development of a human cell-free expression system to generate
TRANSCRIPT
Development of a Human Cell-Free Expression System to Generate Stable-Isotope-Labeled Protein Standards for Development of a Human Cell-Free Expression System to Generate Stable-Isotope-Labeled Protein Standards for Quantitative Mass Spectrometry Quantitative Mass Spectrometry
Ryan D. Bomgarden1, Derek Baerenwald3, Eric Hommema1, Scott Peterman2, John C. Rogers1
1Thermo Fisher Scientific, Rockford, IL; 2Thermo Fisher Scientific, Cambridge, MA; 3University of Iowa, Iowa City, IA1Thermo Fisher Scientific, Rockford, IL; 2Thermo Fisher Scientific, Cambridge, MA; 3University of Iowa, Iowa City, IA
Overview Results FIGURE 5. Heavy BAD protein-protein interaction. A) Coomassie-stained SDS-PAGE gel of recombinant light and heavy BAD-GST-HA-6xHIS purified from
Purpose: To develop a human cell-free expression system for the production of stable isotope-labeled (i.e. heavy) proteins.
Two different approaches for heavy protein production were investigated. One used heavy SILAC-labeled cells to produce a heavy-labeled IVT extract. The other method used normal light IVT extracts supplemented with heavy amino acids.
PAGE gel of recombinant light and heavy BAD-GST-HA-6xHIS purified from HeLa IVT lysates (L) using glutathione resin (E1) and cobalt resin (E2) tandem affinity. Flow troughs (FT) from each column are also indicated. B)Schematic of BAD phosphorylation and protein interactions during cell survival and cell
Methods: HeLa cell lysates supplemented with 13C615N2 L-Lysine and 13C6
15N4 L-arginine were used for in vitro translation (IVT) of recombinant fusion proteins. Labeled proteins were purified using affinity chromatography prior to mass
method used normal light IVT extracts supplemented with heavy amino acids. Although both methods successfully expressed GFP with stable-isotope incorporation of greater than 95%, the light lysates had a significantly higher level
of BAD phosphorylation and protein interactions during cell survival and cell death (i.e. apoptosis).
Labeled proteins were purified using affinity chromatography prior to mass spectrometry (MS) analysis.
Results: We developed a novel, human cell-based IVT system which expresses
incorporation of greater than 95%, the light lysates had a significantly higher level of protein expression and lower cost of production (Figure 2A). Titration and time-course experiments using the light lysate with heavy amino acids showed that amino acid concentrations > 1 mM and incubation times longer than four hours
ALight BAD-GST-HA-HIS Heavy BAD-GST-HA-HIS
Results: We developed a novel, human cell-based IVT system which expresses heavy proteins with 90-97% stable isotope incorporation in less than eight hours.
amino acid concentrations > 1 mM and incubation times longer than four hours were necessary for optimal protein expression and stable-isotope incorporation (Figure 2A and 2B).
Introduction
Stable-isotope-labeled peptides are routinely used as internal standards for the
(Figure 2A and 2B).
In order to validate the use of this human heavy IVT system for production of human-based proteins, six additional recombinant proteins were expressed and Stable-isotope-labeled peptides are routinely used as internal standards for the
quantification of enzymatically-digested protein samples. Stable-isotope-labeled proteins are also the ideal standards for standardization of MS sample
human-based proteins, six additional recombinant proteins were expressed and purified using GST or 6xHIS affinity purification. Although all proteins were expressed as indicated by a Western blot (Figure 3A), only four out of the six
BAD
14-3-3preparation.1 Traditional in vivo expression systems such as 15N-labeled E. coli or SILAC have been use to express recombinant heavy proteins. However, these systems are limited in their expression of toxic or insoluble proteins, require 2-3
expressed as indicated by a Western blot (Figure 3A), only four out of the six proteins were recovered with high yield after purification and sample preparation. All expressed proteins had stable-isotope incorporation equal or greater than 90% as determine by MS analysis of heavy and light peptides (Figure 3B and Figure 4). L FT1 E1 FT2 E2
14-3-3
systems are limited in their expression of toxic or insoluble proteins, require 2-3 days for protein expression, and may have low yield of functional (i.e. properly-folded) proteins. In addition, since these in vivo systems use stable-isotope-labeled cell lines, all proteins in the cell are isotopically-labeled leading to
as determine by MS analysis of heavy and light peptides (Figure 3B and Figure 4).
