p 1% tfa p 546 3477 1747 p standard optimized standard ... · 7 8 microparticle collection (ti-imac...
TRANSCRIPT
Stoyan Stoycheva, Ireshyn Govendera, Previn Naickera, Isak Gerbera, Justin Jordaanb,
Marina Pauwc, David Tabbc, Ignacio Arribas Díezd, Ole Nørregaard Jensend
Tanveer Baathe, Ana Martinez del Vale, Dorte B Bekker-Jensene, Jesper V Olsene
a Council for Scientific and Industrial Research, Pretoria, South Africab Rhodes University, Biotechnology Innovation Centre, Grahamstown, South Africa
c Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, South Africad Institute for Biochemistry and Molecular Biology, University of Southern Denmark, Denmark
e Faculty of Health and Medical Sciences, Novo Nordisk Foundation Center for Protein Research, Denmark
E-mail: [email protected]
CONCLUSIONS & FUTURE WORK• We demonstrate a fully automated workflow for automated phosphoproteome profiling suitable for a range of samples• SAX magnetic beads provide a suitable alternative for high volume but low concentration samples such as urine, with the option of
fractionation (pH) for deep proteome profiling• HILIC chemistry is potentially suitable for high content samples or where precipitation is not desirable • We have optimized protocols and buffers for automated enrichment using three magnetic bead variants• Reduction of glycolic acid in the binding buffer improved the performance of Zr-IMAC, and for the conditions described in this study it
outperformed Ti-IMAC, likely due to increased capture of mono-phosphorylated peptides • Analysis of the properties of enriched peptides showed that dioxide enriched samples showed a vastly different profile than that of IMAC• We are evaluating buffer conditions with the aim of combining dioxide and IMAC chemistry in an attempt to improve coverage for single-shot
phosphoproteomics by enriching single- and multi-phosphorylated peptides• The fully automated platforms will be applied to a range of samples to identify the best conditions for each sample type
AUTOMATED CLEAN-UP TO PHOSPHOPEPTIDE ENRICHMENT
REFERENCES• Batth TS, Tollenaere MAX, Rüther P, Gonzalez-Franquesa A, Prabhakar BS, Bekker-Jensen S, AS Deshmukh, Olsen JV. 2019. Protein Aggregation
Capture on Microparticles Enables Multipurpose Proteomics Sample Preparation. Molecular & Cellular Proteomics. DOI: 10.1074/mcp.TIR118.001270
• Shilov I, Seymour S, Patel A, Loboda A, Tang W, Keating S, Hunter C, Nuwaysir L, Schaeffer D. 2007. The Paragon Algorithm, a Next Generation Search Engine That Uses Sequence Temperature Values and Feature Probabilities to Identify Peptides from Tandem Mass Spectra. Molecular & Cellular Proteomics p1638
• Yángüez E, Hunziker A, Dobay MP, Yildiz S, Schading S, Elshina E, Karakus U, Gehrig P, Grossmann J, Dijkman R, Schmolke M & Stertz S. 2018. Phosphoproteomic-based kinase profiling early in influenza virus infection identifies GRK2 as antiviral drug target. Nature Comms. 9, 3679
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P
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45
6
7
8 Microparticle collection
(Ti-IMAC in 20% EtOH)
Microparticle equilibration(80% ACN, 5% TFA, 1M Glycolic Acid)
Wash 1(Equilibration
buffer)
Wash 2(80% ACN, 1% TFA)
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P
P
P
Disposable plastic comb
collection(Bead drop)
Phosphopeptide Elution
(1% NH4OH)
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P
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P
P
P
P
P
P
Wash 3(10% ACN, 0.2% TFA)
Peptide bind(40µl protein digest + 160µl Equilibration
Buffer)
For example, the use of SAX (Strong Anion Exchange) bead chemistry can be used for capture of proteins and peptides from dilute samples such as urine and ability to potentially apply pH based fractionation, but is not suitable for sample preparation using SDS (accumulation on beads).
HILIC requires lower dilution to capture proteins, and is therefore potentially more suitable for high-content samples, and can be extended to the clean-up of peptides and glycans.
The benefits of coupling of PAC to phosphopeptide enrichment will only require 30 to 40 minutes labour. LC was performed using an EvoSep 1 with 21 minute gradient, coupled to a ThermoFisher Orbitrap HF-X 15000 resolution.
