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: sstoychev@csir.co.za

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

P

P

P

P

1

2

3

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)

P

P

P

P

Disposable plastic comb

collection(Bead drop)

Phosphopeptide Elution

(1% NH4OH)

P

P

P

P

P

P

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.

0%

20%

40%

60%

80%

100%

In-Sol PAC HILIC SAX

Peptides (19121) Proteins (2887)

1% SDC 4% SDS 4% SDS 8M Urea

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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

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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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Isoelectric point (pI)

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-3 -2 -1 0 1 2 3GRAVY Index

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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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Development of a fully automated high throughput magnetic workflow for phosphoproteome profiling

586

175458

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1631 107

133

1428

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1332 187

193

Zr-IMAC

Zr-IMAC

TiO2

TiO2

Standard Buffer

OptimizedBuffers

Ti-IMAC

Ti-IMAC

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Zr-IMAC

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Zr-IMAC

TiO2

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Acidic Residues

Ti-IMAC

Zr-IMAC

TiO2

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<10% 10-20% 20-30% 30-40% 40-50% 50-60% 60-70% 70-80%

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Ti-IMAC

Zr-IMAC

TiO2

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Ti-IMAC

Zr-IMAC

TiO2

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Basic Residues

Ti-IMAC

Zr-IMAC

TiO2

Peptide overlap for sample preparation techniques

Missed Cleavages

98% 98% 99% 97% 98%

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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