Ideal protein standards are identical to their endogenous counterparts. Expression B
L FT1 E1 FT2 E2 L FT1 E1 FT2 E2
labeled cell lines, all proteins in the cell are isotopically-labeled leading to significantly higher waste and cost.
An alternative to in vivo protein expression is in vitro translation (IVT). In this
Ideal protein standards are identical to their endogenous counterparts. Expression of recombinant proteins in human cell-free extract systems has been shown to aid in proper protein folding and post-translational modification.2 During the purification of one mammalian protein, BAD, we observed co-purification of light 14-3-3σ with
BADPP
BAD BADAkt1+
An alternative to in vivo protein expression is in vitro translation (IVT). In this method, a cellular extract system transcribes DNA into mRNA, which is subsequently translated into protein. Most IVT systems utilize prokaryotic (e.g.
of one mammalian protein, BAD, we observed co-purification of light 14-3-3σ with the heavy protein (Figure 5A). This protein-protein interaction is known to be mediated by 14-3-3 binding of serine/threonine phosphorylation motifs (Figure
Bcl-2PP
14-3-3 Bax+
subsequently translated into protein. Most IVT systems utilize prokaryotic (e.g. bacteria) or non-human eukaryotic (e.g. rabbit reticulocyte) cell extracts.2
However, these systems lack components needed for proper folding and modification of human proteins, have lower expression yields, or inefficiently
mediated by 14-3-3 binding of serine/threonine phosphorylation motifs (Figure 5B).5 MS analysis of the IVT-expressed protein identified three out of four Akt consensus phosphorylation sites (Figure 6). Overall, these results indicate that recombinant BAD expressed using this human cell-free expression system has
Cell survival Cell death
modification of human proteins, have lower expression yields, or inefficiently incorporate stable-isotope-labeled amino acids. In this study, we describe a novel human cell-free system3-4 to express stable-isotope-labeled protein standards
recombinant BAD expressed using this human cell-free expression system has functional protein modifications and interactions.
Figure 2. Heavy GFP protein expression. A) GFP was expressed for 20 hr FIGURE 6. Heavy BAD MS analysis. A) BAD proteins sequence coverage showing identified Akt1 consensus phosphorylation sites (red box). B) MS
human cell-free system to express stable-isotope-labeled protein standards (Figure 1) as controls for sample prep loss, digestion efficiency determination, or as quantification standards.
Figure 2. Heavy GFP protein expression. A) GFP was expressed for 20 hr with increasing amounts of heavy arginine (Arg10) and lysine (Lys8) and analyzed using workflow describe in Figure 1. B) GFP expression over time
showing identified Akt1 consensus phosphorylation sites (red box). B) MS spectra of stable-isotope-labeled BAD peptide HSSYPAGTEDDEGmGEEPSPFr.
Methods
Sample Preparation
analyzed using workflow describe in Figure 1. B) GFP expression over time showing corresponding heavy isotope incorporation.
A B
HSSYPAGTEDDEGmGEEPSPFr.
ASample Preparation
Protein expression and purification: Full-length cDNAs for each gene were expressed as C-terminal fusion proteins using the Thermo Scientific 1-Step Heavy
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Heavy Amino Acid Titration
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GFP Expression Time Course
A B
expressed as C-terminal fusion proteins using the Thermo Scientific 1-Step Heavy Protein IVT Kit. Expressed proteins were purified using a Thermo Scientific Pierce GST Spin Purification Kit and/or a Thermo Scientific HisPur Cobalt Purification Kit 60%
80%
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Arg10
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GST Spin Purification Kit and/or a Thermo Scientific HisPur Cobalt Purification Kit depending on the C-terminal affinity tag. Purified protein samples were separated by SDS-PAGE and stained using Thermo Scientific GelCode Blue Stain Reagent. Gel slices containing each protein were destained, reduced, and alkylated before
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Arg10
Lys8 40
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% Heavy
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)
B115
Gel slices containing each protein were destained, reduced, and alkylated before digestion to peptides using trypsin for 4-16 hours. After digestion, the peptides were desalted using Thermo Scientific Proxeon C18 Stage tips and reconstituted
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Amino Acid Concentration (mM)
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Incubation time (hours)
HSSYPAGTEDDEGmGEEPSPFr
95
100
105
110
807.9967R=47700
z=3
807.6632R=47605
z=3were desalted using Thermo Scientific Proxeon C18 Stage tips and reconstituted with 0.1% TFA.
