Automated Immunoaffinity-based Proteomic...
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Automated Immunoaffinity-based Proteomic Methods for the Study of Post-translational Modification Matthew P. Stokes 1 , Jeffrey C. Silva 1 , Steven Murphy 2 , Jason Russell 2 , Xiaoying Jia 1 , Jian Min Ren 1 , Kimberly Lee 1 1: Cell Signaling Technology, Inc., Danvers MA 01923 2: Agilent Technologies, Inc., Santa Clara, CA 95051 Post-translational modification (PTM) of proteins, including phosphorylation, acetylation, methylation, and ubiquitina- tion, are critical events in all aspects of cellular signaling. Antibody-based enrichments of post-translationally modified peptides combined with LC-MS/MS have proven to be powerful methods for the study of PTMs in a wide variety of cells and tissues, and in profiling various disease states (1-4). These antibody-based methods involve complex pro- tocols that necessitate great care to achieve optimal results and reproducibility. Here, manual (batch mode) versus automated protocols have been compared with respect to the number of post-translationally modified peptides iden- tified and the corresponding relative abundance of those peptides between the two sample preparation procedures. The AssayMAP Bravo Platform (Agilent Technologies, Inc.) allows automation of antibody-based peptide enrichments, simplifying the enrichment protocol and providing results that can be superior to manual methods. The AssayMAP Bravo Platform outperformed a traditional batch mode method for immunoaffinity purification of post-translationally modified peptides with all antibodies tested. In each case, the Bravo method resulted in a higher number of modified pep- tides identified along with a lower number of non-specific, unmodified peptides. The improved performance was likely due to the decrease in unmodified peptides in the Bravo samples, as relative abundance of the modified, target peptides changed little between methods (Figures 4F, 5E, 6C), and nearly all modified peptides were present in both methods at the MS1 feature level (Figure 5D). Matthew P. Stokes Cell Signaling Technology 3 Trask Lane, Danvers, MA 01923 email: [email protected] www.cellsignal.com Introduction Conclusions Contact Information Phospho-Serine/Threonine Ubiquitin Branch Acetyl-lysine Phospho-tyrosine © 2014 Cell Signaling Technology. Inc. Cell Signaling Technology ® , CST ™ , PTMScan ® and XP ® are trademarks of Cell Signaling Technology, Inc. Orbitrap Velos ™ is a trademark of Thermo Fisher Scientific Inc. Progenesis ® is a Registered trademark of Nonlinear Dynamics Limited. SORCERER ™ is a trademark of Sage-N Research. 14PSTSHOWPTMS0165ENG_00 Figure 5: Ubiquitin Branch Motif Antibody. Number of ubiquitinated (Green, A) and unmodified (Grey , B) peptides identified in extract from mouse embryo using the Bravo method and the stan- dard batch mode method. Venn diagrams of overlap between Bravo method and standard method at the MS2 (identification, C) and MS1 (feature, D) levels. E. Log2 ratio versus intensity plot comparing Bravo method to batch mode method. Green = ubiquitinated peptides, Grey = unmodified peptides. Figure 6: Acetyl-lysine Antibody. Number of acetylated (Red, A) and unmodified (Grey , B) pep- tides identified using the Bravo method or the standard batch mode method. C. Log2 ratio versus intensity plot comparing Bravo method to batch mode method. Red = acetylated peptides, Grey = unmodified peptides. Figure 4: Phospho-tyrosine (p-Tyr-1000) Antibody. A. Gel Stain and western blot analysis on extract from Jurkat cells treated with pervanadate (PV: high p-Tyr signal) and mouse liver extract (low p-Tyr signal). Western blot performed using phospho-tyrosine (P-Tyr-1000) Rabbit mAb #8954. Num- ber of tyrosine phosphorylated (Blue) and unmodified (Grey) peptides identified with the AssayMAP Bravo Platform or the standard batch mode method in Jurkat cells treated with pervanadate (B & C) or mouse liver (D & E). F. Log2 