New strategies for the enrichment of phosphorylated proteins using precipitation methods by metal cations

Matthias Rainer, Günther Bonn

Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria

PhyNexus User Group Symposium, August 27th, 2014, San Francisco

Major challenges in phosphopeptide analysis

pSer : pThr : pTyr = 1800 : 200 : 1

• 30% of all proteins in eukaryotic cells are phosphorylated

• is involved in biological processes including signaling, transductions, growth and

cell differentiation etc.

• plays a major role in many human diseases

• low abundant proteins • Phosphorylation can occur at multiple residues within a protein • Dynamic regulation of phosphoproteins • weak ionization in positive ionization mode • ion suppression • lability of the phosphoesterbond during fragmentation by CID

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• Antibody-based (a-pY)

phosphoprotein/peptide enrichment relies on immuno-affinity.

Highly specific antibodies exist for phosphotyrosine.

• Affinity-based (IMAC, MOAC)

based on the affinity of the negatively-charged phosphate groups

for positively-charged metal ions.

IMAC: Fe3+, Ga3+ or other metal ions are chelated to nitrilotriacetic

acid (NTA) or iminodiaceticacid (IDA) coated beads.

MOAC: solid metal beads TiO2, ZrO2, Al(OH)3, SnO2 etc.

Overview about different phosphopeptide enrichment strategies

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calcium phosphate precipitation and co-precipitation of phosphorylated peptides/proteins with calcium phosphate.

R … peptide residue

Ca2+… calcium ions

Idea!

Co-precipitation of phosphorylated peptides by calcium ions

X. Zhang, J. Ye, O. N. Jensen, P. Roepstorff. Highly efficient phosphopeptide enrichment by calcium phosphate precipitation combined with subsequent IMAC enrichment. Molecular&Cellular Proteomics 2007, 6, 2032.

3Ca2+ + 2PO43- Ca3(PO4)2

Ca2+ + R – O – PO32- R – O – PO3Ca

(3m+n) Ca2+ + 2mPO4

3- + n R – O – PO32- [Ca3(PO4)2]m •[ R – O – PO3Ca]n

4

5

Highly Efficient Phosphopeptide Enrichment by Calcium Phosphate Precipitation Combined with Subsequent IMAC Enrichment

X. Zhang, J. Ye, O. N. Jensen, P. Roepstorff. Highly efficient phosphopeptide enrichment by calcium phosphate precipitation combined with subsequent IMAC enrichment. Molecular&Cellular Proteomics 2007, 6, 2032.

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Motif-Specific Sampling of Phosphoproteomes

Ba2+/Acetone/pH Precipitation for Phosphopeptide Identification in HeLa Cells Nuclear Extract using MudPIT

Ruse, C. I., McClatchy, D. B., Lu, B., Cociorva, D., Motoyama, A., Park, S. K., & Yates Iii, J. R. (2008). Motif-specific sampling of phosphoproteomes. Journal of proteome research, 7(5), 2140-2150.

Li, X. et al. Geochimica et cosmochimica acta,1997.61(8):p.1625-1633

Lanthanide Phoshates

(Solubility Products)

Precipitation of phosphorylated peptides/proteins by trivalent lanthanide Ions

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Precipitation of phosphorylated molecules by lanthanide ions

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Three major Workflows

1. Precipitation of phosphoproteins (Top-Down)

2. Precipitation of phophopeptides (Bottom-up)

3. Trypsin-assisted digestion of precipitated phosphoproteins

Güzel Y, Rainer M, Mirza MR, Bonn Gk, (2012) Highly efficient precipitation of phosphoproteins using trivalent Europium-,Terbium- and Erbium Ions. Anal Bioanal Chem 403 (5):1323–1331

Precipitation of phosphorylated proteins - workflow

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sig

na

l in

ten

sity

[a

.u.]

m/z

Erbium

Wash 1

Wash 2

Pellet

Supernatent

egg

white

sig

na

l in

ten

sity

[a

.u.]

m/z

Terbium egg

white

Pellet

Wash 2

Supernatent

Wash 1

lysozyme (m/z ~14 kDa), ovomucoid (m/z ~ 28 kDa),

ovoglobulins G2+G3 (m/z ~30-45 kDa), ovotransferrin (m/z ~80 kDa) ovalbumin (m/z ~45 kDa)

phosphoprotein

sig

na

l in

ten

sity

[a

.u.]

