI-CPTAC Network: Toward Quantitative Protein Biomarker Verification
in Human Plasma. Terri Addona, et al. Nature Biotechnology, 27(7):633-41

1.E+09

1.E+08

S erum album in
A polipoprotein A -I
S erotransferrin
Alpha-1-acid glycoprotein 1
Alpha-1-antitrypsin
H aptoglobin beta chain
Transthyretin
Fibrinogen gam m a chain
Fibrinogen alpha chain
Fibrinogen beta chain
Apolipoprotein C -III
A lpha-2 -m acroglobulin
Com plem ent C 3
Antithrom bin-III
C om plem ent factor H
C om plem ent factor B
C eruloplasm in
C om plem ent com ponent C 9
Plasm a pro tease C 1 inhibitor
C om plem ent C 1q subcom ponent, C chain
C om plem ent C 1q subcom ponent, B chain
C om plem ent C 1q subcom ponent, A chain
Prothrom bin
Plasm a retinol-binding protein
C om plem ent C 4 alpha cha in
C om plem ent C 4 gam m a chain
C om plem ent C 4 beta chain
A polipoprotein B -100
Alpha-2-antiplasm in
Plasm ino gen
A polipoprotein E
C om plem ent com ponent C8 gam m a
C om plem ent com ponent C8 alpha chain
Com plem ent com ponent C 8 beta chain
C om plem ent factor I
C om plem ent com ponent C 7
C om plem ent com ponent C 6
Thyroxine-binding globulin
C oagulation factor X IIa light chain
Coagulatio n factor X IIa heavy chain
C om plem ent C 5 alpha cha in
C om plem ent C 5 beta chain
Vitam in K-dependent protein S
A polipoprotein(a)
C oagulation factor X
Adiponectin
C oagulatio n factor XIa light chain
Com plem ent C 2
Beta-2-glycoprotein I
C oagulation facto r IX
C -reactive protein
A L-11
Vitam in K-dependent protein C
C oagulation factor X III A chain
C oagulation factor X III B chain
C om plem ent factor B B b fragm ent
C oagulation factor V
Insulin-like grow th factor IA
C 3a anaphylatoxin
A ngioten sinogen
L-selectin
Vascular cell adhesion protein 1
Factor V II heavy chain
Factor VII light chain
Intercellular adhesion m olecule-1
Fibronectin
Transform ing grow th facto r beta 1
A lpha-1-antichym otrypsin
Throm bospon din 1
Fibrinopeptide A
Platelet factor 4
Plasm inogen activato r inhibitor-1
Throm bom odulin
C oagulation factor VIII
C 5a anaphylatoxin
P-selectin
E-selectin
H eparin-binding grow th factor 2
Insulin-like grow th factor binding
Tissue-type plasm inogen activator chain
Th yroglobulin
Tum or necrosis factor ligand superfam ily
Leptin receptor
Prostate specific antigen
92 kD a type IV collagenase
Interleukin-1 receptor antagonist
Alpha-fetoprotein
Prostatic acid phosphatase
S om atotropin
Inhibin beta A chain
Vitam in K -dependent protein C heavy
V itam in K -dependent protein C light
C arcinoem bryonic antigen-related cell
A trial natriuretic factor
S m all inducible cytokine A2
G am m a-brain natriuretic peptide
M acrophage co lony stim ulating factor-1
Interleukin-8
S m all inducible cytokine A3
H epatocyte grow th factor alpha chain
Interleukin-18
G ranulocyte colony-stim ulating factor
Tissue factor
Interleukin-2
Interleukin-4
V ascu lar endoth elial grow th factor A
Interferon gam m a
R enin
Interleukin-1 beta
Interleukin-5
Tum or necrosis factor, soluble form
Interleukin-10
Interleukin-6
Interleukin-12 alpha chain
Interleu kin-12 beta chain

C o n cen tratio n in a m ol p er 1 0 0 u l
(lo g scale)

Normal Concentrations of 115 Proteins in Plasma

MRM Sensitivity vs
Plasma Proteome Dynamic Range
vs detectability in 2 LC-MS/MS MuDPIT experiments*

MRM alone in 10nl plasma

The human plasma proteome: History, character, and diagnostic prospects. Anderson, N.L.
and Anderson, N.G., Molecular and Cellular Proteomics, 1.11, 845-867 (2002)

2 MS sets
1 MS sets
0 MS sets

1.E+07

1.E+06

1.E+05

1.E+04

1.E+03

1.E+02

1.E+01

1.E+00

1.E-01

Protein

Strengths and Limitations of

MRM Assays
Peptide MRM assays offer
near-absolute structural specificity
true internal standardization
facile multiplexing without cross-talk
none of these available with ELISA

