desalting of proteins why? - universität innsbruck · 2015. 1. 20. · desalting of hydrophilic...

Desalting of Proteins – Why?

Spin Columns

C18-reversed phase

dialysis

Extraction Tips

Spin Columns

ZipTips (Millipore)

N

H

OH

SiHO

SiHO

SiOSi

OH

OH

Fullerene C60-Silica for Desalting Biological Samples

Proteines

MALDI-MS spectra of the peptides and proteins which were eluted from Kovasil 300 Å C60-Silica

breakthrough breakthrough

sample sample

elution elution

Peptides

SPESPESPEPart 2 Part 2

5734.63

8565.63

2867.1016952.4014306.613

0

1.0

2.0

3.0

4.0

0

1.0

2.0

3.0

4.0

8566.35

14291.429

2866.33 16943.00311466.50

0

1.0

2.0

3.0

4.0

8566.83

2866.56

4282.5911466.222 14291.426

0.0

0.2

0.4

0.6

0.8

1.0

2000 4000 6000 8000 10000 12000 14000 16000 18000

A

B

C

D

5734.73

5734.43

4282.79

m/z

4x

10

Inte

ns

ity

. [a

.u.]

129

6.4

4

134

7.5

3

161

9.4

8

209

4.0

2

175

7.7

5

246

6.1

0

0.0

1.0

2.0

0.0

1.0

2.0

134

7.3

2

161

9.3

4

246

5.9

2

209

3.6

6

175

8.4

7

0.0

1.0

2.0

161

9.4

5

134

7.4

2

246

6.0

6

175

8.5

5

209

3.7

8

0.0

1.0

2.0

800 1000 1200 1400 1600 1800 2000 2200 2400

1046.54

1046.72

1047.39

m/z

A

B

C

D

Peptide Proteine

SPESPESPEPart 2 Part 2

5734.63

8565.63

2867.1016952.4014306.613

0

1.0

2.0

3.0

4.0

0

1.0

2.0

3.0

4.0

8566.35

14291.429

2866.33 16943.00311466.50

0

1.0

2.0

3.0

4.0

8566.83

2866.56

4282.5911466.222 14291.426

0.0

0.2

0.4

0.6

0.8

1.0

2000 4000 6000 8000 10000 12000 14000 16000 18000

A

B

C

D

5734.73

5734.43

4282.79

m/z

4x

10

Inte

ns

ity

. [a

.u.]

129

6.4

4

134

7.5

3

161

9.4

8

209

4.0

2

175

7.7

5

246

6.1

0

0.0

1.0

2.0

0.0

1.0

2.0

134

7.3

2

161

9.3

4

246

5.9

2

209

3.6

6

175

8.4

7

0.0

1.0

2.0161

9.4

5

134

7.4

2

246

6.0

6

175

8.5

5

209

3.7

8

0.0

1.0

2.0

800 1000 1200 1400 1600 1800 2000 2200 2400

1046.54

1046.72

1047.39

m/z

A

B

C

D

Peptide Proteine

immobilization washing elution

before

supernatant

elution

before

supernatant

elution

C60-amino-

silica

Vallant Rainer M; Szabo Zoltan; Bachmann Stefan; Bakry Rania; Najam-ul-Haq Muhammad; Rainer Matthias; Heigl

Nico; Petter Christine; Huck Christian W; Bonn Gunther K. Analytical chemistry (2007), 79(21), 8144-53.

Fullerene C60-amino silica for Desalting

C60-Amino Silica vs. ZipTip® C18 (Millipore)

ZipTips C18

supernatant

ZipTips C18

elution

11

40

.52

1.0

2.0

3.0

4.0

0.0

0.2

0.4

0.6

0.8

5.0

0.0

Inte

nsi

ty. [

a.u

.]

m/z

Phosphopeptide in supernatant

No immobilization on commercial

ZipTip ® (Millipore)

Phosphopeptide removed after washing

No immobilization of phosphopeptide

C60-Amino Silica

supernatant

C60-Amino Silica

elution

1.0

2.0

3.0

4.0

5.0

1140

.49

0.0

0.05

0.10

0.15

0.20

0.25

1110 1120 1130 1140 1150 1160 1170 1180

0.0

No Phosphopeptide in supernatant

Quantitative binding of phosphopeptide

on C60-Amino Silica

Phosphopeptide eluted from

C60-Amino Silica

Desalting of hydrophilic Phosphopeptides

Enrichment of Phenolic Compounds using

Hexagonal Boron Nitride

Martin Fischnaller, Rania Bakry, Günther Bonn

Institute of Analytical Chemistry and Radiochemistry

Austrian Drug Screening Institute – ADSI

University of Innsbruck, Austria

Crystalline forms of Boron Nitride

β or cubic BN

• diamond analog

• stable

• very hard

• black

γ or wurzite BN

• lonsdaleit analog

• only stable under high

pressure

α or hexagonal BN

• graphite analog

• chemically inert

• nontoxic

0.1446 nm

Covalent bonds

Van der Waalsbonds

0.3

33

1 n

m

Boron

Nitrogen

Structure of α-BN

Arnold F. Holleman, N. W. Lehrbuch der Anorganischen Chemie; de Gruyter: Berlin, 2007; Vol. 102.

δ 0.93e δ -0.93e

Wettability of α-BN

SEM picture of BN with 60 m² surface area.