45 min per 96 samples
Protein Aggregation Capture Protocol Phosphopeptide Enrichment Protocol
We recently demonstrated the rapid and automated enrichment of phosphopeptides using MagReSyn® Ti-IMAC HP (prototype supplied by ReSyn Biosciences) with clean-up using PAC of proteins extracted with GdHCL using MagReSyn® Amine as the precipitation nucleus.
AUTOMATED PROTEIN AND PEPTIDE CLEAN-UP: SAMPLE DEPENDENT ALTERNATIVES
When comparing the range of workflows for automated protein clean-up we noted little difference in the coverage of samples using the new protocols developed in this study (above left). SAX did show a slightly increased recovery, but we believe similar recovery can be achieved using TFA to aid elution with PAC and HILIC (experiments in progress). On-bead HILIC and SAX digestion showed similar digest efficiency as in solution digestion (above centre), and the further optimisation of this step is currently underway. Venn overlaps (above right) of the data indicated the highest number of unique peptides was identified by SAX, followed by in-solution digestion. The sample preparation techniques do not appear to show any bias with respect to peptide properties (figures below). We intend to investigate the coupling of the optimized protocols to automated phosphopeptide enrichment.
SAX
100mM Borate pH 10
10mM Borate pH 10
LysC/Trp (1-4hr)
1% TFA (frac pH 10→2)
20 µg - 5mg
Urea
Proteins & Peptides
Salt or pH Gradient
Salt or pH Gradient
Disposable plastic comb
collection
1
2
3
45
6
8
7 Microparticle collection
(pre-equilibrated with 70% ACN)
Wash 1(95% ACN)
Wash 3(80% Ethanol)
Protein bind(Sample pH 8,
70% ACN)
On-bead protein
digestion(AmmBic pH 8.5,
Trp 1:20, 4 Hr)
Na+
Cl-Na+
Cl-
Na+
Cl-
Peptide Elution(1%TFA)
Wash 2(80% Ethanol)
Na+
Cl-
37°C
3 to 5 Hrs per 96 samples
HILIC Clean-up Protocol SAX Clean-up Protocol
Although the above developed protocol is suitable for phosphopeptide enrichment from a range of biological samples, we have started investigating the use of alternate clean-up and enrichment tools to further extend the workflow to a diverse range of biological samples, and to try and increase possible coverage offered by this workflow.
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In-Sol PAC HILIC SAX
Peptides (19121) Proteins (2887)
1% SDC 4% SDS 4% SDS 8M Urea
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100%
In-Sol PAC HILIC SAX
% 0miss % 1miss % 2miss
18 hr 4 hr (on-bead)
SAX
PACHILIC
In-Sol
AUTOMATED PHOSPHOPEPTIDE ENRICHMENT: INCREASING SAMPLE COVERAGEMagnetic beads from ReSyn Biosciences have been extensively used phosphopeptide enrichment, with Ti-IMAC being preferred by the majority of research groups (Baath et al., 2019; Niemi et al., 2019., Yángüez et al., 2018). Although the product has been shown to provide a high number of phosphopeptide identifications, specificity can vary based on sample preparation. Recently, the Olsen group has reported >99% specificity using Ti-IMAC by decreasing the bead to protein ratio, thereby improving competition of phosphopeptides for the support material. However, the impact of this approach on total coverage is not yet known. We therefore use the recommended ratio provided by the supplier for the optimization of phosphopeptide enrichment. ReSyn Biosciences offers a range of products offering complimentary sample coverage, the challenges and benefits of which are outlined below.
Sample preparation was automated on a KingFisher® Duo magnetic bead handling station and analysed using an SCIEX TripleTOF 6600 coupled to a Dionex nanoRSLC using 60 minute nano-flow gradients. Data was searched using Sciex Protein Pilot (Shilov et al., 2007) against a Uniprot Swiss Prot H. sapiens database supplemented with sequences of common contaminant proteins. A 1% FDR cut-off was applied at the PSM, peptide and protein levels.
5µg 10µg 20µg 50µg 200µg
Ti-IMAC - HP 1802 3295 3974 5729 8186
0
2000
4000
6000
8000
10000 Phosphopeptides
INTRODUCTIONThe requirement for robust and routine high throughput sample preparation workflows has become a necessity as clinical proteomics reaches maturity. The workflows will enable processing of large sample cohorts with the throughput, robustness and reproducibility required for a routinized laboratory setting. In this study we illustrate a fully automated phosphoproteomics workflow, with further elaboration on options for clean-up from dilute and high content samples, using SAX and HILIC respectively. We further demonstrate improved phosphoproteome coverage using a range of bead enrichment chemistries. Magnetic beads are considered desirable since these are easy to handle, simple to automate, linearly scalable, and high throughput compatible on a range of magnetic bead handling stations.