BUT are limited in sensitivity by

MS detection limits
amol of target peptide on column: currently ~100amol

Matrix effects
max amount of sample on column: currently ~1ug
peptide for nanoflow

Theoretical Detection Limits as a Function

of Sample Size and MS Sensitivity

Attomoles
detectable

Concent rat ion

det ect able for a
50kd prot ein
(pg/ m l)
0.1
1
10
100
1,000
10,000

1
5
50
500
5,000
50,000
500,000

5
1.0
10
100
1,000
10,000
100,000

Sample size (uL)

10
50
0.50
0.100
5.0
1.00
50
10.0
500
100
5,000
1,000
50,000
10,000

100
0.050
0.50
5.0
50
500
5,000

500
0.010
0.10
1.0
10
100
1,000

1,000
0.005
0.05
0.5
5
50
500

Assumes a 50kd target protein and perfect recovery throughout analytical

process
10ul sample volume selected as optimal digest size
At least 100x improvement in MS sensitivity needed to achieve 5pg/ml (best
immunoassay) performance; more if recovery is less than 100%

SISCAPA: Enrich Target Peptide

& Decrease Sample Complexity

~1ug total peptide on column

Anti-Peptide Antibodies As
Analyte-Specific Reagents
Anti-peptide antibody (APA)
Typically recognize 5-8 amino
acid linear epitope in a combining
site groove surrounded by
complementarity-determining
residues of Ig L and H chains
6-8 amino acid sequences are
typically unique in the human
proteome
An anti-peptide antibody has the
potential to select a single tryptic
peptide from the human proteome
The peptide immunogen is easy
to synthesize, and thus the
antibody is easy to make (e.g., in
rabbits)

VL

VH

8-mer peptide bound to antibody groove

N.K. Vyas et al (2003) PNAS 100:1502315028.

Anti-Peptide Antibodies Capture Specific Peptides from

Plasma Digests with Negligible Cross-talk
and Perform Similarly Alone or in Multiplex Panels
1.E+07

9.E+06

8.E+06

6.E+06

5.E+06

4.E+06

Heavy Peak Area

7.E+06

3.E+06

2.E+06

PCI
PC
I

Tf
R

TfR
OPN
FL
C

r2

12 Abs individually

AF
P

An body

He

Tg
1

FLC
CA
12
5

Tg
2

HE
4

LP
SB
P

0.E+00
Meso
LPSBP
HE4
Tg2
Tg1
CA125
Her2
Pep de
AFP

OP
N

12 Abs together

M
es

12

p
le
x

1.E+06

MRM measurements

SISCAPACapturefrom10ulPlasmaDigestExtendsAssay
Sensitivityto~1ng/ml(~1000xbetterthanMRMalone)
StandardAdditionCurvesRevealEndogenousPeptideinPlasmaDigests,
WhileHeavyPeptideCurvesShowNoEndogenousInterferingMaterial
Soluble Mesothelin

Forward and Reverse Curves in 10l

Plasma Digest

Standard addition curve (light peptide)

showing endogenous level of 14 ng/ml

Rabbit Monoclonal Antibodies with Half-offtimes > 30min

Provide Optimum Performance at 0.1-1.0ug Ab per Assay
PCI:Not Xlink:Reverse

TfR:Not Xlink:Reverse

PCI:Xlink:Reverse

10.00

L/H (fwd) or H/L (rev) Area Ratio

L/H (fwd) or H/L (rev) Area Ratio

TfR:Xlink:Reverse
TfR:Not Xlink:Forward
TfR:Xlink:Forward
1.00

0.10

0.01

0.00
0.001

0.010

0.100

1.000

10.000

10

PCI:Xlink:Forward
1

0.1

0.01

0.001
0.001

100.000

PCI:Not Xlink:Forward

0.010

Meso:Not Xlink:Reverse

AFP:Xlink:Reverse

Meso:Xlink:Reverse

AFP:Not Xlink:Forward
AFP:Xlink:Forward

0.1

0.01

0.001
0.010

1.000

AFP:Not Xlink:Reverse
L/H (fwd) or H/L (rev) Area Ratio

L/H (fwd) or H/L (rev) Area Ratio

10

0.100

0.100

1.000

10.000

Spiked Peptide (fmol)

10.000

100.000

Spiked Peptide (fmol)

Spiked Peptide (fmol)

