10
1 Absolute Quantification of Yeast Kinases by LC/MS/MS using QconCAT and MRM Technologies Philip J Brownridge, 1 Victoria Harman, 1 Simon Cubbon, 2 Johannes PC Vissers, 2 Craig Lawless, 3 Simon J Hubbard, 3 Robert J Beynon 1 1 Protein Function Group, Institute of Integrative Biology, University of Liverpool, United Kingdom 2 Waters Corporation, Manchester, United Kingdom 3 Faculty of Life Sciences, University of Manchester, United Kingdom INTRODUCTION Absolute protein quantification by LC/MS/MS is an important tool in assay development and creating data for systems modeling. Enabling predictive biology is one of the primary goals of many system biology studies, achieving detailed knowledge of the cellular constituents, their quantities, dynamics, and interactions. This information can be subsequently embedded in mathematical models that permit simulation of cellular state changes, testable by experiment, and leading to biological process definitions. 1 However, this requires accurate baseline values for the cellular quantities of proteins. The large dynamic range of a proteome is the most challenging barrier to LC/MS/MS-based protein quantification. This application note presents the application of label-mediated targeted mass spectrometry to quantify a range of kinases from yeast, spanning a five-order dynamic range. QconCAT 2 technology was used to create isotopically-labeled internal standard peptides for 138 target proteins and quantification was performed by time-scheduled MRM tandem quadrupole mass spectrometry, investigating the sensitivity of different platforms, assay specificity, and quantitation dynamic range. WATERS SOLUTIONS nanoACQUITY UPLC ® System Symmetry ® Column ACQUITY UPLC ® BEH Column Xevo ® TQ MS Xevo TQ-S MS MassLynx ® Software TargetLynx™ Application Manager KEY WORDS Yeast kinases, absolute quantitation, QconCAT, nanoscale UPLC, MRM quantification, protein abundance APPLICATION BENEFITS Nanoscale separations are combined with quantitative MRM mass spectrometry to accurately determine absolute yeast protein amounts over a wide dynamic range using isotopically labelled standards. More peptides and proteins are more easily quantified and data analysis is more straightforward using elevated analyzer resolution settings and a high sensitivity triple quadrupole mass spectrometer.

Absolute Quantification of Yeast Kinases by LC/MS/MS … · Absolu tificatio Yeas Kna LC/MSMS si Ca MRM Technologies 2 ... BPI: 3.3e5 AVIESENSAER ... Absolute Quantification of Yeast

  • Upload
    vohuong

  • View
    223

  • Download
    0

Embed Size (px)

Citation preview

1

Absolute Quantification of Yeast Kinases by LC/MS/MS using QconCAT and MRM TechnologiesPhilip J Brownridge,1 Victoria Harman,1 Simon Cubbon,2 Johannes PC Vissers,2 Craig Lawless,3 Simon J Hubbard,3 Robert J Beynon1

1 Protein Function Group, Institute of Integrative Biology, University of Liverpool, United Kingdom 2 Waters Corporation, Manchester, United Kingdom 3 Faculty of Life Sciences, University of Manchester, United Kingdom

IN T RO DU C T IO N

Absolute protein quantification by LC/MS/MS is an important tool in assay

development and creating data for systems modeling. Enabling predictive

biology is one of the primary goals of many system biology studies, achieving

detailed knowledge of the cellular constituents, their quantities, dynamics, and

interactions. This information can be subsequently embedded in mathematical

models that permit simulation of cellular state changes, testable by experiment,

and leading to biological process definitions.1 However, this requires accurate

baseline values for the cellular quantities of proteins. The large dynamic

range of a proteome is the most challenging barrier to LC/MS/MS-based

protein quantification.

This application note presents the application of label-mediated targeted mass

spectrometry to quantify a range of kinases from yeast, spanning a five-order

dynamic range. QconCAT2 technology was used to create isotopically-labeled

internal standard peptides for 138 target proteins and quantification was

performed by time-scheduled MRM tandem quadrupole mass spectrometry,

investigating the sensitivity of different platforms, assay specificity, and

quantitation dynamic range.