Wettability of α-BN

Published in: Hui Li; Xiao Cheng Zeng; ACS Nano 2012, 6, 2401-2409.

DOI: 10.1021/nn204661d

Copyright © 2012 American Chemical Society

50-67° high wettability

86° low wettability

Snapshots of a water droplet on (A) an atomically BN sheet in the end of

classical molecular dynamics

Boron Nitride, a Novel Material for the Enrichment and Desalting of Protein Digests and the Protein Depletion

LC-ESI-MS

Incubation

with tryptic

digests

MALDI-MS

Washing

and

enrichment

Elution of

peptides

Analysis of desalted

and protein free

peptide solutions

N

B

N

B

N

B

N

B

N

B

N

B

N

B

N

B

B

N

B

N

B

N

B

B

N

B

N

B

N

N

B

N

BN

Boron Nitride, a Novel Material for the Enrichment and Desalting

Bradykinin Substance P Bombesin LHRH Oxytocin

Recovery rates [%]

A B A B A B A B A B

BN 60 71.65 81.67 70.13 73.59 56.12 90.88 92.13 87.46 85.92 95.24

BN 20 98.83 93.74 96.90 90.13 98.71 96.23 95.18 91.01 84.50 92.66

BN NANO 96.75 94.46 108.52 91.30 89.36 95.70 97.07 93.44 91.39 92.85

Graphite particle

85.54 83.60 31.70 64.17 47.98 52.78 32.15 76.28 76.33 94.39

Graphite 15 17.62 36.90 14.08 16.78 12.65 17.39 25.70 25.21 49.27 77.04

Recovery Study using different Peptides

A = 60% ACN in 1% TFA and 1% H3PO4; B = 60% ACN in 1% formic acid

0

5

10

15

20

25

30

0 10 20 30 40 50 60 70

BN 60 m²/g

BN 20 m²/g

Graphite 15 m²/g

Cequ [µg/ml]

Mads

[µg/m

g]

Langmuir adsorption isotherms of

Bradykinin for BN 60, BN 20 and

graphite 15

max. loading capacity

mads [µg/mg]

mads/surface area

[µg/m2]

BN 20 m²/g 14.61 0.731

BN 60 m²/g 24.13 0.402

Graphite 15 m²/g 8.19 0.546


MALDI Spectra:

The identified peptides are labeled with

asterisk. Sequence Coverage of desalted

BSA > 68 %


0

20

40

60

80

100

Inte

ns.

[a.u

.]

0.0

0.2

0.4

0.6

0.8

1.0

5x10

Inte

ns.

[a.

u.]

1000 1500 2000 2500 3000 3500m/z

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

**

*

*

*

*

*

**

*

*

*

*

*

**

*

**

*

*

*

*

**

*

*

*

*A

B

x 1000

BSA digest in urine

desalted urine sample 1000 fold increase in signal intensity

N

B

N

B

N

B

N

B

N

B

N

B

N

B

N

B

B

N

B

N

B

N

B

B

N

B

N

B

N

N

B

N

Depletion of proteins with BN

0

1000

2000

3000

4000

5000

Inte

ns.

[a.

u.]

*

0

1

2

3

4

5

4x10

Inte

ns.

[a.

u.]

1000 1500 2000 2500 3000 3500m/z

*

***

***

**

* **

*

**

*

*

**

**

**

**

***

**

**

**

**

**

** *

*

** *

**

**

*

*

**

**

2000

4000

Inte

ns.

[a.

u.]

30000 40000 50000 60000 70000m/z

2000

4000

Inte

ns.

[a.

u.]

30000 40000 50000 60000 70000

m/z

A

B

x 10

Ser

um

alb

um

in

Isofo

rm 1

[M+

2H

] 2+

Ser

um

alb

um

in

Isofo

rm 2

(A) MALDI-MS analysis of a human albumin protein and those digest in a ratio of 50000-1 (1 mg/ml-0,02 µg/ml) and a image of the crystallization of the sample and matrix

Sequence coverage of HSA 36%

(B) MALDI spectra of the sample after SPE with BN (sequence coverage of 72%) and an image of the matrix crystallization

asterisk: Mascot identified peptides

15

‐ Pyrrolizidine alkaloids (PA) are secondary plant metabolites (for plant protection) ‐ 400 different PAs in approximately 6000 plant species are known Contamination of plant products during harvest or through animals (for example bees) Examples for contaminated food: herbal teas, honey, salads etc.

‐ Problem: Hepatotoxic for animals as well as for humans Safety values for the maximum dose for drugs in Germany:

‐ Application up to 6 weeks: 1 µg/day oral, 100 µg/day cortically ‐ Application for more than 6 weeks: 0.1 µg/day oral, 10 µg/day cortically

Pyrrolizidine alkaloids Background

16

‐ General structure:

‐ Requirement(s) for toxicity:

‐ 1,2-unsaturated necine ‐ Esterification of at least one OH-group with a branched acid

Nearly all types are in coexistence with their N-oxides

Pyrrolizidine alkaloids Toxicity

17

‐ HPLC-MS (LOD ~ 1 ppb) ‐ GC-MS (LOD = 3 ppb) Otonecines and N-oxides (without derivatisation) not detectable ‐ Double antibody ELISA (LOD = 0.1 – 1.5 ppb) Antibodys only against some specific PAs ‐ HPLC Evaporative Light Scattering Detection (ELSD) (LOD = 40 ppm) ‐ Nonaqueous capillary electrophoresis (NACE) MS (LOD < 7.5 ppm) ‐ Photometric detection with Ehrlich reagent (LOD = 10 ppm)