100.000

10

Meso:Not Xlink:Forward
Meso:Xlink:Forward

0.1

0.01

0.001
0.010

0.100

1.000

10.000

100.000

Spiked Peptide (fmol)

Crosslinking of antibodies to magnetic beads has no effect on standard curves

Multiplexed SISCAPA-MRM assays achieve pg/ml

sensitivity from 1 ml plasma.
Limits* of detection and quantitation are in the low ng/ml for the multiplexed
assay performed on 10 L plasma.
Description
Peptide
Calumenin
SFDQLTPEESK
Disulfide isomerase
VEGFPTIYFAPSGDK
Fibulin-2
IGPAPFAGDTISLTITK
Hypoxia upregulated
LYQPEYQEVSTEEQR
Legumain
DYTGEDVTPENFLAVLR
L-plastin
YTLNILEDIGGGQK
Osteopontin
GDSLAYGLR
Plectin-8
AGTLSITEFADMLSGNAGGFR
Tumor Protein D52
LGISSLQEFK

Protein MW
37064
71973
131818
111209
49373
65000
60000
517328
20059

ng/mL
0.9
2.1
3.9
5.9
3.1
1.1
0.6
101.1
7.5

LOD
fmol
0.3
0.3
0.3
0.5
0.6
0.2
0.1
2.0
3.7

pM
25
29
29
53
64
16
10
196
374

ng/mL
2.4
3.5
9.0
14.8
7.4
2.8
1.6
269.3
20.8

LOQ
fmol
0.7
0.5
0.7
1.3
1.5
0.4
0.3
5.2
10.4

pM
66
48
69
133
150
43
27
520
1038

Limits* of detection and quantitation are in the low pg/ml for the multiplexed
assay performed on 1 mL plasma.
Description
Peptide
Protein MW
Calumenin
SFDQLTPEESK
37064
Disulfide isomerase
VEGFPTIYFAPSGDK
71973
Fibulin-2
IGPAPFAGDTISLTITK
131818
Hypoxia upregulated
LYQPEYQEVSTEEQR
111209
Legumain
DYTGEDVTPENFLAVLR
49373
L-plastin
YTLNILEDIGGGQK
65000
Osteopontin
GDSLAYGLR
60000
Plectin-8
AGTLSITEFADMLSGNAGGFR
517328
Tumor Protein D52
LGISSLQEFK
20059

Jeffrey R. Whiteaker, et al, Mol. Cell. Proteomics 9:184-196 (2010)

pg/mL
22.2
31.5
43.7
18.7
8.6
11.5
3.6
1026
479

LOD
fmol
0.6
0.4
0.3
0.2
0.2
0.2
0.1
2.0
24

pM
60
44
33
17
17
18
6
198
2388

pg/mL
47.6
66.4
116.8
44.3
22.2
24.6
7.2
1973
1186

LOQ
fmol
1.3
0.9
0.9
0.4
0.5
0.4
0.1
3.8
59

pM
128
92
89
40
45
38
12
381
5912

*does not account for losses from trypsin digestion.