WAT E R S SO LU T IO NS

nanoACQUITY UPLC® System

Symmetry® Column

ACQUITY UPLC® BEH Column

Xevo® TQ MS

Xevo TQ-S MS

MassLynx® Software

TargetLynx™ Application Manager

K E Y W O R D S

Yeast kinases, absolute quantitation,

QconCAT, nanoscale UPLC, MRM

quantification, protein abundance

A P P L I C AT IO N B E N E F I T S

Nanoscale separations are combined with

quantitative MRM mass spectrometry to

accurately determine absolute yeast protein

amounts over a wide dynamic range using

isotopically labelled standards. More peptides

and proteins are more easily quantified and

data analysis is more straightforward using

elevated analyzer resolution settings and

a high sensitivity triple quadrupole mass

spectrometer.

2Absolute Quantification of Yeast Kinases by LC/MS/MS using QconCat and MRM Technologies

E X P E R IM E N TA L

Sample preparation

Several yeast kinase QconCATs were designed

to contain two isotopically labelled peptides

for each of the targeted proteins and tryptically

codigested with a native yeast strain as shown

in Figures 1 and 2, respectively. Figure 1

illustrates the QconCAT principle whereby

a synthetic gene is designed to encode

proteotypic peptides of the sample protein

mixture. Summarized in Figure 2 are the

design of a quantitation concatamer and the

high-throughput MRM quantitation workflow.

Yeast kinases, as shown in Figure 3, span the

complete yeast abundance distribution range in

terms of number of copies/cell.

LC/MS/MS conditions

Time-scheduled MRM experiments were

conducted with a nanoACQUITY UPLC System

interfaced to either a Xevo TQ or a Xevo TQ-S3

tandem quadrupole mass spectrometer.

SNVLVVGPSGSGK CopyCAT

(1fmol) Area=836

Yeast 200,000

cells Area=271

QConCAT

•Xevo •Scheduled SRM

digestion

Sample

MxxR* S1 R* Q1 R* Q2 R* Q3 R* Q4 R* Q5 R* S2 R* HisTag

Short sacrificial peptide to protect S1 and provide initiator Met

Quantotypic peptides

Affinity tag for purification

n~50

N-terminal (common) Standard peptide

C-terminal (common) Standard peptide

Time (min)

Figure 1. The principle of QconCAT technology.

A 60-min reversed phase gradient from 3 to

40% acetonitrile (0.1% formic acid) was

employed using a vented trap-configuration

comprising a 5-μm Symmetry C18 2 cm x 180 μm

trap column and a 1.7-μm ACQUITY UPLC HSS T3

C18 15 cm x 75 μm analytical column. Quadrupole

resolution settings of 0.7 Da and 0.4 Da FWHM

were employed, balancing the sensitivity of the

two mass spectrometers, respectively. Elevated

resolution settings for Xevo TQ-S were considered

to investigate MRM assay specificity.

Informatics

The quantitative LC/MS peptide data were

processed and quantified with the TargetLynx

Application Manager for MassLynx Software

version 4.1, Skyline4 version 1.44 (MacCoss Lab,

University of Washington, Seattle, Washington,

U.S.), and/or mProphet (Biognosys AG,

Schlieren, Switzerland).5

3Absolute Quantification of Yeast Kinases by LC/MS/MS using QconCat and MRM Technologies

158 Target Proteins >KAD2_YEAST Adenylate kinase 2 MKADAKQITHLLKPLRLLLLGAPGSGKGTQTSRLLKQIPQLSSISSGDILRQEIKSESTLGREATTYIAQGKLLPDDLITRLITFRLSALGWLKPSAMWLLDGFPRTTAQASALDELLKQHDASLNLVVELDVPESTILERIENRYVHVPSGRVYNLQYNPPKVPGLDDITGEPLTKRLDDTAEVFKKRLEEYKKTNEPLKDYYKKSGIFGTVSGETSDIIFRNY >SMK1_YEAST Sporulation-specific mitogen-activated protein kinase MNCTLTDNTRAINVASNLGAPQQRTIFAKERISIPGYYEIIQFLGKGAYGTVCSVKFKGRSPAARIAVKKISNIFNKEILL............