ratio versus intensity plot comparing Bravo method to batch mode method. Blue = phosphopeptides, Grey = unmodified peptides. The median log2 ratio for each is indicated. Figure 3: Ser/Thr Motif Antibody Mixture. A. List of antibodies included in the Ser/Thr motif antibody mixture with consensus phosphorylation motifs. B. Venn diagram of peptide identifications using the motif antibody mixture and IMAC. Number of phosphorylated (Orange , C) and unmodified (Grey , D) peptide identifications from mouse embryo for the Ser/Thr Motif Antibody mixture using the AssayMAP Bravo Platform or the standard batch mode method. Figure 2: The AssayMAP Bravo System. Antibodies were bound to Protein A cartridges and washed. Peptides were then bound to antibody/Protein A beads, washed, eluted, C18 purified, and analyzed using LC-MS/MS. Figure 1: The Batch Mode PTMScan ® Method. Samples are digested to peptides, run over reverse phase columns, immunoaffinity purified with the appropriate motif antibody, C18 purified, and subjected to LC-MS/MS. 1. Bind Antibody to Protein A 2. Bind Peptides to Protein A/Antibody Antibody Description Motif Akt Substrate RXX(s/t) Akt Substrate RXRXX(s/t) AMPK Substrate LXRXX(s/t) ATM/ATR Substrate (s/t)Q ATM/ATR Substrate (s/t)QG Cdk Substrate (K/R)(s/t)PX(K/R) CK Substrate (s/t)(D/E)X(D/E) MAPK Substrate PX(s/t)P PKA Substrate (K/R)(K/R)X(s/t) PKC Substrate (K/R)X(s/t)X(K/R) PKD Substrate LXRXX(s/t) PLK Binding Motif S(s/t)P tP Motif (s/t)P tPE Motif (s/t)PE tXR Motif (s/t)XR 14-3-3 Binding Motif (R/K)XX(s/t)XP Human cell lines or mouse tissues were lysed, digested with trypsin, and desalted over C18 columns. Peptides from 2 mg of samples were processed using the standard batch-mode PTMScan ® protocol (Figure 1) or the Assay- MAP Bravo system (Figure 2) using Protein A cartridges and the antibody purification application. Motif antibodies (100 μg) were loaded onto the cartridges and washed with PBS. Peptides resuspended in immunoaffinity purification (IAP) buffer were loaded onto Protein A/Antibody cartridges, washed using IAP buffer and water, and eluted in 0.15% TFA. Enriched peptides were purified on StageTips and analyzed by LC-MS/MS on an Orbitrap Velos ™ mass spec- trometer using a top 20 data-dependent analysis method. MS/MS spectra were assigned to peptide sequences using SORCERER ™ (5), and label-free quantification was performed using Progenesis ® (Nonlinear Dynamics). Two indepen- dent immunoprecipitation reactions were performed for all antibodies using the AssayMAP Bravo Platform (Bravo 1 and Bravo 2). Replicate injections were run for each sample. Bars represent average number of identifications across replicate injections, and error bars are –/+ 1 standard deviation. Methods IMAC 6,931 IMAC 6,931 Motif Ab Mixture 3,184 Motif Ab Mixture 3,184 687 3,391 4,000 AssayMAP BRAVO 1 AssayMAP BRAVO 2 Batch Mode Phosphopeptide Identifications 3,500 3,000 2,500 2,000 1,500 1,000 500 0 3,397 2,784 11,709 14,000 AssayMAP BRAVO 1 AssayMAP BRAVO 2 Batch Mode Unmodified Peptide Identifications 12,000 10,000 8,000 6,000 4,000 2,000 0 11,800 12,282 MW Gel Stain p-Tyr-1000 #8954 Jurkat + PV Liver MW Jurkat + PV Liver 5,583 6,000 AssayMAP BRAVO 1 AssayMAP BRAVO 2 Batch Mode 5,000 4,000 3,000 2,000 1,000 0 5,507 4,349 Phosphopeptide Identifications 7,234 12,000 AssayMAP BRAVO 1 AssayMAP BRAVO 2 Batch Mode Unmodified Peptide Identifications 10,000 8,000 6,000 4,000 2,000 0 6,831 10,593 475 600 AssayMAP BRAVO 1 AssayMAP BRAVO 2 Batch Mode 500 400 300 200 100 0 494 184 Phosphopeptide Identifications 6,290 12,000 AssayMAP BRAVO 1 AssayMAP BRAVO 2 Batch Mode Unmodified Peptide Identifications 10,000 8,000 6,000 4,000 2,000 0 6,396 11,104 Jurkat + PV Mouse Liver Complimentary Methods Maximum Abundance 10,000 100,000 1,000,000 10,000,000 100,000,000 1,000,000,000 Log2 Ratio (Bravo : Batch Mode) -10 -5 0 5 Median Phospho = -.09 Median Unmodified = -1.71 2,974 3,500 AssayMAP BRAVO 1 AssayMAP BRAVO 2 Batch Mode