1.E+09

1.E+08

1.E+05

1.E+04

1.E+00

S erum album in
A polipoprotein A -I
S erotransferrin
Alpha-1-acid glycoprotein 1
Alpha-1-antitrypsin
H aptoglobin beta chain
Transthyretin
Fibrinogen gam m a chain
Fibrinogen alpha chain
Fibrinogen beta chain
Apolipoprotein C -III
A lpha-2 -m acroglobulin
Com plem ent C 3
Antithrom bin-III
C om plem ent factor H
C om plem ent factor B
C eruloplasm in
C om plem ent com ponent C 9
Plasm a pro tease C 1 inhibitor
C om plem ent C 1q subcom ponent, C chain
C om plem ent C 1q subcom ponent, B chain
C om plem ent C 1q subcom ponent, A chain
Prothrom bin
Plasm a retinol-binding protein
C om plem ent C 4 alpha cha in
C om plem ent C 4 gam m a chain
C om plem ent C 4 beta chain
A polipoprotein B -100
Alpha-2-antiplasm in
Plasm ino gen
A polipoprotein E
C om plem ent com ponent C8 gam m a
C om plem ent com ponent C8 alpha chain
Com plem ent com ponent C 8 beta chain
C om plem ent factor I
C om plem ent com ponent C 7
C om plem ent com ponent C 6
Thyroxine-binding globulin
C oagulation factor X IIa light chain
Coagulatio n factor X IIa heavy chain
C om plem ent C 5 alpha cha in
C om plem ent C 5 beta chain
Vitam in K-dependent protein S
A polipoprotein(a)
C oagulation factor X
Adiponectin
C oagulatio n factor XIa light chain
Com plem ent C 2
Beta-2-glycoprotein I
C oagulation facto r IX
C -reactive protein
A L-11
Vitam in K-dependent protein C
C oagulation factor X III A chain
C oagulation factor X III B chain
C om plem ent factor B B b fragm ent
C oagulation factor V
Insulin-like grow th factor IA
C 3a anaphylatoxin
A ngioten sinogen
L-selectin
Vascular cell adhesion protein 1
Factor V II heavy chain
Factor VII light chain
Intercellular adhesion m olecule-1
Fibronectin
Transform ing grow th facto r beta 1
A lpha-1-antichym otrypsin
Throm bospon din 1
Fibrinopeptide A
Platelet factor 4
Plasm inogen activato r inhibitor-1
Throm bom odulin
C oagulation factor VIII
C 5a anaphylatoxin
P-selectin
E-selectin
H eparin-binding grow th factor 2
Insulin-like grow th factor binding
Tissue-type plasm inogen activator chain
Th yroglobulin
Tum or necrosis factor ligand superfam ily
Leptin receptor
Prostate specific antigen
92 kD a type IV collagenase
Interleukin-1 receptor antagonist
Alpha-fetoprotein
Prostatic acid phosphatase
S om atotropin
Inhibin beta A chain
Vitam in K -dependent protein C heavy
V itam in K -dependent protein C light
C arcinoem bryonic antigen-related cell
A trial natriuretic factor
S m all inducible cytokine A2
G am m a-brain natriuretic peptide
M acrophage co lony stim ulating factor-1
Interleukin-8
S m all inducible cytokine A3
H epatocyte grow th factor alpha chain
Interleukin-18
G ranulocyte colony-stim ulating factor
Tissue factor
Interleukin-2
Interleukin-4
V ascu lar endoth elial grow th factor A
Interferon gam m a
R enin
Interleukin-1 beta
Interleukin-5
Tum or necrosis factor, soluble form
Interleukin-10
Interleukin-6
Interleukin-12 alpha chain
Interleu kin-12 beta chain

C o n cen tratio n in a m ol p er 1 0 0 u l
(lo g scale)

Extending
MS Access
tointhe
Normal Concentrations
of 115 Proteins
PlasmaFull
Plasma Proteome Dynamic Range
vs detectability in 2 LC-MS/MS MuDPIT experiments*

MRM alone in 10nl plasma

2 MS sets
1 MS sets
0 MS sets

1.E+07

1.E+06

MRM-SISCAPA in 10-100ul plasma

1.E+03

1.E+02

1.E+01

MRM-SISCAPA with larger samples

1.E-01

Protein

Magnetic Bead Implementations

of SISCAPA Assay Technology
Biomarker concentration
Labeled standard
Sample peptide

MRM Chromatogram

Agilent Bravo
liquid handler

Unifying SISCAPA Sample

Preparation on a Single Platform
Combine pipetting, mixing, and magnetic
bead manipulation (washing, elution) on a
single device
Improved control of volume accuracy and
timing
Standardized protocol independent of
analytes selected (Abs & heavy peptides)
Small footprint, manageable cost (<$1/sample
for plastic consumables)

Additional SISCAPA Implementations

Abs on magnetic beads
eluted in LC capillary

A magnetic bead trap replacing a standard 5ul

injection loop allows processing of magnetic
beads on a Spark Holland autosampler
Eksigent 2-D nanochromatography system used
for solvent delivery
N. Leigh Anderson, et al, Mol Cell Proteomics
8:995-1005 (2009)

Abs on POROS
support in LC system

Antibodies immobilized on POROS resin and recycled

>1,000 times
The final assay had <15% interassay relative error
and <15% interassay CV across a range of 4.082980
pmol/L human pepsinogen (0.165120 g/L).
Hendrik Neubert et al (Pfizer) Clinical Chemistry
56:9,14131423 (2010)

Can We Have Access to the Entire Baseline

Human Proteome with Specific Assays?

A Human Proteome Detection and Quantitation Project: hPDQ. N. Leigh Anderson, Norman G. Anderson, Terry W. Pearson,
Christoph H. Borchers, Amanda G. Paulovich, Scott D. Patterson , Ruedi Aebersold and Steven A. Carr, Mol Cell Proteomics 8:883886 (2009)

Acknowledgments

US patent 7,632,686, related applications covering SISCAPA technology, and

the SISCAPA trademark, belong to Anderson Forschung Group (AFG)

SISCAPA Publications

High sensitivity quantitation of peptides by mass spectrometry. Anderson, Norman L., United States Patent 7,632,686. The initial SISCAPA claims.