>KAD2_YEAST Adenylate kinase 2 LLLLGAPGSGK TTAQASALDELLK >SMK1_YEAST Sporulation-specific...... AINVASNLGAPQQR ................

HARD rules 1. Sequence unique 2. Between 700-3000 Da 3. No known PTM 4. No M, NP, DG, N-term Q 5. No dibasic context

Predicting behavior

http://king.smith.man.ac.uk/CONSeQuence/ http://king.smith.man.ac.uk/mcpred/

8 CopyCATSCC54, CC55, CC56, CC57, CC58, CC59, CC60 & CC61

Digestion

Ionization

CopyCAT expressionCC CC CC CC CC CC CC54 56 59 61 55 58 60

Figure 2. Design of a quantitation concatamer (QconCAT) and high-throughput MRM quantitation workflow.

100,000 Broken yeast

cells / L

RapiGest trypsin digestion protocol

Discovery mass spectrometry

SYNAPT G2 Spiked with

100 fmol/ L GluFib, 50 fmol PhosB

Quantification mass spectrometry

Xevo TQ

xorppA100 fmol / L

CopyCAT

1D-SDS PAGE digestion check

Skyline to make spectral library and

first transitions

Theoretical transitions to fill list

Validate defined

transitions

Realign retention

times

Choose transitions with best s/n

Choose good transitions

Optimise collision energy and Cone V

PLGS for database

searching and label-free

quant

Final transition list 3 transitions/peptide

Serially dilute samples to 0.2, 1, 10 fmol/ L

CopyCAT

GluFib ratio to quant CopyCAT

Final Quantification on all 4 bioreplicates in order: yeast blank, 0.2, 1 and 10 fmol/ L

CopyCAT

Tuesday Wednesday Friday

Thursday Monday

4Absolute Quantification of Yeast Kinases by LC/MS/MS using QconCat and MRM Technologies

Figure 3. Yeast kinases span the complete abundance distribution range.

R E SU LT S A N D D IS C U S S IO N

The design and quantification workflow of yeast kinases using QconCAT technology in high-throughput MRM

mode is summarized in Figure 2. The top pane illustrates the design of a QconCAT internal standard protein

and the bottom pane the workflow. The workflow comprised: i) digestion, ii) digestion performance check, iii)

discovery LC/MS plus database searching, iv) creation spectral library, v) preliminary MRM quantitation, vi)

transition validation, vii) experiment optimization, viii) serial dilutions, and ix) final quantitation.

5Absolute Quantification of Yeast Kinases by LC/MS/MS using QconCat and MRM Technologies

Figure 4. Peptide detection/quantification type (top panel), quantification challenges (middle panel) and quantified proteins with nominal quadrupole resolution settings (Xevo TQ) (bottom panel).

TYPE A

Copycat and native peptideboth observed

DIRECT QUANTIFICATION

TYPE B

CopyCAT observed, nativepeptide undetected

INFERRED MAXIMUM

TYPE C

CopyCAT and native peptide undetected at biologically

relevant levels

Peptides were qualified as either type A, B, or C, as

illustrated in the top panel of Figure 4. A Type A

identification infers unambiguous observation of

both the QconCAT and native peptide. In the instance

of Type B identifications, only the QconCAT peptide

is observed and for Type C identifications both the

QconCAT and native peptide were both not detected.

The correlation between the selected peptides for

Xevo TQ quantitation is illustrated in the middle

pane of Figure 4. The majority of the peptides show

good quantitative agreement; however, due to either

low signal or missed tryptic cleavages, certain

peptides cannot be used for quantitation, which are

highlighted with blue, green, and red circles.