Ubiquitinated Peptide Identifications 3,000 2,500 2,000 1,500 1,000 500 0 2,903 1,181 3,789 9,000 AssayMAP BRAVO 1 AssayMAP BRAVO 2 Batch Mode Unmodified Peptide Identifications 8,000 7,000 6,000 5,000 4,000 3,000 2,000 1,000 0 4,082 8,196 Bravo Method 2,608 Bravo Method 2,608 Batch Mode 368 Overlap 1,206 Overlap 1,206 Batch Mode 36 Bravo Method 84 Overlap 3,701 Overlap 3,701 Maximum Abundance 100,000 1,000,000 10,000,000 100,000,000 1,000,000,000 Log2 Ratio (Bravo : Batch Mode) -15 -10 -5 0 5 10 15 Median Ubiquitin = 0.32 Median Unmodified = -2.27 2,313 2,500 AssayMAP BRAVO 1 AssayMAP BRAVO 2 Batch Mode Acetylated Peptide Identifications 2,000 1,500 1,000 500 0 2,220 1,942 3,919 6,000 AssayMAP BRAVO 1 AssayMAP BRAVO 2 Batch Mode Unmodified Peptide Identifications 5,000 4,000 3,000 2,000 1,000 0 4,008 5,368 Maximum Abundance 10,000 100,000 1,000,000 10,000,000 100,000,000 1,000,000,000 Log2 Ratio (Bravo : Batch Mode) -10 -5 0 5 10 Median Acetyl = 0.82 Median Unmodified = 0.20 1. Rush, J. et. al. (2005) Nat Biotechnol. 23, 94–101. 4. Guo, A. et. al. (2013) Mol Cell Proteomics. 13, 372–387. 2. Lee, K.A. et. al. (2011) J Biol Chem. 286, 41530–41538. 5. Lundgren, D. H. et. al. (2009) Curr Protoc Bioinformatics. Chapter 13, Unit 13 13. 3. Stokes, M.P. et. al. (2012) Mol Cell Proteomics. 11, 187–201. References A A B B C C A A D D F E C B B E C D Presentation Posters, Case Studies and Publications
Transcript of Automated Immunoaffinity-based Proteomic...
Automated Immunoaffinity-based Proteomic Methods for the Study of Post-translational ModificationMatthew P. Stokes1, Jeffrey C. Silva1, Steven Murphy2, Jason Russell2, Xiaoying Jia1, Jian Min Ren1, Kimberly Lee1
1: Cell Signaling Technology, Inc., Danvers MA 01923 2: Agilent Technologies, Inc., Santa Clara, CA 95051
Post-translational modification (PTM) of proteins, including phosphorylation, acetylation, methylation, and ubiquitina-tion, are critical events in all aspects of cellular signaling. Antibody-based enrichments of post-translationally modified peptides combined with LC-MS/MS have proven to be powerful methods for the study of PTMs in a wide variety of cells and tissues, and in profiling various disease states (1-4). These antibody-based methods involve complex pro-tocols that necessitate great care to achieve optimal results and reproducibility. Here, manual (batch mode) versus automated protocols have been compared with respect to the number of post-translationally modified peptides iden-tified and the corresponding relative abundance of those peptides between the two sample preparation procedures. The AssayMAP Bravo Platform (Agilent Technologies, Inc.) allows automation of antibody-based peptide enrichments, simplifying the enrichment protocol and providing results that can be superior to manual methods.
The AssayMAP Bravo Platform outperformed a traditional batch mode method for immunoaffinity purification of post-translationally modified peptides with all antibodies tested. In each case, the Bravo method resulted in a higher number of modified pep-tides identified along with a lower number of non-specific, unmodified peptides. The improved performance was likely due to the decrease in unmodified peptides in the Bravo samples, as relative abundance of the modified, target peptides changed little between methods (Figures 4F, 5E, 6C), and nearly all modified peptides were present in both methods at the MS1 feature level (Figure 5D).
Matthew P. Stokes Cell Signaling Technology3 Trask Lane, Danvers, MA 01923email: [email protected]
Introduction
Conclusions
Contact Information
Phospho-Serine/Threonine Ubiquitin Branch Acetyl-lysinePhospho-tyrosine
© 2014 Cell Signaling Technology. Inc. Cell Signaling Technology®, CST™, PTMScan® and XP® are trademarks of Cell Signaling Technology, Inc. Orbitrap Velos™ is a trademark of Thermo Fisher Scientific Inc. Progenesis® is a Registered trademark of Nonlinear Dynamics Limited. SORCERER™ is a trademark of Sage-N Research.