Mass Spectrometric Quantitation of Peptides and Proteins Using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA).
Anderson, N.L., Anderson, N.G., Haines, L.R., Hardie, D.B., Olafson. R.W., and Pearson, T.W. Journal of Proteome Research, 3: 235-44 (2004). Initial proof of
concept results with SISCAPA using 4 peptides & Abs.

An effective and rapid method for functional characterization of immunoadsorbents using POROS beads and flow cytometry. Anderson, N.L. ,
Haines, L.R. and Pearson, T.W. Journal of Proteome Research 3:228-34 (2004). Methods for characterizing SISCAPA immobilized Abs.

The Roles of Multiple Proteomics Platforms in a Pipeline for New Diagnostics. N. Leigh Anderson, Mol Cell Proteomics, 4: 1441 - 1444 (2005). Role of
SISCAPA assays in the Dx pipeline.

Quantitative Mass Spectrometric MRM Assays for Major Plasma Proteins. Leigh Anderson and Christie Hunter, Mol Cell Proteomics, 5: 573-588 (2006).
Capabilities of QqQMS as the MS measurement platform for use in quantitating peptides/proteins in plasma digests: the SISCAPA detector.

Antibody-based enrichment of peptides on magnetic beads for mass-spectrometry-based quantification of serum biomarkers. J. R. Whiteaker, L.
Zhao, H. Y. Zhang, L-C Feng, B. D. Piening, L. Anderson and A. G. Paulovich, Analytical Biochemistry 362:4454 (2007). Implementation of SISCAPA on
magnetic beads.

Protein Quantitation through Targeted Mass Spectrometry: The Way Out of Biomarker Purgatory? Steven A. Carr and Leigh Anderson, Clinical Chemistry
54:11, 17491752 (2008)

Anti-peptide antibody screening: selection of high affinity monoclonal reagents by a refined surface plasmon resonance technique. Matthew E. Pope,
Martin V. Soste, Brett A. Eyford, N. Leigh Anderson and Terry W. Pearson,, J Immunol Methods, 341(1-2):86-96 (2009)

A Human Proteome Detection and Quantitation Project: hPDQ. N. Leigh Anderson, Norman G. Anderson, Terry W. Pearson, Christoph H. Borchers,
Amanda G. Paulovich, Scott D. Patterson , Ruedi Aebersold and Steven A. Carr, Mol Cell Proteomics 8:883-886 (2009)

SISCAPA Peptide Enrichment on Magnetic Beads Using an Inline Beadtrap Device. N. Leigh Anderson, Angela Jackson, Derek Smith, Darryl Hardie,
Christoph Borchers, and Terry W. Pearson, Mol Cell Proteomics 8:995-1005 (2009)

MRM-based, Multiplexed, Absolute Quantitation of 45 Proteins in Human Plasma Michael A. Kuzyk, Derek Smith, Juncong Yang, Tyra Cross, Angela
Jackson, Darryl Hardie, N. Leigh Anderson, and Christoph H. Borchers, Molec. Cell. Proteomics, 8(8):1860-77 (2009)

A Multi-site Assessment of Precision and Reproducibility of Multiple Reaction Monitoring-based Measurements by the NCI-CPTAC Network: Toward
Quantitative Protein Biomarker Verification in Human Plasma. Terri Addona et al. Nature Biotechnology, 27(7):633-41 (2009)

An automated and multiplexed method for high throughput peptide immunoaffinity enrichment and multiple reaction monitoring mass spectrometrybased quantification of protein biomarkers. Jeffrey R. Whiteaker, Lei Zhao, Leigh Anderson, and Amanda G. Paulovich, Mol. Cell. Proteomics 9:184-196
(2010)

Proteomic-Based Multiplex Assay Mock Submissions: Supplementary Material to A Workshop Report by the NCI-FDA Interagency Oncology Task
Force on Molecular Diagnostics, Fred E. Regnier, et al, Clin Chem. 56:2 165171 (2010)

MALDI Immunoscreening (MiSCREEN): A Method for Selection of Anti-peptide Monoclonal Antibodies For Use in Immunoproteomics, Morteza Razavi,
Matthew E. Pope, Martin V. Soste, Brett A. Eyford, N. Leigh Anderson and Terry W. Pearson, in press