‐ General problem for PA analysis: Only about 25 standards are available Validation is limited to a small spectrum of PAs

Isolation of PAs out of plant material with countercurrent chromatography (CCC) to get more standards (Cooperation with Medical University of Lublin)

Pyrrolizidine alkaloids Methods for detection

18

‐ Detection only possible in the low ppb-range (for GC- and HPLC-MS) for lower concentrations and for separation from other plant substances enrichment is necessary ‐ Due to the chemical structure ion exchange is the enrichment method of choice ‐ Automation with PhyNexus MEA 2 possible PhyTip with Toyopearl SP-650 resin

Pyrrolizidine alkaloids Cation exchange

19

Extraction of the plant material

Enrichment of PAs with ion exchange

Detection with UPLC-MS

Pyrrolizidine alkaloids Procedure

20

Pyrrolizidine alkaloids Cation exchange – Comparison of commercial products

• In terms of enrichment of pyrrolizidine alkaloids different commercial materials were tested

• Best recoveries (94-99%) were achieved with a polystyrene-divinylbenzene

resin functionalized with sulfonate groups • Further approach

‐ Polystyrene DVB resin in PhyNexus tips and automation of the cation exchange with PhyNexus MEA 2

‐ Continuing comparison with other resins (in cartridges and tips) ‐ Test of anion exchange resins and Immobilized Metal Ion Affinity

Chromatography (IMAC)

21

Pyrrolizidine alkaloids

• Development of a UPLC-MS/MS method for detection and quantification of standard PA´s

• Improvement of enrichment methodologies with ion-exchange

• Automation of enrichment method

Output

SPE of Phenolic Acids using Bismuth Citrate and Zirconium Silicate Inside Micro Spin Columns

SPE of Phenolic Acids using Bismuth Citrate and Zirconium Silicate Inside Micro Spin Columns

1,3,4,5-tetra-o-galloylquinic acid

Cynarin Chlorogenic acid

mono-caffeoylquinic-acid di-caffeoylquinic-acid

Galloyl- and caffeoylquinic-acids are characterized

by one free carboxylic group in the quinic acid

moiety

Caffeoylquinic acids are the esters of quinic acids

with caffeic, ferulic or p-coumaric acids

Galloylquinic acids are the esters of quinic acid with

gallic acids

Their biological activity includes anti-HIV,

antiasthamatic, bronchial hyper reactivity, allergic

reactions and anti-inflamatory properties

Caffeoylquinic acids are antidiabetic, hypoglycemic,

antihepatitis, hepatoprotective and anti cancer agents

Proposed binding mechanism of bismuth citrate and zirconium silicate with carboxylic group of phenolic acids

Zirconium Silicate

45 µm

Bismuth Citrate

45 µm

Hussain, Shah, et al. Journal of Pharmaceutical and Biomedical Analysis (2013) in press

Tetragalloyl-quinic acids

Crystalline forms of Boron Nitride

β or cubic BN

• diamond analog

• stable

• very hard

• black

γ or wurzite BN

• lonsdaleit analog

• only stable under high

pressure

α or hexagonal BN

• graphite analog

• chemically inert

• nontoxic

Recovery for the enrichment of 5 Phenols

Phenol 4-Nitrophenol 2-Chlorphenol 2-Nitrophenol Dimethylphenol

[12.5 µg/ml] [12.5 µg/ml] [12.5 µg/ml] [12.5 µg/ml] [12.5 µg/ml]

87.51 94.74 108.08 91.29 100.71

93.97 102.95 114.70 95.20 111.82

91.36 101.91 113.24 96.88 105.84

Average 90.95 99.87 112.01 94.46 106.13

RSD 2.66 3.65 2.84 2.34 4.54

5 mg of BN was incubated with a phenol mixture and afterward eluted with

80% acetonitrile in water.

BADGE·2H2O BADGE

BPF BPA

BPZ

Bisphenol derivatives

MW pKa log Kow R2 LOD

[ng/ml] LOQ

[ng/ml]

BADGE·2H2O 376.44 14.7b 2.05b 0.999 27.0 82.0

BPF 200.23 7.5a 2.91a 0.999 27.0 82.0

BPA 228.29 9.6a 3.32a 0.999 33.0 99.0

BPZ 268.35 9.7b 4.53b 0.999 28.0 85.0

BADGE 340.41 - 4.02a 0.999 30.0 90.0

• The French Agency for Food, Environmental and Occupational Health & Safety showed that

there are ‘recognized’ effects in animals (effects on reproduction, on the mammary gland, on

metabolism, the brain and behaviour) and other ‘suspected’ effects in humans (on

reproduction, metabolism and cardiovascular diseases). These effects could be observed, even

at low levels of exposure, during sensitive phases of an individual’s development.

• The French Agency for Food, Environmental and Occupational Health & Safety endorses the

conclusions of the Expert Committee on Assessment of the risks related to chemical

substances relating to the risks associated with BPA for human health, and on toxicological

data and data on the use of bisphenols M, S, B, AP, AF, F and BADGE.

• Endokrine Disruptoren

Risk associated with bisphenols

Tolerable Daily Intake (TDI) of 50 µg BPA/kg body weight (b.w.)/day as set by EFSA in 2006.

The migration limit of BPA set by European Union of 600 µg/kg food.