14PSTSHOWPTMS0165ENG_00
Figure 5: Ubiquitin Branch Motif Antibody. Number of ubiquitinated (Green, A) and unmodified (Grey, B) peptides identified in extract from mouse embryo using the Bravo method and the stan-dard batch mode method. Venn diagrams of overlap between Bravo method and standard method at the MS2 (identification, C) and MS1 (feature, D) levels. E. Log2 ratio versus intensity plot comparing Bravo method to batch mode method. Green = ubiquitinated peptides, Grey = unmodified peptides.
Figure 6: Acetyl-lysine Antibody. Number of acetylated (Red, A) and unmodified (Grey, B) pep-tides identified using the Bravo method or the standard batch mode method. C. Log2 ratio versus intensity plot comparing Bravo method to batch mode method. Red = acetylated peptides, Grey = unmodified peptides.
Figure 4: Phospho-tyrosine (p-Tyr-1000) Antibody. A. Gel Stain and western blot analysis on extract from Jurkat cells treated with pervanadate (PV: high p-Tyr signal) and mouse liver extract (low p-Tyr signal). Western blot performed using phospho-tyrosine (P-Tyr-1000) Rabbit mAb #8954. Num-ber of tyrosine phosphorylated (Blue) and unmodified (Grey) peptides identified with the AssayMAP Bravo Platform or the standard batch mode method in Jurkat cells treated with pervanadate (B & C) or mouse liver (D & E). F. Log2 ratio versus intensity plot comparing Bravo method to batch mode method. Blue = phosphopeptides, Grey = unmodified peptides. The median log2 ratio for each is indicated.
Figure 3: Ser/Thr Motif Antibody Mixture. A. List of antibodies included in the Ser/Thr motif antibody mixture with consensus phosphorylation motifs. B. Venn diagram of peptide identifications using the motif antibody mixture and IMAC. Number of phosphorylated (Orange, C) and unmodified (Grey, D) peptide identifications from mouse embryo for the Ser/Thr Motif Antibody mixture using the AssayMAP Bravo Platform or the standard batch mode method.
Figure 2: The AssayMAP Bravo System. Antibodies were bound to Protein A cartridges and washed. Peptides were then bound to antibody/Protein A beads, washed, eluted, C18 purified, and analyzed using LC-MS/MS.
Figure 1: The Batch Mode PTMScan® Method. Samples are digested to peptides, run over reverse phase columns, immunoaffinity purified with the appropriate motif antibody, C18 purified, and subjected to LC-MS/MS.
1. Bind Antibody to Protein A
2. Bind Peptides to Protein A/Antibody
Antibody Description Motif
Akt Substrate RXX(s/t)
Akt Substrate RXRXX(s/t)
AMPK Substrate LXRXX(s/t)
ATM/ATR Substrate (s/t)Q
ATM/ATR Substrate (s/t)QG
Cdk Substrate (K/R)(s/t)PX(K/R)
CK Substrate (s/t)(D/E)X(D/E)
MAPK Substrate PX(s/t)P
PKA Substrate (K/R)(K/R)X(s/t)
PKC Substrate (K/R)X(s/t)X(K/R)
PKD Substrate LXRXX(s/t)
PLK Binding Motif S(s/t)P
tP Motif (s/t)P
tPE Motif (s/t)PE
tXR Motif (s/t)XR
14-3-3 Binding Motif (R/K)XX(s/t)XP
Human cell lines or mouse tissues were lysed, digested with trypsin, and desalted over C18 columns. Peptides from 2 mg of samples were processed using the standard batch-mode PTMScan® protocol (Figure 1) or the Assay-MAP Bravo system (Figure 2) using Protein A cartridges and the antibody purification application. Motif antibodies (100 μg) were loaded onto the cartridges and washed with PBS. Peptides resuspended in immunoaffinity purification (IAP) buffer were loaded onto Protein A/Antibody cartridges, washed using IAP buffer and water, and eluted in 0.15% TFA. Enriched peptides were purified on StageTips and analyzed by LC-MS/MS on an Orbitrap Velos™ mass spec-trometer using a top 20 data-dependent analysis method. MS/MS spectra were assigned to peptide sequences using SORCERER™ (5), and label-free quantification was performed using Progenesis® (Nonlinear Dynamics). Two indepen-dent immunoprecipitation reactions were performed for all antibodies using the AssayMAP Bravo Platform (Bravo 1 and Bravo 2). Replicate injections were run for each sample. Bars represent average number of identifications across replicate injections, and error bars are –/+ 1 standard deviation.
