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Isotope-labeled Multipeptide Standards for
Quantitative Mass Spectrometry
atlasantibodies.com
QPrEST– Isotope-labeled multipeptide standards for quantitative
mass spectrometry
The QPrEST standards are originally
derived from the Human Protein Atlas
project2,3, where unlabeled (light) recombinant protein fragments are used as
antigens for antibody generation. These
protein epitope signature tags (PrESTs)
can be produced with incorporated heavy
isotope-labeled amino acids to generate proteins with unique properties that
make them suitable as internal standards
for mass spectrometry-based quantification4-6. All QPrESTs contain a stretch of
50-150 amino acids identical to a target
human protein sequence including at least
Production and quality control
Heavy isotope-labeled QPrESTs are expressed in an Escherichia coli BL21(DE3)
derivative, auxotrophic for lysine and arginine8. The cell cultivation is performed
in a minimal autoinduction medium and
isotopic incorporation is achieved by the
addition of heavy isotope-labeled arginine and lysine (13C, 15N) to the culture.
The QTag part of the protein sequence
contains an N-terminal hexahistidine tag
used for affinity purification of the QPrEST
standard. After purification, QPrEST purity is verified using SDS-PAGE and the
5000
4000
QPrEST products
QPrEST products with PAp ID
2000
3000
number of QPrESTs
The QPrEST product catalog contains
over 20,000 products, of which over 70%
have at least one experimentally verified
proteotypic peptide, based on data from
PeptideAtlas7 (Fig 2). In total, the currently available QPrEST standards target more than 13,000 human proteins,
of which more than 40% are covered by
multiple (up to five) QPrEST standards.
The auxotrophic E. coli strain contains deletions in the lysA and argA genes making
the cells unable to survive without the addition of arginine and lysine to the growth
medium (Fig 4A). The auxotrophy results
in a near complete (>99%) isotopic incorporation as verified through the absence
of peaks corresponding to unlabeled peptides in an MS spectrum (Fig 4B).
2
Figure 2.
3
4
5
6
7
8
9
number of unique tryptic peptides
10
11
Distribution of QPrEST products based on number
of unique tryptic peptides (blue bars). The number of
QPrESTs within each group with at least one tryptic
peptide present in PeptideAtlas is also presented (orange bars).
A
B
25000
20000
Counts
QPrEST isotope-labeled standards
Proteome coverage
correct protein molecular weight is confirmed using ESI-MS analysis (Fig 3).
1000
Within the field of proteomics, mass spectrometry (MS) has become the method of
choice for protein analysis1. Using MS,
thousands of proteins from a complex
sample can be identified in a single run.
Apart from enabling protein identification,
MS has also become a key tool for protein quantification. One strategy to obtain
highly accurate measures of protein abundance within a sample is to use a heavy
isotope-labeled standard, preferably
spiked into the sample at an early stage
of the sample preparation procedure. In
order to obtain reliable quantitative data,
it is desirable to quantify multiple peptides
for each target protein. Several types of
isotope-labeled standards exist, one being Atlas Antibodies’ quantitative standard QPrEST. This standard possesses
several beneficial properties such as high
incorporation efficiency of heavy isotopelabeled amino acid residues. It also contains multiple unique tryptic peptides, increasing the reliability of the quantitative
measurement of the corresponding endogenous target protein.
two unique tryptic peptides. The QPrEST
standard can be added to the sample at
an early stage of the sample preparation workflow, decreasing the variation
introduced during for example proteolytic
cleavage. An N-terminal quantification tag
(QTag) is present in all QPrEST standards, and is used for accurate quantification of the QPrEST using an unlabeled
QTag protein as internal reference (Fig
1). The QTag consists of a hexahistidine
tag genetically fused to an albumin binding protein (ABP) sequence derived from
streptococcal protein G. Quantification of
QPrEST standards is based on quantitative analysis of multiple tryptic peptides
spanning the QTag sequence.
0
Introduction
15000
10000
5000
quantification of QPrEST
0
23300
23800
24300
mass (Da)
24800
theoretical MW = 24160 Da
experimental MW = 24158 Da
light QTag
Figure 3.
Quality control of QPrEST standard. (A) SDS-PAGE
is used to determine protein purity and (B) the protein
molecular weight is verified with ESI-MS.
heavy QPrEST
light endogenous protein
quantification of endogenous protein using QPrEST
Figure 1.