Simplifying Targeted Protein

Quantification with Mass Spec:
The SRM Atlas and Multiplexed
MRM Protein Assays

Christine Miller
Senior Application Scientist
Agilent Technologies

Simplifying Targeted
Protein Quantification
with Mass Spec

Improvements in
Instrumentation and
Workflows

Christine Miller
Senior Application Scientist
December 2010

Overview
Improvements in instrumentation
Impact of flow rate and column i.d. on sensitivity
Robustness of analysis with complex samples
Workflow enhancements

Improvements in Instrumentation
Sensitivity is a key requirement for many peptide quantitation
applications such as protein biomarker in biofluids
Improving LC/MS sensitivity can be achieved by enhancing the
sampling and transmission of ions in the mass spectrometer
ESI with thermal gradient focusing
Ion sampling inlet with a multi-bore ion sampling capillary coupled to a dual
ion funnel for improved ion transfer

QQQ with iFunnel Technology

Jet Stream Source
Thermal confinement of
ESI ion plume
Efficient desolvation to
create gas phase ions

Hexabore Capillary

Dual Ion Funnel

6 capillary inlets

Removes the gas but

captures the ions

Samples 12X more ion

rich gas from the source

Helps to remove source

generated noise

High
Pressure
Funnel

Low
Pressure
Funnel
ASMS 2010

JetStream Source vs. ESI at Capillary Flow Rates

(17 L/min)
10 fmol on-column
x10 3

New
AJS

Nebulizer

Heated
Sheath
Gas
Thermal
Gradient
Focusing
Region

ESI

MS Inlet

.5

.5
7
.5
8
.5
Counts vs. Acquisition Time (min)

.5

Impact of Ion Funnel on Sensitivity: Standard Flow

LC/MS with JetStream Source
Calibration Curves

QQQ (6460)
1 fmol on-column
1 fmol 25 pmol

QQQ with ion funnel

100 amol on-column
100 amol 25 pmol

HSA Standard Peptide (LVNEVTEFAK, 575.5 937.5), Poroshell 120 2.1 x 150 mm column at 0.5 mL/min

Impact of Flow Rate/Column ID on Sensitivity

LC system

Standard
Flow

Capillary
Flow

Nano Flow

1290

1200 capLC

1200
cap/nanoLC

LC/MS interface Agilent Jet

New AJS with HPLC-Chip
Stream (AJS) micronebulizer
Column
dimensions
(Zorbax 300SBC18)

2.1 x 150 mm 0.5 x 150 mm

75 m x 43
mm

Flow rate

300 L/min

600 nL/min

Sample

Agilent Peptide Standard (G2455-85001) using

3 MRM transitions for LVNEVTEFAK
(575.5937.5, 575.5694.4, 575.5823.4)

17 L/min

2.1 vs. 0.5 mm ID Columns: JetStream Source

Shows Mass Dependent Behavior
1 fmol on-column
x10 2
1

575.5937.5

0.5 mm
column
2.1 mm column
300 L/min
100 amol 10 pmol

2.1 mm
column

0.5 mm column
17 L/min
100 amol 1 pmol
1

3
4
5
6
7
Counts vs. Acquisition Time (min)

Comparison of Standard (0.3 mL/min), Capillary (17

L/min) and Nanospray (600 nL/min)
100 amol on-column
x10 2

75 m
column

1 fmol on-column

575.5937.5

75 m
column

x10 3

2.1 mm
0.5 mm
column
column

2.1 mm
0.5 mm
column
column
1

3
4
5
6
7
8
9
Counts vs. Acquisition Time (min)

575.5937.5

3 4 5 6
7 8 9
0
Counts vs. Acquisition Time (min)

Standard Flow LC/MS: LVNEVTEFAK Spiked into

Serotransferrin Digest (20 amol on-column)
Quantifier

Qualifiers

Target
Matrix

SNR 602:1

Target
Matrix

Standard Flow LC/MS: Log/Log Plot of Calibration Curve

for LVNEVTEFAK Spiked into Serotransferrin Digest
1% RSD

Curve from 20 amol to 5 pmol on-column

2.1 x 50 mm column at 0.3 mL/min
N=5

6.3% RSD

2% RSD
3.5% RSD

14.7% RSD
5.7% RSD
5.3% RSD

3.5% RSD

12% RSD

Nanoflow LC/MS: LVNEVTEFAK Spiked into

Serotransferrin Digest
x10 2 +ESI MRM Frag=380.0V CID@20.0 (575.3000 -> 937.5000) 5_amolHSA-r003.d
2.668
2014