Expression of isotopically-labeled proteins were performed in IVT reactions using FIGURE 3. Expression of heavy mammalian proteins. A) Anti-GST Western blot of six different GST-fusion proteins expressed using human IVT extract
Incubation time (hours)
>95% isotope 70
75
80
85
90
R=47605z=3
808.3307R=47704
z=3Expression of isotopically-labeled proteins were performed in IVT reactions using a custom amino acid mix supplemented with stable-isotope-labeled amino acids 13C6
15N2 L-Lysine and 13C615N4 L-arginine. All reactions were incubated at 30 C
for 8-16 hours unless otherwise noted. Recombinant HIS-GFP protein
blot of six different GST-fusion proteins expressed using human IVT extract (* indicates an anti-GST cross-reacting band in the lysate). B) Table showing isotope incorporation from peptides derived from proteins show in 3A.
>95% isotope
incorporation
50
55
60
65
70
Rela
tive A
bundance
z=3
for 8-16 hours unless otherwise noted. Recombinant HIS-GFP protein fluorescence was measured using a GFP standard curve with a Safire™ fluorometer (Tecan).
A B
isotope incorporation from peptides derived from proteins show in 3A.
30
35
40
45
50Rela
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bundance
808.6644R=47504
z=3
fluorometer (Tecan).
LC-MS/MS Analysis
A nanoflow HPLC with a Thermo Scientific PepMap C18 column (75 µm ID x 20
Sample MW % Heavy10
15
20
25
30
808.9987R=48004
z=3
807.3299804.6613 809.3320804.9958804.3274A nanoflow HPLC with a Thermo Scientific PepMap C18 column (75 µm ID x 20 cm) was used to separate peptides using a 5-40% gradient (A: water, 0.1% formic acid; B: acetonitrile, 0.1% formic acid) at a flow rate of 300 nL/min for 40 min. A Thermo Scientific LTQ Orbitrap XL mass spectrometer equipped with electron-
GFP 27 kDa 95%
BAD 19 kDa 92%Light Heavy
803.0 803.5 804.0 804.5 805.0 805.5 806.0 806.5 807.0 807.5 808.0 808.5 809.0 809.5 810.0m/z
0
5
807.3299R=44104
z=?
804.6613R=45000
z=3
809.3320R=48204
z=3
804.9958R=46704
z=3
804.3274R=43705
z=3
805.8584R=42604
z=?
806.9905R=43104
z=?
806.3579R=48504
z=?
809.6719R=50604
z=3
803.6577R=33704
z=?
Conclusions
Thermo Scientific LTQ Orbitrap XL mass spectrometer equipped with electron-transfer dissociation (ETD) was used to detect peptides using a top six experiment consisting of single-stage MS followed by acquisition of six MS/MS spectra with
*
CyclinD1 36 kDa 97%
p53 53 kDa 91%
RB 110 kDa 96% Conclusions
� Six different stable-isotope-labeled proteins were produced using a modified non-SILAC human IVT system.
consisting of single-stage MS followed by acquisition of six MS/MS spectra with collision induced dissociation (CID) for protein identification.
Data Analysis GFP BAD Cyclin D1 p53RB GAPDH
RB 110 kDa 96%
GAPDH 37 kDa 94%
non-SILAC human IVT system.
� Isotope incorporation efficiency of > 95% was observed for IVT reactions containing stable-isotope amino acids at concentrations > 50mM and incubated
Data Analysis
MS spectra were searched using Thermo Scientific Proteome Discoverer software version 1.3 with using the SEQUEST® search engine against a custom human FIGURE 4. MS spectra of peptides derived from proteins show in 3A. A) MS
GFP BAD Cyclin D1 p53RB GAPDH
containing stable-isotope amino acids at concentrations > 50mM and incubated for longer than four hours.
� Co-purification of heavy BAD protein with light 14-3-3 proteins suggests proper
version 1.3 with using the SEQUEST search engine against a custom human SWISSProt database. Static modification included carbamidomethyl with methionine oxidation, Lysine-8 and Arginine-10 used as dynamic modifications. SILAC ratios were based on the area under the curve (AUC) for each heavy and
FIGURE 4. MS spectra of peptides derived from proteins show in 3A. A) MS spectra of stable-isotope-labeled GAPDH peptide AGAHLQGGAk. B) MS spectra of stable-isotope-labeled cyclin D1 peptide AYPDANLLNDr.