Schematic figure of a QPrEST standard (green). The N-terminal part of the
sequence consists of the QTag sequence, used for purification and accurate
quantification of the QPrEST using an unlabeled QTag (orange). The C-terminal
part of the sequence is identical to a portion of a human protein (blue). This part
is used for absolute quantification of the endogenous target protein.
m/z
3.5E5
1.8E5
3E5
1.5E5
6E4
5E4
3E4
4
8
12
16
20
24
0
595
0
time (min)
3.5E5
2.5E5
m/z
ISEATDGLSDFLK 2+
L/H ratio 0.64
2E5
2.5E5
intensity
intensity
3E5
613
1E5
610
9E4
607
1.5E4
1.2E4
604
2E4
TVEGVDKLQAQVVESAK 3+
L/H ratio 0.63
601
2.5E5
intensity
intensity
time (min)
376
0
24
374
20
598
16
364
12
372
2E4
370
4E4
368
6E4
2E4
8
GSHMASLEAEAK 2+
L/H ratio 0.65
8E4
4E4
4
2E4
1.5E4
1.5E5
1E5
1E5
time (min)
709
707
0
705
24
703
20
701
16
699
12
697
8
693
4
695
5E4
5E4
0
m/z
GSSHHHHHHSSGLVPRGSHMASLAEAKVLANRELDKYGVSDYHKNLINNAKTVEGVKDLQAQVVE
SAKKARISEATDGLSDFLKSQTPAEDTVKSIELAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVK
Figure 5.
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Verification of isotopic incorporation. (A) The Lys and Arg auxotrophic E. coli BL21(DE3) strain is dependent on
addition of Arg and Lys when grown on minimal medium. (B) Analysis of QPrEST tryptic digests using ESI-MS
shows that no peaks corresponding to unlabeled peptides can be detected.
1E5
0
()+"
)*+(
Figure 4.
1.2E5
6E4
ELDKYGVSDYHK 3+
Heavy: 490.6 m/z
Light: 485.2 m/z
$&&"
Rosetta
ΔArgA
ΔLysA
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Rosetta
1.2E5
8E4
)*+(
+,-./012-/3."45"
- LYS
- ARG
$&%"
- LYS
+ ARG
ISEATDGLSDFLK 2+
Heavy: 702.3 m/z
Light: 698.3 m/z
$&("
Rosetta
ΔArgA
ΔLysA
,-./0123-1435"%6"
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Rosetta
1.4E5
1E5
B
+ LYS
- ARG
()("
+ LYS
+ ARG
The QPrEST standards are stable and
no effect on the QPrEST concentration
has been observed after repeated freezethaw cycles.
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A
intensity
intensity
Labeled QPrEST standards are accurately quantified using the QTag part of
the protein. An unlabeled version of the
QTag is used as internal reference in an
MS-based setup where multiple QTagderived peptides are used to quantify the
QPrEST standard5. A high purity of the
QTag is ensured through multiple affinity
purification steps based on both N- and
C-terminal purification handles and the
QTag concentration is accurately quantified using amino acid analysis. The
heavy isotope-labeled QPrEST is mixed
with the unlabeled QTag protein and the
sample is digested. Peptides are further
analyzed using an LC-ESI-QTOF setup
where ratios between light and heavy
QTag-derived peptides are used to determine the absolute QPrEST concentration
(Fig 5). The analysis is not limited to fully
cleaved tryptic peptides, as the equal digestion efficiency between QPrEST and
QTag results in very similar peptide ratios
for fully cleaved tryptic peptides and peptides containing one or two intact tryptic
cleavage sites. Accurate quantification of
the endogenous target protein depends
on the quantitative precision of the added
standard. Based on data from three replicate experiments, the concentration is determined with an imprecision below 10%.
366
Accurate QPrEST quantification
QPrEST quantification using three example QTag peptides. Extracted ion chromatograms are shown to the left. Extracted spectra with peaks
corresponding to both labeled and unlabeled peptides are shown to the right. The determined L/H ratio for each peptide is also presented.
The three peptides are marked in the QTag sequence at the bottom of the figure.
copy number of UGDH in HeLa
●
●
●
●
●
●
●
●
AQVEIVTDGEEPAEMIQVLGPKPALK
Figure 6.