Matrix peak

5 amol

Target peak
2.905
557

+ESI MRM Frag=380.0V CID@20.0 (575.3000 -> 937.5000) 2_amolHSA-r003.d

x10 2 Noise (RMS) = 0.09; SNR (2.905min) = 1442.0

2 amol
* 2.602
0

2.905
427

x10 2 +ESI MRM Frag=380.0V CID@20.0 (575.3000 -> 937.5000) blank2-r003.d

blank
2.911
335

.2

.4

.6

.8

.2

.4

.6

.8

2
.2
.4
.6
.8
3
.2
Counts vs. Acquisition Time (min)

.4

.6

.8

.2

.4

.6

.8

Nanoflow LC/MS: Log/Log Plot of Calibration Curve for

LVNEVTEFAK Spiked into Serotransferrin Digest
1.3% RSD

1.3% RSD

Curve from 2 amol to 500 fmol on-column

75 m x 43 mm column at 0.6 L/min
N=5
4.2% RSD

3.5% RSD
4.6% RSD

2.5% RSD
3.8% RSD
4.1% RSD

2.8% RSD

Robustness of LC/MS System

Goal: Demonstrate the performance of the instrumentation for
the analysis of peptides in a complex matrix
Instrumentation: 1290 Infinity LC system with 6490 QQQ
Sample: Stable isotope-labeled standard (SIS) peptides (12
total) in undepleted plasma digest

These samples were kindly provided by Derek Smith and Christoph H. Borchers from the UVic-Genome BC Proteomics Centre
6490QQQ_Peptides.ppt Agilent restricted
12/8/2010

Injecting Less Sample to Reduce Matrix Effects

Plasminogen: LFLEPTR
25 g
2.5 g

von Willebrand Factor: ILAGPAGDSNVVK

25 g
2.5 g

x10 2 +ESI MRM Frag=380.0V CID@14.0 (4 x10 2 +ESI MRM Frag=380.0V CID@14.0 (4
* 10.634

Heavy

* 10.709

Heavy

x10 2 +ESI MRM Frag=380.0V CID@20.0 (4 x10 2 +ESI MRM Frag=380.0V CID@20.0 (4
* 10.578

* 10.727

Heavy

Heavy

x10 3 +ESI MRM Frag=380.0V CID@10.0 (4 x10 2 +ESI MRM Frag=380.0V CID@10.0 (4
* 10.720

Heavy

Heavy

x10 5 +ESI MRM Frag=380.0V CID@14.0 (4 x10 4 +ESI MRM Frag=380.0V CID@14.0 (4
* 10.721

* 10.638

Light
0
0.5
1
.5
Counts vs. Acquisition Time (min)

Light
0
0.5
1
.5
Counts vs. Acquisition Time (min)

These samples were kindly provided by Derek Smith and Christoph H. Borchers from the UVic-Genome BC Proteomics Centre

Standard Flow LC/MS: Plasminogen Peptide in 2.5

g of Plasma Digest Matrix
50 amol on-column

250 amol on-column

10 fmol on-column

These samples were kindly provided by Derek Smith and Christoph H. Borchers from the UVic-Genome BC Proteomics Centre
Confidentiality Label
December 8, 2010

Standard Flow LC/MS: Quantitation of the

Plasminogen Peptide in Plasma
Linear plot

Log-Log plot

Calibration from
5 10000 amol/L
in 250 ng/uL plasma
digest (n=3)

ESTD Calibration standards (amol/uL)

441.3>621.4
5

10

25

50

75

100

500

750

1000

5000

10000

%Accuracy (average, n =3)

98.2

97.5

104.6

98.1

99.2

97.4

101.3

101.8

101.3

101.8

98.8

Reproducibility (%RSD, n = 3)

11.85

10.78

7.21

1.17

6.22

2.74

1.41

0.48

1.52

2.58

3.24

Response factor

8.33

8.28

8.87

8.32

8.42

8.27

8.60

8.64

8.60

8.64

8.39

Precision (%RSD, n = 11)

2.30

The samples were provided by Derek Smith and Christoph H. Borchers from the UVic-Genome BC Proteomics Centre

Reproducibility for 110 Injections (10 fmol SIS

Peptides and 2.5 g Plasma Digest On-column)
Plasminogen_LFL_h - 6 Levels, 6 Levels Used, 15 Points, 15 Points Used, 110 QCs
y = 3.602471E-004 * x - 4.854952E-004
R^2 = 0.99909469

Plasminogen LFLEPTR
2.2% RSD
n=110

4.7% RSD, n=4

7.9% RSD, n=4

12.3% RSD, n=4
5

00

00

00
00
000
Relative Concentration

The samples were provided by Derek Smith and Christoph H. Borchers from the UVic-Genome BC Proteomics Centre