Tolerable Daily Intake (TDI) of 150 µg BADGE and its hydrolytic products/kg body weight (b.w.)/day.

The migration limit of BADGE and its hydrolytic products 9000 µg/kg food.

http://www.efsa.europa.eu/en/topics/topic/bisphenol.htm

http://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htm

Analysis of 5 bisphenol derivatives after enrichment

with BN by LC and fluorescence detector

HPLC-FLD chromatogramms of (A) standard solution containing 5 bisphenol

derivatives [100 ng/mL] and (B) bisphenol leached from baby bottle after

enrichment with h-BN-60. Chromatographic conditions: Shimadzu LC-10 Acvp, Phenomenex Luna 5u C18 250*4,6 mm, isocratic 3 min 30% B,

gradient 30-70% B in 15 min, flow rate 1mL/min, 50°C, inj.: 30 µL,FLD: 230/303 nm

Recovery rates for the enrichment of 5 bisphenol

derivatives

Recovery rates ± SD [%]

Concentration

[ng/ml] BADGE·2H2O BPF BPA BPZ BADGE

0.5 90.01 ± 5.65 96.43 ± 8.17 111.93 ± 3.8 95.40 ± 5.87 85.38 ± 11.53

1 93.69 ± 2.57 98.88 ± 2.57 98.13 ± 4.05 100.90 ± 1.84 91.43 ± 4.47

2 99.47 ± 1.91 100.92 ± 1.13 95.88 ± 4.21 102.11 ± 3.23 97.48 ± 1.97

10 97.39 ± 2.41 102.47 ± 2.42 103.03 ± 4.21 101.67 ± 2.56 99.17 ± 4.15

100 98.67 ± 5.78 101.55 ± 4.45 101.54 ± 4.47 100.23 ± 2.64 87.51 ± 7.78

Found ± SD [ng/ml] Recovery rates ± SD [%]

BADGE·2H2O BPF BPA BPZ BADGE

river watera nd

(92.13 ± 2.69)

nd

(102.62 ± 3.73)

nd

(107.14 ± 6.50)

nd

(104.95 ± 1.92)

nd

(98.74 ± 2.57)

drinking watera nd

(90.16 ± 2.40)

nd

(98.56 ± 3.51)

nd

(99.16 ± 1.60)

nd

(101.14 ± 2.58)

nd

(96.68 ± 3.35)

colab 4.63 ± 0.14

(103.41 ± 4.39)

2.23 ± 0.21

(96.98 ± 2.39)

10.34 ± 0.41

(96.93 ± 5.72)

nd

(92.46 ± 6.58)

1.49 ± 0.04

(96.57 ± 3.84)

apple juiceb 1.93 ± 0.15

(96.43 ± 3.63)

3.11 ± 0.30

(90.77 ± 3.63)

nd

(84.22 ± 2.20)

nd

(98.74 ± 1.23)

1.36 ± 0.22

(98.22 ± 2.71)

lemon sodab 8.14 ± 0.09

(82.46 ± 3.01)

1.40 ±0.07

(84.45 ± 5.44)

nd

(97.60 ± 1.81)

nd

(87.61 ± 2.94)

5.70 ± 0.04

(94.00 ± 2.61)

citrus soft drinkb 5.32 ± 0.71

(103.90 ± 2.58)

nd

(97.82 ±5.82)

1.04 ± 0.31

(98.31 ± 5.67)

nd

(98.05 ± 4.04)

1.56 ± 0.042

(99.63 ± 4.42)

herbal sodab nd

(100.45 ± 5.15)

nd

(99.07 ± 5.09)

nd

(104.43 ± 6.20)

nd

(105.07 ± 7.21)

nd

(108.42 ± 4.83)

energy drinkb 4.46 ± 0.07

(98.54 ± 9.21)

1.87

(91.55 ± 4.21)

5.57 ± 0.81

(93.55 ± 5.84)

nd

(100.76 ± 2.04)

nd

(103.60 ± 1.91)

canned mushroom liquidb 20.42 ± 1.19

(99.06 ± 6.88)

nd

(91.35 ±0.79)

8.60 ± 0.37

(98.47 ± 8.37)

nd

(103.71 ± 6.17)

1.81 ± 0.12

(99.63 ± 0.62)

pickled cucumber liquidb 1.81 ± 0.15

(97.53 ± 1.12)

2.23 ± 0.21

(96.02 ± 2.45)

125.00 ± 2.18

(109.12 ± 0.77)

nd

(103.36 ±3.80)

1.44 ± 0.06

(92.67 ± 0.71)

pickled onion liquidb 2.98 ± 0.08

(105.80 ± 3.90)

nd

(96.53 ± 8.20)

13.45 ± 0.63

(92.03 ± 4.70)

nd

(97.91 ± 3.12)

1.74 ± 0.00

(95.20 ± 0.63)

urineb nd

(100.45 ± 3.13)

nd

(105.67 ± 0.68)

nd

(92.80 ± 2.52)

nd

(105.26 ± 4.00)

nd

(103.70 ±3.45)

Determination of bisphenol derivatives

Glas

Dose

Leaching of BPA from polycarbonate products

leached BPA [ng/ml] ± SD (%)

Babybottle

1. treatment 20.79 1.05

Babybottle

2. treatment 19.82 0.72

muffin form 2.26 0.07

Syringe 0.85 0.03

Treatment conditions: boiling water for 1 h

Langmuir adsorption isotherms of Bisphenol A

on h-BN-60

M a

ds

[µg/m

g]

0

10

20

30

40

50

60

70

0 5 10 15 20 25 30

Cequ[µg/ml]

max. loading capacity of Bisphenol A = 60 µg/mg

Aufgabe 1

Analyt: Disaccharide

Welche Festphase? Warum?