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3 5 7 9 11
Isoelectric point (pI)
In-sol
HILIC
PAC
SAX
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600 1100 1600 2100 2600 3100 3600
Molecular weight (Da)
In-sol
HILIC
PAC
SAX
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-3 -2 -1 0 1 2 3GRAVY Index
In-sol
HILIC
PAC (SP3)
SAX
We recently reported (ASMS 2019) on extensive optimization of binding buffer for phosphopeptide enrichment, in particular the effect of hydroxy acids on the capacity and specificity. Initially performed on a simple protein mixture, we confirm the effect of the new binding buffers using a complex lysate. Proteins from HEPG2 cells were extracted by sonication with pre-chilled OPB (50mM TEAB, 1% SDC, 10mM TCEP, 40mM CAA). Proteins were precipitated overnight with acetone, and resuspended in 50mM AmmBic containing 1% SDC. Protein (10mg) was digested overnight and peptides desalted using Oasis HLB cartridges.
Phosphopeptide enrichment was performed from 200 µg of lyophilized protein digest on a KingFisher Duo, and samples analysed using a Dionex nanoRSLC using 60 minute nano-flow gradients, coupled to a SCIEX 6600 TripleTOF. Data was searched using Proteome Discoverer (MASCOT) with 50 ppm peptide tolerance, 0.2 Da tolerance, and 2 miss cleavages. Peptide properties were extracted using a custom R-script.
Buffer Binding Buffer
Standard 80% ACN, 5% TFA, 1M Glycolic Acid
Ti-IMAC 80% ACN, 5% TFA, 0.1M Lactic Acid
Zr-IMAC 80% ACN, 5% TFA, 0.1M Glycolic Acid
TiO2 50% ACN, 0.1% Acetic Acid, 0.1M Glycolic Acid
Product Active Group Advantages Disadvantages Notes
Ti-IMAC Titanium ions High Coverage Sensitive to contaminants Selective towards mono-phosphorylated peptides
Zr-IMAC Zirconium ions High Coverage (New Buffer) Sensitive to chelating agents Selective towards mono-phosphorylated peptides
Ti-Dioxide Titanium dioxide nanoparticles Resistant to Contaminants Lower capacity Bias towards multiply phosphorylated peptides
Zr-Dioxide Zirconium dioxide nanoparticles Resistant to Contaminants Low capacity Bias towards multiply phosphorylated peptides
Ti-IMAC Zr-IMAC TiO2
Standard Buffer
OptimizedBuffers
% 1 PO3
% 2 PO3
% 3 PO3
593 7783328
546 17473477
543 12341571
Ti-IMAC
Zr-IMAC
TiO2
Standard Buffer
OptimizedBuffers
The use of optimized buffers for Ti-IMAC and Zr-IMAC increase sample coverage, with Zr-IMAC increasing by 20%, while retaining a high specificity of over 95% (above). The increased coverage offered by TiO2 also resulted in a decrease in the specificity to around 75% (above). Standard enrichment with TiO2 captures multi- phosphorylated peptides (left), while the optimized buffer system normalizes to the standard IMAC profile. It appears that the increased coverage for Zr-IMAC results from improved capture of mono-phosphorylated peptides at the expense of multi-phosphorylated.
When comparing the phosphopeptide overlaps for the standard and optimized buffers, we can see that the Zr-IMAC and TiO2 show significant improvement in coverage with the new buffer systems (above left). Analysis of peptide properties (above right for standard buffer, below right optimized buffers) further elucidates selectivity of the three phosphopeptide enrichment chemistries. With standard buffers Zr-IMAC shows higher selectivity for hydrophobic residues, while the use of optimized buffer for Ti-IMAC increases hydrophobic residues (a possible reason for improved coverage using this new buffer system). Optimized buffers for Ti-IMAC and Zr-IMAC normalized their selectivity for the peptides (highly overlapping profiles, below right). The new buffer system for TiO2 provides a vastly different peptide property profile, increasing selectivity for acidic peptides and shifting towards hydrophilic peptides.