The proteins were indexed based on determined

amounts and correlated with the copies per cell

number (CPC) values available from pax-db

(http://pax-db.org) for type A and type B quantified

peptides, shown in the lower pane of Figure 4. Type B

quantified peptides are shown at CPC equivalent

to the lowest observed loading of the CopyCAT

protein. As can be seen, the protein amounts are

distributed reasonably well over the expected

CPC/abundance values/dynamic range.

• Error in native peptide: • Error in CopyCAT peptide

Very low signal/noise

Miscleave in CopyCAT

Miscleave in Target

Probable

LOW CPC HIGH CPC

Type B are shown at CPC equivalent to lowest observed

loading of CopyCAT

6Absolute Quantification of Yeast Kinases by LC/MS/MS using QconCat and MRM Technologies

Next, MRM assay specificity was investigated by

running the same yeast sample on a Xevo TQ-S

Mass Spectrometer. The top pane of Figure 5

compares the mass extracted chromatograms for

a number of QconCAT peptides, illustrating the

same chromatographic behavior/profile for both MS

platforms. The middle pane of Figure 5 shows the

benefits of improved specificity afforded by running

Xevo TQ-S at elevated resolution, while maintaining

the same sensitivity provided by Xevo TQ,

annotating peptides as type A that were previously

qualified as type B using Xevo TQ. The number of

quantifiable type A peptides increased from 147 to

212 out of a possible 276 peptides (44% increase)

and type A proteins from 98 to 124 out of a possible

138 proteins (26% increase), respectively,

illustrated in the bottom pane of Figure 5.

Xevo TQ Xevo TQ-SQ1 0.7 Q2 0.7

Q1 0.4 Q2 0.4

IGDYAGIKBPI: 3.3e5

AVIESENSAERBPI: 7.7e5

IQNAGTEVVEAKBPI: 1.0e6

BPI: 4.8e4NVNDVIAPAFVK

IGDYAGIKBPI: 4.8e5

AVIESENSAERBPI: 4.0e6

IQNAGTEVVEAKBPI: 2e6

BPI: 8.2e4NVNDVIAPAFVK

Xevo TQ Xevo TQ-S

HEAVY

LIGHT

HEAVY

LIGHT

HEAVY

LIGHT

HEAVY

LIGHT

P416

95 B

UB1

ETTE

TDVVPI

IQTP

KP2

7636

CD

C15

N

IDTI

IPFI

DD

AK

TYPE A TYPE B

276 Target Peptides: 147 Type A

> 77 Type B 52 Type C

138 Target Proteins: 98 with at least one Type A peptide

> 35 with at least one Type B peptide 5 both peptides were Type C

Xevo TQ

Xevo TQ-S

276 Target Peptides: 212 Type A

> 23 Type B 41 Type C

138 Target Proteins: 124 with at least one Type A peptide > 8 with at least one Type B peptide

6 both peptides were Type C Figure 5. MRM chromatograms of selected peptides contrasting nominal and elevated quadrupole resolution settings acquired

with Xevo TQ at and Xevo TQ-S, respectively (top pane), increased confidence in peptide detection with improved MRM acquisition specificity (left middle panel (Q1 and Q2 at 0.7 Da FWHM) vs.

right middle panel (Q1 and Q2 at 0.4 Da FWHM)) and contrasting the peptide detection types for the two applied LC-MS platforms

(two bottom panes), illustrating a marked increase in Type A detections vs. the results shown in the middle pane of Figure 4.

7Absolute Quantification of Yeast Kinases by LC/MS/MS using QconCat and MRM Technologies

The MRM transitions were visually inspected with Skyline and statistically validated with mProphet prior to

final quantitation of the yeast kinases. The top pane of Figure 6 illustrates a comparison based on the Skyline

results of the relative intensities of the transitions of a number of native and QconCAT peptides, showing great

similarity between the two peptide types. The bottom pane of Figure 6 illustrates, in general, the increased

statistical confidence in the ability to quantify peptides and proteins by using quadrupole resolution settings

of 0.4 Da vs. 0.7 Da.