Aufgabe 2

Analyt: Proteine


Aufgabe 3

Analyt: Pestizide


Grundlagen und Anwendung moderner Trennverfahren

Günther K. Bonn Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens

University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria

40

Teil 3 - Fällung

Komplexität von biologischen Proben

1. enorme Komplexität der Proben

2. geringe Konzentration der Zielanalyten

3. beschränkter dynamischer Bereich der analytischen Messgeräte

4. Störkomponenten (Detergenzien, Puffersysteme etc.)

MS

biologische Probe

Herausforderungen:

41

Einleitung Analytik der Biomoleküle

Matthias Rainer

42

22 Proteine machen 99% des gesamten Serumproteoms aus!

90% 10%


1. Enorme Komplexität von biologischen Proben

z.B. Blutserum

Matthias Rainer

43 Anderson, N. L. (2002) Mol. Cell. Proteomics 1: 845-867

Dynamischer Bereich von Blutplasma


2. Geringe Konzentration vieler Zielanalyten

Selektivität

Empfind-lichkeit

Geschwin-digkeit

Hoher Probendurchsatz

Verbesserte Detektion

Gezielte Analyse

Effizienz

Effiziente Probenvorbereitung


Matthias Rainer

Zusätzliche Komplexität durch Variationen von Proteinen

• Bei einem Pentapeptid (Länge 5 Aminosäuren) gibt es 205 = 3.200.000

Kombinationsmöglichkeiten

• Proteine haben eine Länge von 100 bis 1000 Aminosäuren, also 20100 -

201000 Kombinationsmöglichkeiten

• Drehbarkeit der C-C Bindung in der Kette

• Peptidbindung normalerweise in trans-, kann aber auch in cis-

Konformationen vorliegen

• Seitengruppen können modifiziert sein

• Posttranslationale Modifikationen (z.B. Glykosylierung, Phosphorylierung)

45


Das Verhalten von Proteinen in wässriger Lösung hängt von

der Art des Lösungsmittels ab, denn in Gegenwart von Salzen,

Säuren oder Laugen ändert sich das Löslichkeitsverhalten der

Proteine stark. Viele Methoden zur Trennung von Proteinen

machen sich diese Eigenschaft zunutze, so z.B. bei der Fällung

von Proteinen aus einer Lösung.

Fällung von Proteinen

46

Proteine können auf verschiedene Art in ihrer nativen Form gefällt werden:

• Fällung durch Aussalzen

• isoelektrische Fällung (Fällung am IEP)

• Fällung mit organischen Lösungsmitteln

• Co-Präzipitation


47


• Fällung durch Aussalzen


Das Aussalzen von Proteinen ist ein Spezialfall einer Fällungsreaktion, die als Trennverfahren bei der Reinigung von Proteinen eingesetzt wird. Dabei nutzt man aus, dass Proteine nur dann in Lösung vorliegen, wenn sie über eine ausreichende Hydrathülle aus Wassermolekülen verfügen. Werden der Lösung Salze zugesetzt, so binden die dissoziierten Ionen ihrerseits Wassermoleküle in ihrer Hydrathülle und entziehen diese den Proteinmolekülen. Ab einer bestimmten Salzkonzentration, die von der Art des Salzes und dem Protein abhängt, wird dieser Effekt so stark, dass das Protein nicht mehr in Lösung gehalten werden kann, d.h. es fällt aus der Lösung aus und bildet einen Niederschlag.

48


• isoelektrische Fällung (Fällung am IEP)


Praktisch lässt sich der IEP auch zur Fällung von Proteinen aus einer Lösung nutzen. Die Löslichkeit eines Proteins wird sehr stark durch den pH-Wert der Umgebung beeinflusst und erreicht am isoelektischen Punkt ein Minimum. Ober- oder unterhalb des IEP tragen alle Moleküle die gleiche Ladung (positiv oder negativ) und stoßen sich daher ab. Eine Zusammenballung zu unlöslichen Aggregaten ist durch die Abstoßung der Moleküle untereinander verhindert und das Protein bleibt in Lösung.

49


• Fällung mit organischen Lösungsmitteln


Aceton Precipitation

TCA Precipitation

Wessel Flügge Precipitation

50

51


Fällung mit organischen Lösungsmitteln

52



53




• Co-Präzipitation


54

Precipitation of Phosphoproteins by

Trivalent Lanthanide Ions

A Top-Down Approach

61

Protein phosphorylation

Protein dephosphorylation

Berg JM et al. 2010

Protein separation and isolation

2. Chromatographic techniques

IMAC Immobilized Metal ion Affinity Chromatography

MOAC Metal Oxide Affinity Chromatography

Berg JM et al. 2010 According to: Leitner A. 2010

Proposed Precipitation Mechanism

64

Lanthanide Phoshates (Solubility Products)

solubility products

[logKsp(M)]

ErPO4 -25.13 ± 0.11

EuPO4 -25.96 ± 0.03

TbPO4 -25.39 ± 0.04

Solubility products of the rare-earth phosphates

References:

Xuewu Liu and Robert H. Bryne, Geochimica et Cosmichimica Acta, 1997, Vol. 61,No8,pp. 1625-1633

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

65

Dissolvation

of pellet

MALDI-MS

on-pellet

digest

denaturation, tryptic digestion

30% formic acid

washing

precipitation

10 min, 70W microwave-assisted digest

MALDI-MS

Peptides Proteins

Scheme for Precipitation of Phosphoproteins by

Trivalent Europium-, Terbium- and Erbium- Ions

Precipitant

KH2PO4

Yüksel et al. Anal Bioanal Chem (2012) 403:1323–1331 66

Methods and instruments

MALDI-TOF MS: linear mode


MALDI-TOF MS: reflector mode

sign

al in

ten

sity

[a.

u.]

m/z

Erbium Protein

Standard

Wash 1

Wash 2

Pellet

Supernatent

sig

na

l in

ten

sity

[a

.u.]

m/z

Terbium

Wash 1

Wash 2

Pellet

Supernatent

Protein

Standard

αS1-casein (m/z ~24.5 kDa)

β-casein (m/z ~25.1 kDa)

Phosphoproteins cytochrom c (m/z ~11.7 kDa)

lysozyme (m/z ~14.4 kDa) myoglobin (m/z ~17 kDa)

BSA (m/z ~ 69 kDa)

Standardproteins

sig

na

l in

ten

sity

[a

.u.]

m/z

Europium

Wash 1

Wash 2

Pellet

Supernatent

Protein

Standard

Precipitation of Phosphoproteins from Standards by



Standart Eu

0

1

2

3

4

5

4x1

0In

tens.

[a.u

.]

1000 2000 3000 4000 5000 6000 Dam/z

β

β β

β

β

α1

β

ββ

α1

α1

α1

α1

α1

α1

α1

α1

α1

α1

α1

α-casein seq. Coverage

[%] Mascot Search

Score Error [ppm]

Er 94 90 117

Eu 94 79 106

Tb 92 98 117

β-casein seq. Coverage

[%] Mascot Search

Score Error [ppm]

Er 56 76 32

Eu 67 54 94

Tb 53 75 147

Europium

Peptide Mass-Fingerprint-Analyse

α1... αS1-Casein

β … β-Casein

On-Pellet Digest of Precipitated Phosphoproteins by



Bovine milk composition

87,1%

4,9% 3,9%

3,4% 0,7%

water

carbohydrates

fats

proteins

minerals

38%

9% 29%

9%

4% 9% 2%

α-S1-casein

α-S2-casein

β-casein

κ-casein

α-lactalbumin

β-lactoglobulin

others

according to Pavia DL. 1990 / Eigel WN et al. 1984

Milk proteins

protein (variant) mol. weight /

Da amino acids

c / g∙L-1 P

per mol SH | S-S per mol

pI

αS1-casein (B 8P) 23,614 199 12 – 15 8 0 | 0 4.4 – 4.8

αS2-casein (A 11P) 25,230 207 3 – 4 11 2 | 0 −

β-casein (A2 5P) 23,983 209 9 – 11 5 0 | 0 4.8 – 5.1

κ-casein (B 1P) 19,023 169 2 – 4 1 2 | 0 5.3 – 5.8

α-lactalbumin (B) 14,176 123 0.6 – 1.7 0 0 | 4 4.2 – 4.5

β-lactoglobulin (B) 18,363 162 2 – 4 0 1 | 2 5.1

serum albumin 66,267 582 0.4 0 1 | 17 4.7 – 4.9

according to: Eigel WN et al. 1984 / Fox PF et al. 1998

sig

na

l in

ten

sity

[a

.u.]

m/z

Terbium

Wash 1

Wash 2

Pellet

Supernatent

milk

sig

na

l in

ten

sity

[a

.u.]

m/z

Erbium

Wash 1

Wash 2

Pellet

Supernatent

milk

sig

na

l in

ten

sity

[a

.u.]

m/z

Europium

PPC-5 (m/z ~12–13 kDa)

α-lactalbumin (m/z ~14.1 kDa)

β-lactoglobulin (m/z ~18.3 kDa)

non-phosphorylated milk-proteins


β-casein (m/z ~25.1 kDa)

phosphoproteins


κ-casein (m/z ~20.0 kDa)

Wash 1

Wash 2

Pellet

Supernatent

milk

Precipitation of Phosphoproteins from Bovine Milk by



0.0

0.5

1.0

1.5

2.0

2.5

4x1

0In

tens.

[a.u

.]

1000 2000 3000 4000 5000 6000m/z

α1

κ β

κ

Da

κ

α1

α1α1

α1

α2

α1

α1

α1

ββ β

ββ

α2

α2

α2

α2

α2

α2

β

Milk Tb

Eu seq. Coverage [%] Mascot Search Score Error [ppm]

α(S1)-casein 95 74 81 α(S2)-casein 76 68 55

β-casein 25 72 53 κ-casein 50 68 46

Tb seq. Coverage [%] Mascot Search Score Error [ppm]


β-casein 46 69 77 κ-casein 50 56 118

Er seq. Coverage [%] Mascot Search Score Error [ppm]


β-casein 25 55 44 κ-casein 50 60 68

Terbium

α1... αS1-Casein

α2... αS2-Casein

β … β-Casein

κ … κ-Casein

Peptide Mass-Fingerprint-Analysis

On-Pellet Digest of precipitated Phosphoproteins from Bovine Milk

by Trivalent Europium-, Terbium- and Erbium- Ions


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)

non phosphorylated egg-white proteins 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



ov

ov

ov

ov

ov

ov

ot

ov

ov

ot

otov

ot

ov

0.0

0.2

0.4

0.6

0.8

1.0

x10

Inte

ns. [

a.u.