DLALAIR
EGNPEEDLTADK
2e+05
1e+05
2e+06
1e+06
5e+05
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
2e+05
1e+05
EQIVVDLSHPGVSEDDQVSR
●
●
●
●
5e+06
IAILGFAFK
2e+06
1e+06
5e+05
●
●
●
●
1e+07
IIDSLFNTVTDK
2e+07
1e+07
5e+06
copy number per cell
5e+07
EGNPEEDLTADKANAQAAALYK
copy number per cell
copy number of CAPG in HeLa
IIDSLFNTVTDKK
Protein quantification using QPrEST standards can be performed either in single-plex,
or in in a multi-plex format where a set of
QPrEST standards is added to the sample for parallel analysis of multiple target
proteins. QPrEST standards have been
used as internal references to determine the copy number of proteins in cell
lines4,5. A set of QPrEST standards was
spiked into a HeLa cell lysate directly after cell lysis and the sample was digested
using the filter-aided sample preparation
(FASP) methodology. Generated peptides
were further fractionated into six samples
using strong anion exchange chromatography in a pipette tip format. Peptide
fractions were desalted and separated
using a 3 h LC gradient prior to injection
on a QExactive mass spectrometer. Fig 6
shows two examples of protein quantification using QPrEST standards4. The protein UGDH was quantified using a total of
five peptides generated from two separate QPrEST standards, resulting in a determined copy number of 890,000 copies
per cell. CAPG was quantified using one
QPrEST and a total of four peptides. The
copy number was for this protein determined to be 2.5 million copies per cell.
MLKPAFIFDGR
Application example of QPrESTs
as internal standards
Absolute quantification of two human proteins using QPrEST standards. The copy numbers of CAPG and UGDH
were determined in HeLa cells. Quantified peptides are shown on the x axis and determined copy numbers on
the y axis. Each circle represents data from one of three replicate analyses.
Summary
REFERENCES
1. Aebersold R and Mann M. (2003) Mass spectrometry-based proteomics. Nature 422, 198-207
2. Uhlén M et al. (2015) Proteomics. Tissue-based
map of the human proteome. Science 23;347(6220).
3. Uhlén M, Oksvold P, Fagerberg L, Lundberg E,
Jonasson K, Forsberg M, Zwahlén M, Kampf C, Wester
K, Hober S, Wernérus H, Björling L and Pontén F.
(2010) Towards a knowledge-based Human Protein
Atlas. Nat Biotechnol 28, 1248-1250
4. Edfors F, Boström T, Forsström B, Zeiler M,
Johansson H, Lundberg E, Hober S, Lehtiö J, Mann
M and Uhlén M. (2014) Immuno-proteomics using
polyclonal antibodies and stable isotope labeled
affinity-purified recombinant proteins. Mol Cell
Proteomics 13, 1611-1624
5. Zeiler M, Straube WL, Lundberg E, Uhlén M and
Mann M. (2012) A Protein Epitope Signature Tag
(PrEST) library allows SILAC-based absolute quantification and multiplexed determination of protein
copy numbers in cell lines. Mol Cell Proteomics 11,
O111 009613
6. Zeiler M, Moser M and Mann M. (2014) Copy
number analysis of the murine platelet proteome
spanning the complete abundance range. Mol Cell
Proteomics 13, 3435-3445
7. Farrah T, Deutsch EW, Omenn GS, Sun Z, Watts
JD, Yamamoto T, Shteynberg D, Harris MM and
Moritz RL. (2014) State of the human proteome in
2013 as viewed through PeptideAtlas: comparing the
kidney, urine, and plasma proteomes for the biology- and disease-driven Human Proteome Project. J
Proteome Res 13, 60-75
8. Matic I, Jaffray EG, Oxenham SK, Groves MJ,
Barratt CL, Tauro S, Stanley-Wall NR and Hay R.
T. (2011) Absolute SILAC-compatible expression
strain allows Sumo-2 copy number determination
in clinical samples. J Proteome Res 10, 4869-4875
• QPrESTs are recombinantly produced, heavy isotope-labeled, multipeptide standards for MS-based absolute quantification
• The product catalog contains over 20,000 bioinformatically selected QPrEST standards with proteome-wide coverage
• An accurate MS-based setup with an unlabeled internal QTag reference standard is used for precise QPrEST quantification to
ensure reliable downstream results
• QPrEST standards have been used to determine absolute protein copy numbers in different cell lines
atlasantibodies.com
Our website provides you with easy access to all
characterization data, and online ordering via our
web shop. You can also send your order to
[email protected].
Or send an e-mail to [email protected]
to discuss any matters regarding protein
quantification. You’ll find we’re Totally Human.
2015-04-02
Atlas Antibodies AB
AlbaNova University Center
SE-106 91 Stockholm, Sweden
atlasantibodies.com
Phone +46(0)8 54 59 58 50
Fax
+46(0)8 54 59 58 51
[email protected]
[email protected]