Figure 6. Skyline processed relative intensities evaluation of the three selected transition of QconCAT and native peptides (top) and mProphet probabilistic scoring (bottom) of the Xevo TQ and Xevo TQ-S transitions (blue = pass; grey = fail).

8Absolute Quantification of Yeast Kinases by LC/MS/MS using QconCat and MRM Technologies

The results in Figure 7 show a comparison between the quantitative data obtained with both LC/MS/MS

platforms, i.e. Xevo TQ operated at 0.7 Da resolution vs. Xevo TQ-S operated at 0.4 Da resolution, with molar

amounts converted to CPC values for both platforms, illustrating good quantitative agreement. Shown inset

is the increased number of quantifiable proteins by MRM using elevated resolution settings in combination

with Xevo TQ-S whereby it was feasible to balance specificity due to the increased sensitivity afforded by the

StepWave™ source of the instrument.

Xevo TQ

Xev

o T

Q-S

Pearson’s correlation 0.93 r2 0.88 Spearman Rank correlation 0.85 p<0.0001

Figure 7. Quantitative comparison of the results obtained at nominal (x-axis) vs. elevated (y-axis) quadrupole resolution MRM measurements. The inset demonstrates the number of quantifiable proteins at elevated quadrupole resolution.

9Absolute Quantification of Yeast Kinases by LC/MS/MS using QconCat and MRM Technologies

Figure 8. Quantitative comparison elevated quadrupole MRM results vs. number of copies/cell according to Pax-db.

The quality of quantification was assessed by contrasting the elevated quadrupole resolution Xevo TQ-S

MRM results with the number of protein copies per cell, shown in Figure 8, illustrating reasonable agreement.

Moreover, the combination of QconCAT and elevated resolution MRM afforded the detection and quantitation of

20 yeast kinases that have not been previously quantified.

20 proteins that have not been previously quantified Pearson’s correlation w/PaxDB 0.96 r2 0.92 Spearman Rank correlation w/PaxDB 0.7 p<0.0001

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

AcknowledgementThis work was supported by BBSRC grants BB/G009112/1

(Liverpool) and BB/G009058/1 (Manchester). The authors are

grateful to Dr. Karin Lanthaler for provision of chemostat-grown

yeast cell preparations.

References

1. Brownridge P et al. Global absolute quantification of a proteome: Challenges in the deployment of a QconCAT strategy. Brownridge P et al. Proteomics. 2011 Aug;11(15):2957-70.

2. Beynon RJ, Doherty MK, Pratt JM, Gaskell SJ. Multiplexed absolute quantification in proteomics using artificial QCAT proteins of concatenated signature peptides. Nat Methods. 2005 Aug;2(8):587-9.

3. Giles K and Gordon D. A new conjoined RF ion guide for enhanced ion transmission. ASMS 2010 poster (described in Travelling Wave ion mobility, Int J Ion Mobility Spectrom, 2013; 16(1):1-3.

4. MacLean B et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics. 2010 Apr 1;26(7):966-8.

5. Reiter L et al. mProphet: automated data processing and statistical validation for large-scale SRM experiments. Nat Methods. 2011 May;8(5):430-5.

CO N C LU S IO NS■■ A combination of QconCAT and MRM with the Xevo TQ-S

can quantify proteins across the dynamic range of yeast

■■ Equivalent mammalian range would be

to 1000 to 35,000,000 CPC

■■ Deciding factor in quantification success is peptide choice

■■ Still unpredictable parameters

■■ If predicting quantotypic peptides, use multiple peptides

to infer protein abundance (at least N=3)

■■ Many ways for a peptide to produce incorrect

protein quantification

■■ Xevo TQ-S shows a significant increase in performance

over Xevo TQ

■■ More and better quantifications, more straightforward

data analysis

Waters, T he Science of What’s Possible, nanoACQUITY UPLC, ACQUITY UPLC, Symmetry, MassLynx, and Xevo are registered trademarks of Waters Corporation. TargetLynx and StepWave are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2014 Waters Corporation. Produced in the U.S.A.January 2014 720004925EN AG-PDF