MRM-triggered MRMs for Confirmation +

Quantitation

Secondary MRMs Only Acquired If Primary MRMs

Exceed User Specified Threshold
x10 4 +ESI TIC MRM Frag=120.0V CID@20.0 (** -> **) HSA-tDMRM-1pep-0

x10 3 +ESI MRM:1 (3.516-3.607 min, 20 scans) Frag=120.0V CID@20.0 (575.3000 -> 937.5000) HSA-tDMRM-1pep
937.5000

Primary
x10 4 +ESI MRM Frag=120.0V CID@20.0 (575.3000 -> 937.5000) HSA-tDM

Composite Spectrum
from all MRMs

Primary
x10 3 +ESI MRM Frag=120.0V CID@20.0 (575.3000 -> 694.4000) HSA-tDM

Primary
x10 3 +ESI MRM Frag=120.0V CID@20.0 (575.3000 -> 823.4000) HSA-tDM

Secondary
x10 2 +ESI MRM Frag=120.0V CID@20.0 (575.3000 -> 469.2000) HSA-tDM

Secondary

694.4000
595.3000

x10 3 +ESI MRM Frag=120.0V CID@20.0 (575.3000 -> 595.3000) HSA-tDM

Secondary
x10 2 +ESI MRM Frag=120.0V CID@20.0 (575.3000 -> 1036.5000) HSA-tD

Secondary

823.4000

1036.5000

469.2000

.5

.5
2
.5
3
.5
Counts vs. Acquisition Time (min)

.5

50

00

50

00

50

00
50
00
50
Counts vs. Mass-to-Charge (m/z)

00

50

00

50

Interfacing to the MRM Atlas

Conclusions
The ion funnel system shows at least a 5x improvement in sensitivity
compared to a standard QQQ
ESI with thermal gradient focusing shows mass dependent behavior
for capillary to standard LC flow rates
Nanoflow is still significantly more sensitive than cap or standard flow.
The QQQ + iFunnel system demonstrated robust performance for
peptide quantitation in plasma digests using standard flow LC/MS
Data dependent MRM acquisition offers the possibility of achieving
confirmation plus quantitation in a single analysis
Interfacing acquisition software to the MRM Atlas will facilitate the use
of this public resource

Acknowledgements

Agilent

UVic-Genome BC Proteomics
Centre

Yanan Yang

Derek Smith

Paul Momoh

Christoph H. Borchers

Anabel Fandino
Paul Goodley
Craig Love
Alex Mordehai

Acknowledgements
KenMiller
ChristineMiller
UlrikeKusebauch
EricDeutsch
DavidCampbell
MiYounBrusniak
DouglasSpicer
CarolineChu
JeffreyStevens
Zhi Sun
JoeSlagel

RuediAebersold
ChristineCarapito
PaolaPicotti

LeroyHood

ARRA1RC2HG00580501

Duchyof
Luxembourg

www.systemsbiology.org

Simplifying Targeted Protein

Quantification with Mass Spec:
The SRM Atlas and Multiplexed
MRM Protein Assays

Your Moderator

Tamlyn Oliver
Managing Editor
Genetic Engineering & Biotechnology News

Simplifying Targeted Protein

Quantification with Mass Spec:
The SRM Atlas and Multiplexed
MRM Protein Assays

Simplifying Targeted Protein Quantification with Mass Spec:

The SRM Atlas and Multiplexed MRM Protein Assays

Simplifying Targeted Protein

Quantification with Mass Spec:
The SRM Atlas and Multiplexed
MRM Protein Assays

Robert L. Moritz, Ph.D.

Associate Professor and Director of Proteomics
Institute for Systems Biology

Simplifying Targeted Protein

Quantification with Mass Spec:
The SRM Atlas and Multiplexed
MRM Protein Assays

Leigh Anderson, Ph.D.

Founder and CEO
Plasma Proteome Institute

Simplifying Targeted Protein

Quantification with Mass Spec:
The SRM Atlas and Multiplexed
MRM Protein Assays

Christine Miller
Senior Application Scientist
Agilent Technologies

Simplifying Targeted Protein

Quantification with Mass Spec:
The SRM Atlas and Multiplexed
MRM Protein Assays

Thank You For Attending

Simplifying Targeted Protein Quantification with

Mass Spec: The SRM Atlas and Multiplexed MRM
Protein Assays
Broadcast Date: Thursday, December 16, 2010
Time: 1:00 PM EDT
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