� Co-purification of heavy BAD protein with light 14-3-3 proteins suggests proper protein folding and post-translational modification of in vitro expressed protein.
References
SILAC ratios were based on the area under the curve (AUC) for each heavy and light peptide and used to determine stable-isotope incorporation. A
95
100
459.2536R=62601
z=2
References
1.Hanke, S., et al. Absolute SILAC for accurate quantitation of proteins in complex mixtures down to the attomole level. J. Proteom Res, 2007, 7:1118-30.
FIGURE 1. Heavy recombinant protein expression, purification and MS analysis A) IVT lysates are combined with reaction mixture, vector DNA and
AGAHLQGGAk
>95% isotope
incorporation70
75
80
85
90
complex mixtures down to the attomole level. J. Proteom Res, 2007, 7:1118-30.
2.Ciccimaro E., et al. Absolute quantification of phosphorylation on the kinase activation loop of cellular focal adhesion kinase by stable isotope dilution liquid
analysis A) IVT lysates are combined with reaction mixture, vector DNA and stable isotope-labeled amino acids to express recombinant proteins. B) Expressed proteins are then purified and digested into peptides for LC-MS
incorporation
45
50
55
60
65
Rela
tive A
bundance
459.7548R=62604
z=2 activation loop of cellular focal adhesion kinase by stable isotope dilution liquid chromatography/mass spectrometry. Anal. Chem. 2009, 81(9):3304-13.
3.Mikami, S., et al. A human cell-derived in vitro coupled transcription/translation
Expressed proteins are then purified and digested into peptides for LC-MS analysis.
PurificationA B 20
25
30
35
40
45
Rela
tive A
bundance
R=62604z=2
3.Mikami, S., et al. A human cell-derived in vitro coupled transcription/translation system optimized for production of recombinant proteins. Protein Expr. Purif.
2008, 62(2):190-8.
Purification
Expressed
A B
454.0 454.5 455.0 455.5 456.0 456.5 457.0 457.5 458.0 458.5 459.0 459.5 460.0 460.5 461.0 461.5 462.0
m/z
0
5
10
15
20
460.2561R=63904
z=2455.2468R=64001
z=2
458.7525R=59904
z=?
455.7486R=63704
z=2
460.7569R=52204
z=2
461.2581R=64504
z=2
456.7495R=55604
z=2
456.2470R=45204
z=2
454.4142R=51604
z=?
2008, 62(2):190-8.
4.Stergachis, A., et al. Rapid empirical discovery of optimal peptides for targeted proteomics. Nat. Meth. 2011, 8(12):1041-1043.
BHarvest
Cells Digestion
Expressed
ProteinPurified Protein
636.3123R=54201
Light Heavym/z
proteomics. Nat. Meth. 2011, 8(12):1041-1043.
5.Tzivion G, et al. 14-3-3 proteins; bringing new definitions to scaffolding. Oncogene. 2001, 20(44):6331-8.
Cells Digestion
LC-MS AYPDANLLNDr80
85
90
95
100
R=54201z=2
Oncogene. 2001, 20(44):6331-8.
Cell Lysate Reaction Mixture:
LC-MS AYPDANLLNDr
>95% isotope
incorporation
55
60
65
70
75
80
Rela
tive A
bundance 636.8135
R=53804z=2
Safire is a trademark of Tecan. SEQUEST is a registered trademark of the University of Washington. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.
Reaction Mixture:
- stable isotope-labeled amino acids
- accessory proteins
- energy mix, IVT 30
35
40
45
50
55
Rela
tive A
bundance
- energy mix, nucleotides, salts
Expressed
IVT
Isotope
incorporation 5
10
15
20
25
30
637.3152R=53904
z=2
631.6829R=52704
z=?635.8120R=45504
z=?631.1301R=54104
637.8175R=45004
632.2360R=48004
630.3022R=46204
632.7896R=51504
633.3446R=47704Expressed
Protein
incorporation
determination
Light Heavy
630.0 630.5 631.0 631.5 632.0 632.5 633.0 633.5 634.0 634.5 635.0 635.5 636.0 636.5 637.0 637.5 638.0m/z
0
5z=?R=54104
z=?R=45004
z=2R=48004
z=?R=46204
z=?R=51504
z=?R=47704z=?