784
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25191085
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27981088
307
25191182
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4082
928
214
1021
309
1182
211
Development of a fully automated high throughput magnetic workflow for phosphoproteome profiling
586
175458
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1631 107
133
1428
148391
2277
1332 187
193
Zr-IMAC
Zr-IMAC
TiO2
TiO2
Standard Buffer
OptimizedBuffers
Ti-IMAC
Ti-IMAC
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<10% 10-20% 20-30% 30-40% 40-50% 50-60% 60-70% 70-80%
% T
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l Pep
tid
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Hydrophobic Residues
Ti-IMAC
Zr-IMAC
TiO2
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40%
<10% 10-20% 20-30% 30-40% 40-50% 50-60% 60-70% 70-80%
% T
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Hydrophilic Residues
Ti-IMAC
Zr-IMAC
TiO2
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40%
<10% 10-20% 20-30% 30-40% 40-50% 50-60% 60-70% 70-80%
% T
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tid
es
Hydrophobic Residues
Ti-IMAC
Zr-IMAC
TiO2
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40%
<10% 10-20% 20-30% 30-40% 40-50% 50-60% 60-70% 70-80%
% T
ota
l Pep
tid
es
Hydrophilic Residues
Ti-IMAC
Zr-IMAC
TiO2
0%
10%
20%
30%
<10% 10-20% 20-30% 30-40% 40-50% 50-60% 60-70% 70-80%
% T
ota
l Pep
tid
es
Acidic Residues
Ti-IMAC
Zr-IMAC
TiO2
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10%
20%
30%
40%
50%
60%
70%
<10% 10-20% 20-30% 30-40% 40-50% 50-60% 60-70% 70-80%
% T
ota
l Pep
tid
es
Basic Residues
Ti-IMAC
Zr-IMAC
TiO2
0%
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20%
30%
40%
<10% 10-20% 20-30% 30-40% 40-50% 50-60% 60-70% 70-80%
% T
ota
l Pep
tid
es
Acidic Residues
Ti-IMAC
Zr-IMAC
TiO2
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% T
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l Pep
tid
es
Basic Residues
Ti-IMAC
Zr-IMAC
TiO2
Peptide overlap for sample preparation techniques
Missed Cleavages
98% 98% 99% 97% 98%
75%
40%
60%
80%
100%
0
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6000
Standard Optimized Standard Optimized Standard Optimized
Ti-IMAC Zr-IMAC TiO2
# P
ho
sph
epti
des
Pu
rity (%)
Conditions Amine (PAC)
Bind 50-80% ACN (or EtOH) pH 8
Wash 80% EtOH & 95% ACN
Digest LysC/Trp (1-4hr)
Elute H2O or 0.5% TFA or DMSO
Range (automated) sub 1µg - 200µg
Extraction SDS, NP40, Tween, SDC
Clean-up of Proteins
Fractionation NA
Top-down NA
HILIC
15% ACN, 100mM NH4Ac pH 4.5
95% ACN
LysC/Trp (1-4hr)
1% TFA
5µg - 0.5mg
SDS, NP40, Tween, SDC
Proteins, Peptides & Glycans
NA
NA
Disposable plastic comb
collection 96 well
1
2
3
45
6
7
8 Microparticle collection:20% Ethanol
Storage Solution
HILIC Microparticle equilibration:100mM NH4Ac pH
4.5; 15% ACN (1min)
Wash 1:95% ACN (1min)
Wash 2:95% ACN (1min)
Protein bind:
Mix sample 1:1 with 200 mM NH4Ac in 30% ACN (15 min)
On-bead protein
digestion:
50mM NH4 Bicarb pH 8; 37°C (4hr)
Trypsin 1:10
Na+
Cl-
Na+
Cl-
Na+
Cl-
Bead Drop & Storage
Disposable plastic comb
collection: 96 well
1
2
3
45
6
7
8Microparticle collection and
storage:20% Ethanol Storage
Solution
SAX Microparticle equilibration:
100mM Borate pH 10 (1min)
Wash 1:10 mM BoratepH 10 (1min)
Wash 2:10 mM Borate pH 10 (1min)
Protein bind:
Mix sample 1:9 with 100 Borate pH
10 (15 min)
On-bead protein
digestion:
50mM NH4 Bicarb pH 8; 37°C (4hr)
Trypsin 1:10
Na+
Cl-
Na+
Cl-
Na+
Cl-
Peptide Elution:
1% TFA