m/z

Europium

Wash 1

Wash 2

Pellet

Supernatent

egg

white

Precipitation of phosphoproteins from egg-white by

trivalent europium-, terbium- and erbium- Ions

MALDI-TOF mass spectra recorded after phosphoprotein precipitation from 1:4 diluted egg-white by trivalent europium-, terbium- and erbium-ions using sinapinic acid as matrix. Samples before precipitation (a), supernatant after precipitation (b), wash with 80 mM precipitating agent (c), wash with DHB solution (d), dissolved pellet (e). Signal intensities of all mass spectra were brought to the same level to allow direct comparison

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0

20

40

60

80

100

0,5 1 1,5 2 2,5 3

Re

co

ve

ry [

%]

Volume Precipitant [µl]

Recovery Study

Lanthanum

Europium

Terbium

Erbium

100%

ccasein = 300µg/ml

cprecip. = 2M

Quantification of precipitated phosphoproteins

Recovery Study

∼1.67×10−7 mol TbCl3 per μg α-casein ∼9.98×10−8 mol ErCl3 per μg α-casein ∼1.66×10−7 mol EuCl3 per μg α-casein

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A Novel Strategy for Phosphopeptide Enrichment using Lanthanide Phosphate Precipitation – A Bottom-Up Approach

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Mirza MR, Rainer M, Güzel Y, Choudhary, IM, Bonn Gk, (2012) A Novel Strategy for Phosphopeptide Enrichment using Lanthanide Phosphate Precipitation. Anal Bioanal Chem (accepted)

MALDI mass spectra taken from digested milk peptides after precipitation with trivalent lanthanide ions. A, phosphopeptide enriched by precipitation with Er3+. B, phosphopeptide enriched by precipitation using Ho3+. C, phosphopeptide enriched by precipitation using Ce3+. α-S1 and β-S2 refers to first and second subunits of α-casein respectively. β-C refers to peptides form β-casein

Erbium

Holmium

Cerium

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A Novel Strategy for Phosphopeptide Enrichment using Lanthanide Phosphate Precipitation – A Bottom-Up Approach

MALDI mass spectra taken from egg white peptides after precipitation with trivalent lanthanide ions. A, phosphopeptide enriched by precipitation with Er3+. B, phosphopeptide enriched by precipitation using Ho3+. C, phosphopeptide enriched by precipitation using Ce3+. Only phosphorylated peptides are labeled

Erbium

Holmium

Cerium

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A Novel Strategy for Phosphopeptide Enrichment using Lanthanide Phosphate Precipitation – A Bottom-Up Approach

MALDI mass spectra of a sensitivity study using two synthetic phosphopeptides. A, representing 500 fmol/µL; B, 10 fold dilution (50 fmol/µL) and C, 100 fold dilution (5 fmol/µL)

500 fmol/µL

50 fmol/µL

5 fmol/µL

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A Novel Strategy for Phosphopeptide Enrichment using Lanthanide Phosphate Precipitation – A Bottom-Up Approach

[M+H]+ Da Phosphopeptide Sequencesa Phosho-

groups ErCl3 HoCl3 CeCl3 LaCl3 EuCl3 TmCl3 TbCl3 TiO2

1254.52

1331.53

1411.50

1466.61

1594.70

1660.79

1832.83

1847.69

1927.69

1951.95

2061.83

2088.89

2432.05

2511.13

2556.10

2619.04

2678.01

2703.50

2720.91

2747.10

2856.50

2901.32

2935.15

2966.16

3008.01

3042.27

3087.99

3122.27

3132.20

EVVGSpAEAGVDAA (Ov-(340–352))

EQLSpTSpEENSK (α-S2-(141–151))

EQLSpTSpEENSK (α-S2-(141–151))

TVDMESpTEVFTK (α-S2-(153–164))

TVDMESpTEVFTKK (α-S2-(153–165))

VPQLEIVPNSpAEER α(-S1-(121–134))

YLGEYLIVPNSpAEER (α-S1)

DIGSESpTEDQAMEDIK (α-S1-(58–73))

DIGSESpTEDQAMEDIK (α-S1-(58–73))

YKVPQLEIVPNSpAEER (α-S1-(119–134))

FQSpEEQQQTEDELQDK (β-C-(33–48))

EVVGSpAEAGVDAASVSEEFR (Ov-(340–359))

IEKFQSpEEQQQTEDELQDK (β-C-(33–48))