]

1000 1500 2000 2500 3000 3500 4000 4500 5000 5500

m/zDa

1.2

ot

ov

ov

ot

ov

ov ov

ot

ot

ov

Eu seq. Coverage [%] Mascot search

Score Error [ppm]

ovalbumin 74 62 169

ovotransferrin 52 118 153

Tb seq. Coverage [%] Mascot search

Score Error [ppm]

ovalbumin 59 75 169


Er seq. Coverage [%] Mascot search

Score Error [ppm]

ovalbumin 60 113 172


Europium Peptide Mass-Fingerprint-Analysis

ov... Ovalbumin

ot... Ovotransferrin

On-Pellet Digest of precipitated Phosphoproteins from Egg-

White by Trivalent Europium-, Terbium- and Erbium- Ions


2M Ln3+-Chloride

UV/VIS

take supernatant

bicinchoninic acid (BCA)

chelation of two bicinchoninic acid molecules with Cu+1

reduction of Cu+2 to Cu+1 in the presence of proteins (alkaline conditions)

colorimetric detection 562 nm

Recovery Study of Phosphoprotein

77


Colorimetric assays: Bradford and BCA

Bradford reagent: Coomassie Brilliant Blue G-250

BCA assay reaction

Lottspeich F. 2012

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%

Precipitation of Phosphoproteins by


Recovery Study

ccasein = 300µg/ml cprecip. = 2M

79

Precipitation of Phosphopeptides by

Trivalent Lanthanide Ions

A Bottum-Up Approach

80

A Novel Strategy for Phosphopeptide Enrichment using Lanthanide Phosphate Precipitation

Workflow for the precipitation of phosphorylated peptides 81


MALDI mass spectra taken from peptide mixture (α-casein, β-casein and ovalbumin) 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+. Phosphorylated peptides are labeled.

Erbium

Holmium

Cer

82


MALDI mass spectra taken from peptide mixture (α-casein, β-casein and ovalbumin) after precipitation with trivalent lanthanide ions. A, phosphopeptide enriched by precipitation with La3+. B, phosphopeptide enriched by precipitation using Eu3+. C, phosphopeptide enriched by precipitation using Tm3+. Only phosphorylated peptides are labeled

Lanthanum

Europium

Terbium

83


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

Cer

84


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

Cer

85


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

86

[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))

Mono

Mono

Di

Mono

Mono

Mono

Mono

Mono

Di

Mono

Mono

Mono

Mono

Mono

Mono

Tetra

Tri

Mono

Penta

Tetra

Di

Mono

Tri

Tetra

Tetra

Tetra

Penta

Tetra

Tetra

-

-

-

+

+

+

+

-

+

+

+

+

+

-

+

+

+

+

+

+

+

+

+

-

+

+

+

+

+

-

-

-

+

+

+

+

+

+

+

+

+

+

-

+

+

-

+

-

-

+

-

+

-

+

+

+

+

+

-

-

-

+

+

+

+

+

+

+

+

+

+

-

+

+

-

+

-

-

+

+

-

+

+

+

-

+

+

-

-

-

+

+

+

+

-

+

+

+

+

+

-

+

+

-

+

+

-

-

+

+

-

+

+

+

+

+

-

-

-

+

+

+

+

+

+

+

+

+

+

-

+

+

-

+

+

-

+

-

+

-

+

+

+

+

+

-

-

-

+

+

+

+

+

+

+

+

+

+

-

+

-

-

-

-

-

-

+

-

-

-

-

-

-

-

-

-

-

+

+

+

+

-

+

+

+

+

-

-

-

-

-

+

+

-

-

-

-

-

+

+

-

+

+

-

-

-

+

+

+

+

-

+

+

+

+

-

-

+

-

+

+

+

-

+

-

+

-

+

-

+

+

+

Recovery of Phosphopeptides

23 20 19 20 21 12 14 18

P P

P

P P

3.6% HCl

P P

P P

P

trypsin

Tryptic On-Pellet Digest of Precipitated Phosphoproteins

Inte

nsity

m/z 88

Workflow

Development of New Bioanalytical Tools and Methods for the Enrichment of Phosphorylated Peptides and Proteins

Güzel, Y.; Rainer, M. (); Mirza, M.R.; Messner, C.B.; Bonn, G.K. Highly Selective Recovery of Phosphopeptides using Trypsin-Assisted Digestion of Precipitated Lanthanide-Phosphoprotein Complexes. Analyst (2013) 138(10), 2897-2905.

