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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
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