LPGFGDSpIEAQCGTSVNVHSSLR (Ov-(62–84))

FQSpEEQQQTEDELQDKIHPF (β-C-(48-67))

NTMEHVSpSpSpEESpIISQETYK (α-S2-(17–36))

VNELSpKDIGSpESpTEDQAMEDIK (α-S1-(52–73))

LRLKKYKVPQLEIVPNSpAEERL(α-S1-(114–135))

QMEAESpISpSpSpEEIVPNSVEAQK (α-S1-(74–94))

NTMEHVSpSpSpEESpIISQETYKQ (α-S2-(17–37))

EKVNELSpKDIGSpESTEDQAMEDIK (α-S1-(50–73))

FDKLPGFGDSpIEAQCGTSVNVHSSLR (Ov-(59–84))

EKVNELSpKDIGSpESpTEDQAMEDIK (α-S1-(50–73))

ELEELNVPGEIVESpLSpSpSpEESITR (β-C-(17–40))

NANEEEYSIGSpSpSpEESpAEVATEEVK (α-S2-(61–85))

RELEELNVPGEIVESLSpSpSpEESITR (β-C-(16–40))

NANEEEYSIGSpSpSpEESpAEVATEEVK (α-S2-(61–85))

RELEELNVPGEIVESpLSpSpSpEESITR (β-C-(16–40))

KNTMEHVSpSpSpEESpIISQETYKQEK (α-S2-(16–39))

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Recovery of Phosphopeptides from a Protein Mixture

23 20 19 20 21 12 14 18

Tryptic On-Pellet Digest of Precipitated Phosphoproteins

Inte

nsity

m/z

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Güzel, Y., Rainer, M., Mirza, M. R., Messner, C. B., & Bonn, G. K. (2013). Analyst, 138(10), 2897-2905.

Workflow

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Overview of recovered phosphopeptides from a proteinmixture (lysozyme, cytochrome c, myoglobin, bovine serum albumin, a- and b-casein) and bovine milk

34 15 31 16 22 Sum

Glygen Glygen

Tryptic On-Pellet Digest of Precipitated α-casein from spiked cell lysates

Dephosphorylated HeLa cell lysate (1 mg/mL) with spiked α-casein (5 μg/mL) before and after enzymatic on-pellet digestion using trivalent cerium cations. α1, α2 and β correspond to the tryptic phosphopeptides deriving from αS1-, αS2, and β-casein, respectively

spiked cell lysate before precipitation

spiked cell lysate after precipitation

Tryptic On-Pellet Digest of Precipitated Human Saliva using CeCl3

Automation using Bench Top Robotic MEA 2

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PhyTipTM

screen (permeable membrane)

2.5 µL LnCl3

Ln… lanthanides

no chromatography (no stationary phase) no unspecific binding due to absence of any sorbent or resin

patent pending

dissolvable bed column

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Sample

well plate

patent pending

Sample Loading

1. PO4 containing sample

2. precipitation of PP

3. pellet formation

dissolvable bed column

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well plate

patent pending

Sample Loading

1. PO4 containing sample

2. precipitation of PP

3. pellet formation

dissolvable bed column

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well plate

patent pending

Sample Loading

1. PO4 containing sample

2. precipitation of PP

3. pellet formation

4. washing of unspecific

compounds

dissolvable bed column

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well plate

patent pending

Sample Loading

1. PO4 containing sample

2. precipitation of PP

3. pellet formation

4. washing of unspecific

compounds

5. dissolving pellet

dissolvable bed column

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well plate

patent pending

Sample Loading

1. PO4 containing sample

2. precipitation of PP

3. pellet formation

4. washing of unspecific

compounds

5. dissolving pellet

6. recovery of PP

dissolvable bed column

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Conclusion

simple and fast method

highly selective for phosphorylated peptides and proteins

enables top-down and bottum-up phosphoproteomics

no stationary phase or resin required (reduced unspecific binding)

trypsin was observed to be not affected by the lanthanide ions

the amount of precipitant can be adjusted to each application

MS and LC-MS compatible

allows automation using liquid handling robotics

Highly efficient precipitation of phosphorylated peptides and proteins using trivalent lanthanide ions

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Acknowledgements

o.Univ.-Prof. Dr. Günther K. Bonn

Yüksel Güzel

Christof Messner

Munazza Raza

Douglas Gjerde, PhyNexus