LaPO4

P P

P

P P

washing

LaPO4

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

proteinmixture milk 1:100 dilution

[M+H]+ Position Protein Phosphopeptide sequences Phospho groups LaCl3 CeCl3 TiO2 LaCl3 CeCl3 TiO2 LaCl3 CeCl3 1466.6 153-164 α-S2 TVDMESpTEVFTK mono + + - + + + + +

1482.6 153-164 α-S2 TVDM*ESpTEVFTK mono + + - - + - + +

1594.7 153-165 α-S2 TVDMESpTEVFTKK mono + + + + + + + +

1610.7 153-165 α-S2 TVDM*ESpTEVFTKK mono + + - - + - + +

1660.7 121-134 α-S1 VPQLEIVPNSAEER mono + + + + + + + +

1832.8 104-119 α-S1 YLGEYLIVPNSpAEER mono + + + + + + + +

1847.6 58-73 α-S1 DIGSESpTEDQAMEDIK mono + + - - - - - -

1927.6 58-73 α-S1 DIGSpESpTEDQAMEDIK di + + - + + + + +

1943.6 58-73 α-S1 DIGSpESpTEDQAM*EDIK di + + + + + + + +

1951.9 119-134 α-S1 YKVPQLEIVPNSpAEER mono + + + + + + + +

1981.8 48-63 β FQSpEEQQQTEDELQDK mono + + + + - + + -

2000.0 118-134 α-S1 KYKVPQLEIVPNSpAEER mono + + - + + - - +

2061.8 48-63 β FQSpEEQQQTEDELQDK mono + + + + + + - -

2080.0 118-134 α-S1 KYKVPQLEIVPNSpAEER mono + + + + + + + +

2432.0 45-63 β IEKFQSpEEQQQTEDELQDK mono + + - + + - - -

2548.2 119-139 α-S1 YKVPQLEIVPNSpAEERLHSMK mono + + - + + - + +

2560.1 44-63 β KIEKFQSpEEQQQTEDELQDK mono + + - + + - - -

2564.6 119-139 α-S1 YKVPQLEIVPNSpAEERLHSM*K mono + + - + + - + +

2598.0 52-73 α-S1 VNELSpKDIGSpESpTEDQAMEDIK di + + - + + - + +

2678.0 52-73 α-S1 VNELSpKDIGSpESpTEDQAMEDIK tri + + - + - + + +

2703.5 114-135 α-S1 LRLKKYKVPQLEIVPNSpAEERL mono + + - + + + - -

2716.2 130-152 α-S2 NAVPITPTLNREQLSpTSpEENSKK di + + - + + - + +

2720.9 74-94 α-S1 QMEAESpISpSpSpEEIVPNSpVEQK penta + + - + + - - -

2747.1 17-37 α-S2 NTMEHVSpSpSpEESpIISQETYKQ tetra + + + + + + - -

2856.5 50-73 α-S1 EKVNELSpKDIGSESTEDQAMEDIK di + + - + + - + +

2935.1 50-73 α-S1 EKVNELSpKDIGSpESTEDQAMEDIK tri + + - + - - + +

2966.1 17-40 β ELEELNVPGEIVESpLSpSpSpEESITR tetra + + + + + - - -

3008.0 61-85 α-S2 NANEEEYSIGSpSpSpEESpAEVATEEVK tetra + + + + + + + +

3024.2 16-40 β RELEELNVPGEIVESLSpSpSpEESITR tri + + + + + - - -

3042.2 16-40 β RELEELNVPGEIVESLSpSpSpEESITR tri + + + + + - - -

3087.9 61-87 α-S2 NANEEEYSpIGSpSpSpEESpAEVATEEVK penta + + - + + - - -

3122.2 16-40 β RELEELNVPGEIVESpLSpSpSpEESITR tetra + + + + + + + +

3132.2 16-39 α-S2 KNTMEHVSpSpSpEESpIISQETYKQEK tetra + + - + + + + +

34 15 31 16 22

Dephosphorylated HeLa cell lysate (1 mg/mL) with spiked α- and ß-casein (5 μg/mL) 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

HeLa cell lysate - digest

spiked cell lysate after precipitation


On-pellet digest of precipitated α-/ß-casein from spiked cell lysates

crude saliva digest

precipitated fraction CeCl3

Rainer, M. (), Güzel, Y., Messner, C., & Günther Bonn (2014). Co-Precipitation of Phosphorylated Proteins Using Trivalent Cerium-, Holmium-, and Thulium Cations. Current Pharmaceutical Analysis, 10(3), 175-184.


On-pellet digest of precipitated proteins from Human Saliva

92

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

Gelelektrophorese

Structure of the Repeating Unit of Agarose, 3,6-anhydro-L-galactose

Basic disaccharide

repeating units of

agarose,

G: 1,3-β-d-galactose

and

A: 1,4-α-l-3,6-

anhydrogalactose

Gel Structure of Agarose

Crosslinking Acrylamide Chains

Improvements in 2D-PAGE

- +

- IPG (Immobilised Ph Gradient) strips for the first dimension

pH-forming chemical groups are grafted onto the polyacrylamide matrix, creating a mechanically stable pH gradient

+ + mechanical stability

+ + reproducibility

+ + loadable amounts

+ + „zoom“ pI ranges

pH 3.0 10.0

1st dimension

Protein sample can be applied at sample application well following

the rehydration step if the protein sample was not included in the

rehydration solution.

Place assembled strip holder on electrophoresis platform

Equilibration step 1 :

• SDS

• Buffer pH 6.8

• DTT (reduce –S-S-)

• Iodoacetamide

(alkylate –S-S-)

After IEF : equilibration and 2nd dimension

+ -

6.5 10 3.0 5.5 8.5

pI

100

75

50

37

20

10

150

kDa

Size

horizontal gel electrophoresis

Electrophoresis Equipment

Vertical gel

Electrophoresis Equipment

Combs are used to put wells in the cast gel for sample loading.

– Regular comb: wells separated by an “ear” of gel

– Houndstooth comb: wells immediately adjacent

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