Methods
IMAC6,931IMAC6,931
Motif AbMixture3,184
Motif AbMixture3,184
687
3,391
4,000
AssayMAPBRAVO 1
AssayMAPBRAVO 2
Batch Mode
Pho
spho
pep
tide
Iden
tifica
tions 3,500
3,000
2,500
2,000
1,500
1,000
500
0
3,397
2,784
11,709
14,000
AssayMAPBRAVO 1
AssayMAPBRAVO 2
Batch Mode
Unm
od
ified
Pep
tide
Iden
tifica
tions
12,000
10,000
8,000
6,000
4,000
2,000
0
11,800 12,282
MW
Gel Stain p-Tyr-1000 #8954
Jurk
at +
PV
Live
r
MW
Jurk
at +
PV
Live
r
5,5836,000
AssayMAPBRAVO 1
AssayMAPBRAVO 2
Batch Mode
5,000
4,000
3,000
2,000
1,000
0
5,507
4,349
Pho
spho
pep
tide
Iden
tifica
tions
7,234
12,000
AssayMAPBRAVO 1
AssayMAPBRAVO 2
Batch Mode
Unm
od
ified
Pep
tide
Iden
tifica
tions
10,000
8,000
6,000
4,000
2,000
0
6,831
10,593
475
600
AssayMAPBRAVO 1
AssayMAPBRAVO 2
Batch Mode
500
400
300
200
100
0
494
184
Pho
spho
pep
tide
Iden
tifica
tions
6,290
12,000
AssayMAPBRAVO 1
AssayMAPBRAVO 2
Batch Mode
Unm
od
ified
Pep
tide
Iden
tifica
tions
10,000
8,000
6,000
4,000
2,000
0
6,396
11,104
Jurkat + PV
Mouse Liver
Complimentary Methods
Maximum Abundance
10,000 100,000 1,000,000 10,000,000 100,000,000 1,000,000,000
Log2
Rat
io (B
ravo
: Ba
tch
Mod
e)
-10
-
5
0
5
Median Phospho = -.09 Median Unmodified = -1.71
2,9743,500
AssayMAPBRAVO 1
AssayMAPBRAVO 2
Batch Mode
Ub
iqui
tinat
ed P
eptid
e Id
entifi
catio
ns
3,000
2,500
2,000
1,500
1,000
500
0
2,903
1,181
3,789
9,000
AssayMAPBRAVO 1
AssayMAPBRAVO 2
Batch Mode
Unm
od
ified
Pep
tide
Iden
tifica
tions 8,000
7,000
6,000
5,000
4,000
3,000
2,000
1,000
0
4,082
8,196
Bravo Method2,608
Bravo Method2,608
Batch Mode368
Overlap1,206
Overlap1,206
Batch Mode36
Bravo Method84
Overlap3,701
Overlap3,701
Maximum Abundance
100,000 1,000,000 10,000,000 100,000,000 1,000,000,000
Log2
Rat
io (B
ravo
: Ba
tch
Mod
e)
-15
-1
0
-5
0
5
10
1
5
Median Ubiquitin = 0.32 Median Unmodified = -2.27
2,3132,500
AssayMAPBRAVO 1
AssayMAPBRAVO 2
Batch Mode
Ace
tyla
ted
Pep
tide
Iden
tifica
tions
2,000
1,500
1,000
500
0
2,220
1,942
3,919
6,000
AssayMAPBRAVO 1
AssayMAPBRAVO 2
Batch Mode
Unm
od
ified
Pep
tide
Iden
tifica
tions
5,000
4,000
3,000
2,000
1,000
0
4,008
5,368
Maximum Abundance
10,000 100,000 1,000,000 10,000,000 100,000,000 1,000,000,000
Log2
Rat
io (B
ravo
: Ba
tch
Mod
e)
-10
-5
0
5
1
0
Median Acetyl = 0.82 Median Unmodified = 0.20
1. Rush, J. et. al. (2005) Nat Biotechnol. 23, 94–101. 4. Guo, A. et. al. (2013) Mol Cell Proteomics. 13, 372–387.
2. Lee, K.A. et. al. (2011) J Biol Chem. 286, 41530–41538. 5. Lundgren, D. H. et. al. (2009) Curr Protoc Bioinformatics. Chapter 13, Unit 13 13.
3. Stokes, M.P. et. al. (2012) Mol Cell Proteomics. 11, 187–201.
References
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Presentation Posters, Case Studies and Publications
